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Toxicity of α-Fe2O3 nanoparticles to Artemia salina cysts and three stages of larvae
Science of the Total Environment 598 (2017) 847–855
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Toxicity of α-Fe2O3 nanoparticles to Artemia salina cysts and three stages of larvae Chunjie Wang ⁎, Huali Jia ⁎, Lili Zhu, Hui Zhang, Yunsheng Wang College of Chemistry and Chemical Engineering, Zhoukou Normal University, Zhoukou, Henan Province 466000, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Significant effects on hatchability, mortality, and other end-points • Effects are accounted for α-Fe2O3-NPs and mediated by oxidative stress. • Instar II larvae show the greatest sensitivity to α-Fe2O3-NPs. • NPs were distributed in nephridial duct, primary body cavity and intestine. • The uptake kinetics was shown.
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Article history: Received 25 February 2017 Received in revised form 14 April 2017 Accepted 24 April 2017 Available online xxxx Editor: D. Barcelo Keywords: Toxicity Brine shrimp Iron oxide nanoparticles Oxidative stress Uptake
a b s t r a c t Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) were exposed to α-Fe2O3 nanoparticles (α-Fe2O3-NPs) to evaluate the effects on marine ecosystems. Hatchability, mortality and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Results indicate that the hatchability of capsulated and decapsulated cysts was significantly decreased (p b 0.01) following exposure to 600 mg/L at 12, 18, 24 and 36 h. Both increases of mortality and decreases of swimming speed were shown concentration-dependent manners. The LC50 values for instar II and III were 177.424 and 235.495 mg/L, respectively (not calculable for instar I), the EC50 values for instar I, II and III were 259.956, 99.064 and 129.088 mg/L, respectively. Instar II larvae show the greatest sensitive to α-Fe2O3-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths and individual dry weight of instar I, II and III larvae were decreased following exposure. α-Fe2O3-NPs attached onto the gills and body surface of larvae, resulting in irreversible damages. All of malondialdehyde content, total antioxidant capacity, reactive oxygen species and antioxidant enzymes activities were substantially increased in dose-dependent manners after exposure to α-Fe2O3-NPs suspensions, indicating that toxic effects were mediated by oxidative stress. Finally, the uptake result indicated that α-Fe2O3-NPs were ingested and distributed in the nephridial duct, primary body cavity and intestine of A. salina. Moreover, the uptake kinetics data show that the maximum α-Fe2O3-NPs content (8.818 mg/g) was reached at 36 h, and a steady state was reached after 60 h. The combined results indicate that α-Fe2O3-NPs have the potential to affect aquatic life when released into the marine ecosystems. © 2017 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author. E-mail addresses: [email protected] (C. Wang), [email protected] (H. Jia).
http://dx.doi.org/10.1016/j.scitotenv.2017.04.183 0048-9697/© 2017 Elsevier B.V. All rights reserved.
With the rapid development of nanotechnology, tremendous interest has arisen in the field of nano-materials. Due to their distinctive
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characteristics, nanomaterials have broad applications in various fields (Aragay and Merkoçi, 2012; Giersig and Khomutov, 2008; Xu et al., 2012). As one of the most important magnetic nanomaterials, α-Fe2O3 nanoparticles (α-Fe2O3-NPs) are being used in broad areas, such as sewage treatment, biomedical, electrochemical and photocatalytic applications (Alagiri and Hamid, 2014; Gao et al., 2009; Predescu and Nicolae, 2012; Wu et al., 2008). According to a previous report, the global market for magnetic nanoparticles in electronic, magnetic, and optoelectronic applications was exceeded $1.7 billion by 2012 (http://vww. nanotechwire.com/news.asp?nid=5395). With the rapidly increasing production and application of α-Fe2O3NPs worldwide, they will be likely released into the environment at significant levels. Considerable α-Fe2O3-NPs release could occur during their applications, such as wastewater treatment (Predescu and Nicolae, 2012), biomedical and architectural application (Basilevsky and Shamov, 2003; Khoshakhlagh et al., 2012). In addition, α-Fe2O3NPs could be introduced into the environment with the discharge of wastewater from the production processes. Eventually, most of the released nanoparticles will enter into the aquatic environment, especially into marine environment (Chen et al., 2012; Scown et al., 2010). Therefore, the subsequent impacts of α-Fe2O3-NPs to marine ecosystem have drawn significant attentions. In recent years, using aquatic invertebrates as models to assess toxicological effects of environmental contaminants has become prevalent (Hu et al., 2012). Artemia salina (A. salina) is an invertebrate zooplankton found in various marine ecosystems. As one of the most popular live foods for fish larvae, A. salina plays a pivotal role in the energy flow of the food chains (Nunes et al., 2006). A. salina is a non-selective filter feeder, and filters a lot of water per hour. Therefore, it has significant interactions with aquatic environment, causing it faces a higher risk exposure to environmental contaminants compared with other aquatic species (Ates et al., 2015; Nunes et al., 2006). The intrinsic features of A. salina turn it into a suitable organism for studies in toxicology. For example, according to the differences in tissue differentiation and morphological characteristics, many stages are divided along the development process of A. salina. Previous studies showed that A. salina larvae exhibit discrepant sensitivity to pollutants in relation to the stages (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003; Sorgeloos et al., 1979). Besides, varied end-points can be selected as criterions for toxicological evaluation, such as hatchability, mortality, and a number of ethological, morphological and biochemical parameters (Ates et al., 2016; Caldwell et al., 2003). For aquatic organisms, swimming represents an ethological response determinant that can be directly affected by physiological status (Gambardella et al., 2014). Biochemical parameters of oxidative stress have been proposed for the evaluation of potential toxic effects of NPs, such as reactive oxygen species (ROS), malondialdehyde (MDA) and antioxidant enzymes activities (Ates et al., 2013b; Gambardella et al., 2014). In recent years, an increasing number of studies have investigated the effects of nanoparticles (e.g., TiO2, Al2O3 and NiO NPs) on A. salina (Ates et al., 2013a, 2016, 2015). Nevertheless, the related information in concerning the effects of α-Fe2O3-NPs on A. salina is currently limited. The present study was conducted to evaluate the acute toxicity of αFe2O3-NPs on both cysts (capsulated and decapsulated) and larvae (instar I, II and III) of A. salina. Hatchability, mortality, and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Microscope and transmission electron microscope (TEM) were used to observe the uptake and distribution of α-Fe2O3-NPs in A. salina. Moreover, the uptake kinetics of α-Fe2O3-NPs in A. salina was assessed. 2. Materials and methods 2.1. Preparation and characterization of α-Fe2O3-NPs The α-Fe2O3-NPs were purchased from Beijing Dk Nano technology Co., Ltd. (Beijing, China), and the structural parameters are listed in
Table S1. Scanning electron microscopy (SEM) analysis was carried out on a Hitachi S-4800 electron microscope (Japan) with an accelerating voltage of 15 kV. TEM observations were made on a JEM-1200EX electron microscope (Japan) operating at 80–100 kV. Fourier transform infrared (FTIR) spectra were recorded from 400 to 800 cm− 1 with a Bruker Vetex70 spectrophotometer (Germany) using KBr pellet technique (Wang et al., 1998). X-ray diffraction (XRD) analysis was performed on a Bruker D8 Advance diffractometer (Germany) with CuKα radiation (λ = 1.54060 Å), and the sample was scanned from 10° to 80° (2θ) with a scanning rate of 1° min−1. Diffraction peaks were compared with those of standard compounds reported in the JCPDS data file. α-Fe2O3-NPs were suspended in filtered natural seawater (FNSW; 30‰ m/v; pH 8.6) to create suspensions with concentration as required. To assess Fe3+ released from α-Fe2O3-NPs, the suspensions were centrifuged at 12,000 rpm for 30 min to pellet α-Fe2O3-NPs. The Fe3+ content in supernatants were then determined using inductively coupled plasma mass spectrometry (ICP-MS, Jarrell-Ash, MA). A dynamic light scattering (DLS, Brookhaven BI-200SM, USA) was used to estimate the hydrodynamic size distribution of α-Fe2O3-NPs in FNSW. 2.2. Model organism The A. salina cysts were purchased from Binzhou Haifa Biological Technology Co., Ltd. (Shandong, China). The dehydrated cysts were first hydrated in distilled water at 4 °C for 12 h, and the sunken cysts were collected on a Buchner funnel. In order to acquire decapsulated cysts, a solution of NaOCl, NaOH and water was used as described by Sorgeloos et al. (1986). Approximately 2 g of cysts were incubated in 1 L FNSW in a hatcher at 28 °C, with a continuous 1300 lx light regime and strong aeration. Instar I, II and III larvae were obtained by using the procedure described by Sorgeloos et al. (1979). Briefly, to obtain a population consisting only of instar I, the larvae were separated from the unhatched cysts within 2 h after the first free-swimming larva was observed. One-third of the population was used immediately for the tests on the instar I larvae, and the other larvae were maintained for another 24 and 48 h to obtain instar II and III larvae, respectively. 2.3. Hatching assay The capsulated and decapsulated cysts were cultivated in α-Fe2O3NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) to study the effects of α-Fe2O3-NPs on the hatchability. In order to evaluate the influence of Fe3 + released from α-Fe2O3-NPs on the hatchability, the αFe2O3-NPs suspensions were centrifuged at 12,000 rpm for 30 min and the capsulated and decapsulated cysts were cultivated in the supernatants. Hatching assay was performed in 24-well plates, and each well contained 1 mL test solution. Ten capsulated/decapsulated cysts were introduced into each well, and each treatment was taken out in octuplicate. All plates were incubated under a continuous illumination with shaking at 28 °C. The hatchability was detected using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan) at 12, 18, 24 and 36 h. 2.4. Acute toxicity test The acute toxicity test was performed by adding 10 larvae (instar I, II and III) to each well of 24-well plates that contained 1 mL of α-Fe2O3NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or supernatants. The plates were incubated at 28 °C with shaking under a 16:8 h light/dark cycle. The larvae were not fed during the test. All of the tests were taken out in octuplicate. After 24 h, the numbers of dead larvae (completely motionless) were counted using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Larvae (instar I, II and III) were also randomly distributed into beakers (approximately 1000 larvae in each beaker) containing 100 mL of α-Fe2O3-NPs suspensions, and cultured as described above. After 24 h, the larvae were randomly sampled immediately prepared for
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morphological and behavioral analyses and ROS measurements. Samples were fixed in 2.5% glutaraldehyde for SEM and TEM analysis. Specimens for MDA content, total antioxidant capacity (T-AOC) and enzyme activity analysis were frozen in liquid nitrogen and stored at −80 °C.
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Fe by ICP-MS (Thermo Elemental X7, USA). The ferric contents were calculated according to the standard curve and then translated into corresponding α-Fe2O3-NPs contents. 2.10. Statistical analysis
2.5. Morphological and behavioral analysis After exposure for 24 h, 20 surviving larvae (instar I, II and III) were randomly selected for morphological and behavioral analysis. The swimming was recorded using a swimming behavior recorder that consisted of a video camera (Nikon, Japan) fixed on a microscope (Leica, Germany). The behavior recorder was placed in a dark room to exclude external sources of light. All larvae were dark-adapted for 5 min before the video recording to reach a steady speed, and then the swimming behavior was recorded. The result was shown as swimming inhibition, normalized to the average swimming speed of the control. Body length of A. salina was also recorded using the recorder. Individual dry weight of A. salina larvae was weighed following completely dried. In addition, a SEM (Hitachi S-4800, Japan) was used to investigate the attachment of NPs and the surface damage to A. salina. 2.6. ROS detection The fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) (Beyotime Biotech, Nantong, China) was used to determine the generation of ROS in larvae (instar I, II and III) following exposure to α-Fe2O3NPs for 24 h. In brief, approximately 500 larvae from each treatment were homogenized on ice in 500 μL ice-cold Tris–HCl buffer (100 mM, pH 7.4) using a homogenizer. The homogenates were then centrifuged at 12, 000 g for 15 min, after which the supernatants were collected for ROS detection. The detection was carried out in black 96-well plates and performed following the manufacturer's instructions. Fluorescence was measured on a microplate reader (Multiskan MK3, Thermo Labsystems Co., Beverly, MA) with an excitation and emission wavelength of 485 and 530 nm, respectively. All of the tests were carried out in triplicate. 2.7. MDA content and T-AOC and antioxidant enzymes activities After exposure for 24 h, approximately 500 larvae (instar I, II and III) were homogenized in 0.5 mL of ice-cold phosphate buffer for 5 min, and then centrifuged (12000g; 15 min) at 4 °C. The supernatants were collected for biochemical analysis. Total protein, MDA content, T-AOC and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] activities were measured using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. All of the tests were carried out in triplicate. 2.8. Uptake of α-Fe2O3-NPs The uptake of α-Fe2O3-NPs by A. salina was qualitatively examined using a microscope (Leica, Germany) at the end of the exposure. Images were captured by a digital camera (Nikon, Japan) from surviving larvae in petri dishes. At the same time, a TEM (JEOL, Tokyo, Japan) was also used to examine the uptake and distribution of α-Fe2O3-NPs in A. salina. 2.9. Uptake kinetics study To determine the uptake kinetics of α-Fe2O3-NPs in A. salina, 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. The cleaned samples were then dried using a freeze dryer (FD5-3, GOLD-SIM). To determine the ferric contents, 0.1 g of dry larvae were weighed and digested in 2 mL of trace metal grade nitric acid at 160 °C. The solutions were diluted to 5 mL with distilled water after completely digested and analyzed for
All experiments were repeated at least three times, and data were recorded as mean ± standard deviation (SD). The median effective concentration (EC50) for swimming speed alteration, median lethal concentration (LC50) and related 95% confidence limits were calculated using the Probit method. To perform statistical analysis, the SPSS Version 11.0 software package (SPSS Inc., Chicago, IL) was used. Data were analyzed for differences between the control and treatments using oneway ANOVA followed by Tukey's test, where p b 0.05 is considered significant and p b 0.01 extremely significant. 3. Results and discussion 3.1. α-Fe2O3-NPs characterization The physicochemical properties of nanoparticles, such as size and particulate state, have been proposed to be related to their uptake, bioaccumulation and toxicity (Ates et al., 2013b, 2015). In the study, αFe2O3-NPs were characterized by SEM, TEM, FTIR, XRD and DLS analysis (Fig. 1A–E). The SEM and TEM images of α-Fe2O3-NPs are shown in Fig. 1A and B, respectively, indicating that the α-Fe2O3-NPs are spherical with varying sizes. The FTIR spectra of α-Fe2O3-NPs are represented in Fig. 1C, and the characteristic absorption bands at 481 cm−1 and 574 cm−1 are assigned to α-Fe2O3 (Suresh et al., 2010). Fig. 1D shows the XRD spectrum of α-Fe2O3-NPs, all of the diffraction peaks are in accordance with the standard XRD card of rhombohedral α-Fe2O3 (JCPDS No. 87-1165). The peaks are sharp and no characteristic peak of impurities can be observed, indicating that the α-Fe2O3-NPs are wellcrystallized and high purity. Fe3+ released from α-Fe2O3-NPs was quantitatively measured using ICP-MS, and the data are shown in Table S2. Only a small fraction of the NPs was dissolved due to the high pH of the suspensions (pH = 8.6). The DLS data show that the hydrodynamic diameter of α-Fe2O3-NPs was ranged from 316 nm to 34.674 μm with a mean diameter of 8.134 μm (Fig. 1E). The hydrodynamic diameter is larger than the diameter which estimated by TEM (mean diameter: 44.56 nm; Fig. 1F). This result is due to the reduction of electrostatic repulsion in the FNSW and hydration of α-Fe2O3-NPs surfaces, and is consistent with previous findings (Ates et al., 2013a, 2015). Although aeration was provided to maintain homogeneity of the suspensions in the study, the agglomeration was inevitable. 3.2. Effects of α-Fe2O3-NPs and Fe3+ on hatchability Hatching rate of A. salina is a reliable and sensitive end-point, and has been used in many acute toxicity tests (Alyuruk and Cavas, 2013; Rotini et al., 2015). In this study, both capsulated and decapsulated cysts were conducted to evaluate the effects of α-Fe2O3-NPs on hatchability. As shown in Fig. 2, on the whole, for both capsulated and decapsulated cysts at 12, 18, 24 and 36 h, dose-dependent decreases in hatching rates are observed relative to the controls. Moreover, significant differences (p b 0.01) of hatching rates were found in 600 mg/L for capsulated and decapsulated cysts at 12, 18, 24 and 36 h compared to the controls. However, the data of dissolution test substantiate that αFe2O3-NPs did release a small amount of Fe3 + into the suspensions (Table S2), indicating that A. salina cysts and larvae were exposed to Fe3+ and aggregates of the NPs. Thus, the toxic effects may be attributed to Fe3+ from the dissolution of NPs. In order to elucidate the contribution of Fe3 + to the toxic effects, capsulated and decapsulated cysts were exposed to the supernatants. As shown in Fig. S1, there was no obvious influence of Fe3 + on the hatchability of capsulated and decapsulated cysts. Therefore, the results indicated that the toxic effects
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Fig. 1. The SEM image (A), TEM image (B), FTIR spectrum (C) and XRD pattern (D) of α-Fe2O3-NPs. Size distributions of α-Fe2O3-NPs detected by using DLS (E) and TEM (F).
Fig. 2. Hatching percentages of capsulated (A) and decapsulated (B) cysts exposed to different α-Fe2O3-NPs concentrations. Values are presented as mean ± SD. Values that are significantly different from the control are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
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are accounted for α-Fe2O3-NPs rather than Fe3+ from the dissolution of NPs. 3.3. Mortality rate, behavioral and morphological analysis The end-point selection for acute toxicity tests is sometimes a vital factor to be considered, most frequently, mortality of larvae, behavioral and morphological parameters and biomarkers are selected as criterions (Nunes et al., 2006). In this study, instar I, II and III larvae of A. salina were conducted to elucidate the toxic effects of α-Fe2O3-NPs. As shown in Fig. 3A, dose-dependent increases in mortality rates of instar I, II and III larvae following exposure to α-Fe2O3-NPs suspensions. Mortality rates of all control groups were 2%–5%, indicating that absence of food did not induce any lethal effects on larvae even up to 96 h. Following treatments, the mortalities was significantly increased (p b 0.01) in 200, 400 and 600 mg/L, and the mean mortality rates in 600 mg/L treatment groups were 48.85%, 77.63% and 71.90% for instar I, II and III, respectively. The LC50 values for instar II and III are 177.424 and 235.495 mg/L, respectively (not calculable for instar I; Table S3). A. salina are relatively resistant to metal ions toxicity, and can tolerate a wide range of Fe3+ concentration (Gajbhiye, 1990; Kokkali et al., 2011). Gajbhiye (1990) systematically investigated the toxic effects of metal ions to A. salina and demonstrated that the LC50 values for Fe3+ were 18.2 and 13.9 mg/L in 24 and 48 h, respectively. Apparently, the concentrations of Fe3 + from the dissolution of NPs were all lower than the 24 h LC50 value, ranging from 0.47 to 1.98 mg/L (Table S2). Larvae (instar I, II and III) were exposed to the supernatants to elucidate the contribution of Fe3+ to the mortality rates. As shown in Fig. S2, all the
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controls and treatments showed about 2 to 5% mortality, and no statistically significant (p N 0.05) was found. Therefore, the results indicated that the toxic effects of α-Fe2O3-NPs on mortality are accounted for NPs rather than Fe3+ from the dissolution of NPs. Behavior and morphology are determinants that results from physiological and ecological aspects of toxicology. In the study, the swimming speed of instar I, II and III larvae showed concentration–dependent decreases following exposure to α-Fe2O3-NPs suspensions, and was significantly decreased (p b 0.01) in 100, 200, 400 and 600 mg/L. Likewise, Gambardella et al. (2014) demonstrated that swimming speed was significantly decreased in A. salina larvae following exposure to CeO2 NPs (Gambardella et al., 2014). The EC50 values for swimming speed inhibition of instar I, II and III were 259.956, 99.064 and 129.088 mg/L, respectively (Table S3). Moreover, instar II and III larvae showed significantly (p b 0.01) lower EC50 compared with instar I larvae. As shown in Fig. 3C, the body lengths were decreased with the rising of α-Fe2O3NPs concentrations, and significant reduction (p b 0.01) was observed at 400 and 600 mg/L for instar II and III larvae. For individual dry weight, significant reduction (p b 0.01) was only observed at 600 mg/L compared with the controls (Fig. 3D). A small amount of studies investigated the toxic effects of some materials on different stages of A. salina larvae (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003). Caldwell et al. (2003) assessed the toxicity of algal extracts and short chain aldehydes to cysts and different stages of A. salina, and demonstrated that hatching assay shows a lower sensitivity compared with the mortality assays. In addition, Barahona and Sánchezfortún (1996) compared the sensitivity of three stages of A. salina larvae to several compounds, and
Fig. 3. The mortality rate (A), swimming inhibition (SI; B), body length (C) and individual dry weight (D) for instar I, II and III larvae exposure to different concentrations of α-Fe2O3-NPs. Values are presented as mean ± SD. Values that are significantly different from the control are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
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demonstrated that 48-hours larvae (instar II) are more sensitive than the 24 (instar I) and 72-hours (instar III) larvae. In the study, the results of hatching assay, mortality assay and behavioral and morphological analysis revealed that the sensitivity of cysts and larvae to α-Fe2O3NPs is in the order of instar II N instar III N instar I N decapsulated cysts N capsulated cysts. The conclusion is somewhat similar to the results of previous studies (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003). 3.4. Attachment of NPs and damages on the body surface Interactions of NPs with A. salina can be external, such as NPs attached onto the skin or exoskeleton, which cause direct damage to the lipid membranes. Mesarič et al. (2015) reported that carbon-based nanomaterials were extensively attached to the gills and caused the gill branches to fuse together, they also found the nanomaterials were attached onto the entire body surface of A. salina (Mesarič et al., 2015). In the study, the attachment of α-Fe2O3-NPs and damages on the body surface was checked by using a SEM, and the representative images are displayed in Fig. 4. Images of instar I, II and III larvae treated without α-Fe2O3-NPs are shown in Fig. 4A, B and C, respectively, indicating that the body surface was clean and undamaged. After exposure, α-Fe2O3-NPs were attached onto the gill (Fig. 4D) and body surface (Fig. 4E). In addition, some irreversible damages were observed, such as created “holes” (Fig. 4F–H) in the body surface and rupture of body surface (Fig. 4I).
derived from lipid peroxidation, and has been widely used as an indicator of oxidative damages to membranes and oxidative stress (Ates et al., 2013a, 2013b, 2015). The production of ROS following NPs treatment seems to be a key event of the toxic effects, and the imbalance between ROS formation and the T-AOC result in oxidative stress occurs. SOD, CAT and GPx are antioxidants that catalyze the decomposition of ROS, and can prevent organisms from adverse effects of oxidative stress (Cazenave et al., 2006). In the study, on the whole, all of the MDA content, T-AOC, ROS and antioxidant enzymes (SOD, CAT and GPx) activities were increased in dose-dependent manners after exposure to α-Fe2O3-NPs suspensions (Fig. 5). Significant increases (p b 0.01) were observed at 400 and 600 mg/L for all of the metabolites, suggesting that the physical decline of larvae following exposure to α-Fe2O3-NPs was related to the oxidative damages. Increases of MDA contents confirm that α-Fe2O3-NPs induced oxidative stress and caused damages on the body surface of A. salina. The production of ROS causes an elevation of SOD, CAT and GPx activities as defense mechanisms against oxidative stress. Consistent with the result, induction of oxidative stress has also been reported in other studies (Ates et al., 2013b; Gambardella et al., 2014; Taze et al., 2016). For example, Taze et al. (2016) investigated the toxicity of iron oxide nanoparticles in Mytilus galloprovincialis. They demonstrated that the iron oxide nanoparticles induced significant increase in ROS production, lipid peroxidation, protein carbonylation, ubiquitin conjugates and DNA damage. Moreover, they concluded that iron oxide nanoparticles caused adverse effects on physiology by causing oxidative stress in hemocytes of exposed mussels.
3.5. MDA content, T-AOC, ROS and antioxidant enzymes activities 3.6. Uptake of α-Fe2O3-NPs Some studies reported that the toxic effects of NPs on A. salina were due to oxidative stress (Ates et al., 2013a, 2013b). Changes on the levels of certain metabolites, such as MDA, ROS and antioxidant enzymes activities have been described as biomarkers of oxidative stress (Ates et al., 2015; Mesarič et al., 2015; Zhu et al., 2016). MDA is a metabolite
Interactions of NPs with A. salina also can be internal, such as NPs intake. A. salina is non-selective filter feeder, and can ingest particles smaller than 50 μm (Ates et al., 2013a, 2013b). A. salina has a very primitively ingestive behavior compared with other crustaceans, it is a
Fig. 4. SEM images of instar I (A), II (B) and III larvae (C) treated without α-Fe2O3-NPs. Attachment of α-Fe2O3-NPs (red arrows) onto gills (D) and body surface (E) of A. salina. Created “holes” (F–H) in body surface and rupture of body surface (I) after being directly contacted with α-Fe2O3-NPs, the black arrows are pointed to the morphological damages. Scale bars in D and G are of 2 μm, and 10 μm for remaining images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Measurement of MDA content (A) and T-AOC (B), ROS (C) and the changes in SOD (D), CAT (E) and GPx (F) activities in A. salina larvae (instar I, II and III) following exposure to different concentrations of α-Fe2O3-NPs. Values are presented as mean ± SD. Values that are significantly different from the controls are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
Fig. 6. Ingestion of α-Fe2O3-NPs (red arrows) by A. salina larvae. (A) The gut is empty in the control. (B) A. salina larvae start to ingest α-Fe2O3-NPs. (C) α-Fe2O3-NPs is visible as a dark line inside the gut of treatment. (D–F) TEM characterization of intracorporal localization of α-Fe2O3-NPs in A. salina larvae. Scale bars in A–C are of 300 μm. nd, nephridial duct; pbc, primary body cavity; inc, intestinal cell; in, intestine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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continuous non-selective, obligate phagotrophic filter-feeder (Provasoli and Shiraishi, 1959). Suspended particles (no matter what their nature is) with suitable size are continuously ingested by A. salina (Reeve, 1963). In the study, uptake of α-Fe2O3-NPs was checked under a microscope, and representative images are shown in Fig. 6. The gut for the control was empty (Fig. 6A), and larvae started to ingest α-Fe2O3-NPs (Fig. 6B) following exposure to the NPs. Gradually, the gut was almost entirely filled with α-Fe2O3-NPs, verified by a dark line inside the gut (Fig. 6C) of the larvae. Several studies also reported the uptake of NPs by A. salina (Ates et al., 2013a; Gambardella et al., 2014; Ozkan et al., 2015). TEM was used to check the distribution of NPs in A. salina. αFe2O3-NPs were visible within the nephridial duct (nd; Fig. 6D), primary body cavity (pbc; Fig. 6E) and intestine (in; Fig. 6F). 3.7. Uptake kinetics As shown above, α-Fe2O3-NPs were ingested and well distributed in A. salina. In order to quantitatively assess the uptake profile, the iron contents in A. salina were measured using ICP-MS. The contents were based on the dry weight of A. salina, reflecting the total body burden from 1 to 72 h. As shown in Fig. 7, result revealed a general increase during the first 36 h followed by a decrease from 36 to 60 h, and a steady state was reached after 60 h. Average NPs contents were ranged from 0.178 to 8.818 mg/g. Uptake of NPs was slowly increased during the first 12 h (instar I), probably due to the mouth and anus of larvae are not yet completely opened, and the digestive tract is not fully formed (yolk sac consumption period). Moreover, mouth size is generally correlated with body size, thus instar I larvae have a smaller mouth size compared with other stages. The small mouth size restricts the size of particles which can be ingested (Sorgeloos et al., 1986). After molt into the instar II, tissues and organs of A. salina are fully formed, and the mouth size are larger. Therefore, a fast accumulation of NPs was occurred. The maximum content was reached at 36 h (instar II). As mentioned before, instar II larvae show the greatest sensitivity to α-Fe2O3NPs. The high content may be responsible for the strong toxic responses. There is a decrease from 36 to 60 h, probably because of redistribution and discharge of α-Fe2O3-NPs. As shown above, NPs were visible within the nephridial duct, indicating that a portion of NPs may be eliminated by A. salina. A steady state was achieved after 60 h may be due to the accumulation and elimination was finally balanced. 4. Conclusion In this study, we evaluated the toxic effects of α-Fe2O3-NPs on A. salina to elucidate the impacts to marine ecosystems. The results so far pointed to the fact that the acute exposure of cysts (capsulated and
Fig. 7. The contents of α-Fe2O3 uptake by A. salina at different time points.
decapsulated) and larvae (instar I, II and III) to α-Fe2O3-NPs causes significant changes in hatchability, mortality, and ethological, morphological and biochemical parameters. The toxic effects were mediated by oxidative stress. Instar II larvae show the greatest sensitivity to αFe2O3-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts, indicating that instar II would be a suitable candidate for toxicological test. Although α-Fe2O3-NPs rapidly aggregate in seawater to form large particles, there is no effect on the uptake of the NPs. α-Fe2O3-NPs were accumulated in the gut and well distributed in nephridial duct and primary body cavity of A. salina. Moreover, the uptake kinetics data show that the accumulation of α-Fe2O3-NPs in A. salina was firstly increased and decreased then, and a steady state was reached in the end. The results revealed short-term effects (24 h) of α-Fe2O3-NPs on A. salina, for safe and commercial purposes, longterm treatments and other complementary studies must be undertaken. Acknowledgements This work is supported by the Natural Science Foundation of Henan Province (Program No. 162300410197), the Doctoral Scientific Research Foundation of Zhoukou Normal University (Program No. ZKNUB2013001) and the Scientific Research Innovation Foundation of Zhoukou Normal University (Program No. ZKNUA201701). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.04.183. References Alagiri, M., Hamid, S.B.A., 2014. Green synthesis of α-Fe2O3 nanoparticles for photocatalytic application. J. Mater. Sci. Mater. Electron. 25, 3572–3577. Alyuruk, H., Cavas, L., 2013. Toxicities of diuron and irgarol on the hatchability and early stage development of Artemia salina. Turk. J. Biol. 37, 151–157. Aragay, G., Merkoçi, A., 2012. Nanomaterials application in electrochemical detection of heavy metals. Electrochim. Acta 84, 49–61. Ates, M., Daniels, J., Arslan, Z., Farah, I.O., 2013a. Effects of aqueous suspensions of titanium dioxide nanoparticles on Artemia salina: assessment of nanoparticle aggregation, accumulation, and toxicity. Environ. Monit. Assess. 185, 3339–3348. Ates, M., Daniels, J., Arslan, Z., Farah, I.O., Rivera, H.F., 2013b. Comparative evaluation of impact of Zn and ZnO nanoparticles on brine shrimp (Artemia salina) larvae: effects of particle size and solubility on toxicity. Evnviron. Sci. Process. Impacts 2013, 225–233. Ates, M., Demir, V., Arslan, Z., Daniels, J., Farah, I.O., Bogatu, C., 2015. Evaluation of alpha and gamma aluminum oxide nanoparticle accumulation, toxicity, and depuration in Artemia salina larvae. Environ. Toxicol. 30, 109–118. Ates, M., Demir, V., Arslan, Z., Camas, M., Celik, F., 2016. Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater. Water Air Soil Pollut. 227, 1–8. Barahona, M.V., Sánchezfortún, S., 1996. Comparative sensitivity of three age classes of Artemia salina larvae to several phenolic compounds. Bull. Environ. Contam. Toxicol. 56, 271–278. Basilevsky, M.V., Shamov, A.G., 2003. Topical review: functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D. Appl. Phys. 36, R198–R206(199). Caldwell, G.S., Bentley, M.G., Olive, P.J.W., 2003. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. Toxicon 42, 301–306. Cazenave, J., Bistoni, M.L., Pesce, S.F., Wunderlin, D.A., 2006. Differential detoxification and antioxidant response in diverse organs of Corydoras paleatus experimentally exposed to microcystin-RR. Aquat. Toxicol. 76, 1–12. Chen, X., Zhu, X., Li, R., Yao, H., Lu, Z., Yang, X., 2012. Photosynthetic toxicity and oxidative damage induced by nano-Fe3O4 on in aquatic environment. Open J. Ecol. 02, 21–28. Gajbhiye, S.N., 1990. Toxicity of heavy metals to brine shrimp Artemia. J. Indian Fish. Assoc. 20. Gambardella, C., Mesarič, T., Milivojević, T., Sepčić, K., Gallus, L., Carbone, S., Ferrando, S., Faimali, M., 2014. Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses. Environ. Monit. Assess. 186, 4249–4259. Gao, J., Gu, H., Xu, B., 2009. Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc. Chem. Res. 42, 1097–1107. Giersig, M., Khomutov, G.B., 2008. Nanomaterials for Application in Medicine and Biology. Hu, J., Wang, D., Wang, J., Wang, J., 2012. Bioaccumulation of Fe2O3(magnetic) nanoparticles in Ceriodaphnia dubia. Environ. Pollut. 162, 216–222. Khoshakhlagh, A., Nazari, A., Khalaj, G., 2012. Effects of Fe2O3 nanoparticles on water permeability and strength assessments of high strength self-compacting concrete. J. Mater. Sci. Technol. 28, 73–82.
C. Wang et al. / Science of the Total Environment 598 (2017) 847–855 Kokkali, V., Katramados, I., Newman, J.D., 2011. Monitoring the effect of metal ions on the mobility of Artemia salina nauplii. Bios 1, 36–45. Mesarič, T., Gambardella, C., Milivojević, T., Faimali, M., Drobne, D., Falugi, C., Makovec, D., Jemec, A., Sepčić, K., 2015. High surface adsorption properties of carbon-based nanomaterials are responsible for mortality, swimming inhibition, and biochemical responses in Artemia salina larvae. Aquat. Toxicol. 163, 121–129. Nunes, B.S., Carvalho, F.D., Guilhermino, L.M., Van, S.G., 2006. Use of the genus Artemia in ecotoxicity testing. Environ. Pollut. 144, 453–462. Ozkan, Y., Altinok, I., Ilhan, H., Sokmen, M., 2015. Determination of TiO2 and AgTiO2 nanoparticles in Artemia salina: toxicity, morphological changes, uptake and depuration. Bull. Environ. Contam. Toxicol. 125, 1–7. Predescu, A., Nicolae, A., 2012. Adsorption of Zn, Cu and Cd from waste waters by means of maghemite nanoparticles. Upb Sci. Bull. 74. Provasoli, L., Shiraishi, K., 1959. Axenic cultivation of the brine shrimp Artemia salina. Biol. Bull. 117, 347–355. Reeve, M.R., 1963. The Filter-feeding of Artemia: II. In Suspensions of Various Particles. 40 p. 207. Rotini, A., Manfra, L., Canepa, S., Tornambè, A., Migliore, L., 2015. Can Artemia hatching assay be a (sensitive) alternative tool to acute toxicity test? Bull. Environ. Contam. Toxicol. 95, 745–751. Scown, T.M., Van, A.R., Tyler, C.R., 2010. Review: do engineered nanoparticles pose a significant threat to the aquatic environment. Crit. Rev. Toxicol. 40, 653–670.
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Sorgeloos, P., Wielen, R.V.D., Persoone, G., 1979. The use of Artemia nauplii for toxicity tests—a critical anaysis. Ecotoxicol. Environ. Saf. 2, 249–255. Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W., Versichele, D., 1986. Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Suresh, R., Vijayalakshmi, L., Stephen, A., Narayanan, V., 2010. Hydrothermal Synthesis and Characterization of Cobalt Doped α-Fe2O3. 1276 pp. 362–367. Taze, C., Panetas, I., Kalogiannis, S., Feidantsis, K., Gallios, G.P., Kastrinaki, G., Konstandopoulos, A.G., Václavíková, M., Ivanicova, L., Kaloyianni, M., 2016. Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis. Aquat. Toxicol. 172, 9–20. Wang, Y., Muramatsu, A., Sugimoto, T., 1998. FTIR analysis of well-defined α-Fe2O3 particles. Colloids Surf. A Physicochem. Eng. Asp. 134, 281–297. Wu, X.L., Guo, Y.G., Wan, L.J., Hu, C.W., 2008. α-Fe2O3 nanostructures: inorganic salt-controlled synthesis and their electrochemical performance toward lithium storage. J. Phys. Chem. C 112, 16824–16829. Xu, P., Zeng, G.M., Huang, D.L., Feng, C.L., Hu, S., Zhao, M.H., Lai, C., Wei, Z., Huang, C., Xie, G.X., 2012. Use of iron oxide nanomaterials in wastewater treatment: a review. Sci. Total Environ. 424, 1–10. Zhu, S., Zhu, B., Huang, A., Hu, Y., Wang, G., Ling, F., 2016. Toxicological effects of multiwalled carbon nanotubes on Saccharomyces cerevisiae: the uptake kinetics and mechanisms and the toxic responses. J. Hazard. Mater. 318, 650–662.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Toxicity of α-Fe2O3 nanoparticles to Artemia salina cysts and three stages of larvae Chunjie Wang ⁎, Huali Jia ⁎, Lili Zhu, Hui Zhang, Yunsheng Wang College of Chemistry and Chemical Engineering, Zhoukou Normal University, Zhoukou, Henan Province 466000, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Significant effects on hatchability, mortality, and other end-points • Effects are accounted for α-Fe2O3-NPs and mediated by oxidative stress. • Instar II larvae show the greatest sensitivity to α-Fe2O3-NPs. • NPs were distributed in nephridial duct, primary body cavity and intestine. • The uptake kinetics was shown.
a r t i c l e
i n f o
Article history: Received 25 February 2017 Received in revised form 14 April 2017 Accepted 24 April 2017 Available online xxxx Editor: D. Barcelo Keywords: Toxicity Brine shrimp Iron oxide nanoparticles Oxidative stress Uptake
a b s t r a c t Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) were exposed to α-Fe2O3 nanoparticles (α-Fe2O3-NPs) to evaluate the effects on marine ecosystems. Hatchability, mortality and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Results indicate that the hatchability of capsulated and decapsulated cysts was significantly decreased (p b 0.01) following exposure to 600 mg/L at 12, 18, 24 and 36 h. Both increases of mortality and decreases of swimming speed were shown concentration-dependent manners. The LC50 values for instar II and III were 177.424 and 235.495 mg/L, respectively (not calculable for instar I), the EC50 values for instar I, II and III were 259.956, 99.064 and 129.088 mg/L, respectively. Instar II larvae show the greatest sensitive to α-Fe2O3-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths and individual dry weight of instar I, II and III larvae were decreased following exposure. α-Fe2O3-NPs attached onto the gills and body surface of larvae, resulting in irreversible damages. All of malondialdehyde content, total antioxidant capacity, reactive oxygen species and antioxidant enzymes activities were substantially increased in dose-dependent manners after exposure to α-Fe2O3-NPs suspensions, indicating that toxic effects were mediated by oxidative stress. Finally, the uptake result indicated that α-Fe2O3-NPs were ingested and distributed in the nephridial duct, primary body cavity and intestine of A. salina. Moreover, the uptake kinetics data show that the maximum α-Fe2O3-NPs content (8.818 mg/g) was reached at 36 h, and a steady state was reached after 60 h. The combined results indicate that α-Fe2O3-NPs have the potential to affect aquatic life when released into the marine ecosystems. © 2017 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author. E-mail addresses: [email protected] (C. Wang), [email protected] (H. Jia).
http://dx.doi.org/10.1016/j.scitotenv.2017.04.183 0048-9697/© 2017 Elsevier B.V. All rights reserved.
With the rapid development of nanotechnology, tremendous interest has arisen in the field of nano-materials. Due to their distinctive
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characteristics, nanomaterials have broad applications in various fields (Aragay and Merkoçi, 2012; Giersig and Khomutov, 2008; Xu et al., 2012). As one of the most important magnetic nanomaterials, α-Fe2O3 nanoparticles (α-Fe2O3-NPs) are being used in broad areas, such as sewage treatment, biomedical, electrochemical and photocatalytic applications (Alagiri and Hamid, 2014; Gao et al., 2009; Predescu and Nicolae, 2012; Wu et al., 2008). According to a previous report, the global market for magnetic nanoparticles in electronic, magnetic, and optoelectronic applications was exceeded $1.7 billion by 2012 (http://vww. nanotechwire.com/news.asp?nid=5395). With the rapidly increasing production and application of α-Fe2O3NPs worldwide, they will be likely released into the environment at significant levels. Considerable α-Fe2O3-NPs release could occur during their applications, such as wastewater treatment (Predescu and Nicolae, 2012), biomedical and architectural application (Basilevsky and Shamov, 2003; Khoshakhlagh et al., 2012). In addition, α-Fe2O3NPs could be introduced into the environment with the discharge of wastewater from the production processes. Eventually, most of the released nanoparticles will enter into the aquatic environment, especially into marine environment (Chen et al., 2012; Scown et al., 2010). Therefore, the subsequent impacts of α-Fe2O3-NPs to marine ecosystem have drawn significant attentions. In recent years, using aquatic invertebrates as models to assess toxicological effects of environmental contaminants has become prevalent (Hu et al., 2012). Artemia salina (A. salina) is an invertebrate zooplankton found in various marine ecosystems. As one of the most popular live foods for fish larvae, A. salina plays a pivotal role in the energy flow of the food chains (Nunes et al., 2006). A. salina is a non-selective filter feeder, and filters a lot of water per hour. Therefore, it has significant interactions with aquatic environment, causing it faces a higher risk exposure to environmental contaminants compared with other aquatic species (Ates et al., 2015; Nunes et al., 2006). The intrinsic features of A. salina turn it into a suitable organism for studies in toxicology. For example, according to the differences in tissue differentiation and morphological characteristics, many stages are divided along the development process of A. salina. Previous studies showed that A. salina larvae exhibit discrepant sensitivity to pollutants in relation to the stages (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003; Sorgeloos et al., 1979). Besides, varied end-points can be selected as criterions for toxicological evaluation, such as hatchability, mortality, and a number of ethological, morphological and biochemical parameters (Ates et al., 2016; Caldwell et al., 2003). For aquatic organisms, swimming represents an ethological response determinant that can be directly affected by physiological status (Gambardella et al., 2014). Biochemical parameters of oxidative stress have been proposed for the evaluation of potential toxic effects of NPs, such as reactive oxygen species (ROS), malondialdehyde (MDA) and antioxidant enzymes activities (Ates et al., 2013b; Gambardella et al., 2014). In recent years, an increasing number of studies have investigated the effects of nanoparticles (e.g., TiO2, Al2O3 and NiO NPs) on A. salina (Ates et al., 2013a, 2016, 2015). Nevertheless, the related information in concerning the effects of α-Fe2O3-NPs on A. salina is currently limited. The present study was conducted to evaluate the acute toxicity of αFe2O3-NPs on both cysts (capsulated and decapsulated) and larvae (instar I, II and III) of A. salina. Hatchability, mortality, and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Microscope and transmission electron microscope (TEM) were used to observe the uptake and distribution of α-Fe2O3-NPs in A. salina. Moreover, the uptake kinetics of α-Fe2O3-NPs in A. salina was assessed. 2. Materials and methods 2.1. Preparation and characterization of α-Fe2O3-NPs The α-Fe2O3-NPs were purchased from Beijing Dk Nano technology Co., Ltd. (Beijing, China), and the structural parameters are listed in
Table S1. Scanning electron microscopy (SEM) analysis was carried out on a Hitachi S-4800 electron microscope (Japan) with an accelerating voltage of 15 kV. TEM observations were made on a JEM-1200EX electron microscope (Japan) operating at 80–100 kV. Fourier transform infrared (FTIR) spectra were recorded from 400 to 800 cm− 1 with a Bruker Vetex70 spectrophotometer (Germany) using KBr pellet technique (Wang et al., 1998). X-ray diffraction (XRD) analysis was performed on a Bruker D8 Advance diffractometer (Germany) with CuKα radiation (λ = 1.54060 Å), and the sample was scanned from 10° to 80° (2θ) with a scanning rate of 1° min−1. Diffraction peaks were compared with those of standard compounds reported in the JCPDS data file. α-Fe2O3-NPs were suspended in filtered natural seawater (FNSW; 30‰ m/v; pH 8.6) to create suspensions with concentration as required. To assess Fe3+ released from α-Fe2O3-NPs, the suspensions were centrifuged at 12,000 rpm for 30 min to pellet α-Fe2O3-NPs. The Fe3+ content in supernatants were then determined using inductively coupled plasma mass spectrometry (ICP-MS, Jarrell-Ash, MA). A dynamic light scattering (DLS, Brookhaven BI-200SM, USA) was used to estimate the hydrodynamic size distribution of α-Fe2O3-NPs in FNSW. 2.2. Model organism The A. salina cysts were purchased from Binzhou Haifa Biological Technology Co., Ltd. (Shandong, China). The dehydrated cysts were first hydrated in distilled water at 4 °C for 12 h, and the sunken cysts were collected on a Buchner funnel. In order to acquire decapsulated cysts, a solution of NaOCl, NaOH and water was used as described by Sorgeloos et al. (1986). Approximately 2 g of cysts were incubated in 1 L FNSW in a hatcher at 28 °C, with a continuous 1300 lx light regime and strong aeration. Instar I, II and III larvae were obtained by using the procedure described by Sorgeloos et al. (1979). Briefly, to obtain a population consisting only of instar I, the larvae were separated from the unhatched cysts within 2 h after the first free-swimming larva was observed. One-third of the population was used immediately for the tests on the instar I larvae, and the other larvae were maintained for another 24 and 48 h to obtain instar II and III larvae, respectively. 2.3. Hatching assay The capsulated and decapsulated cysts were cultivated in α-Fe2O3NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) to study the effects of α-Fe2O3-NPs on the hatchability. In order to evaluate the influence of Fe3 + released from α-Fe2O3-NPs on the hatchability, the αFe2O3-NPs suspensions were centrifuged at 12,000 rpm for 30 min and the capsulated and decapsulated cysts were cultivated in the supernatants. Hatching assay was performed in 24-well plates, and each well contained 1 mL test solution. Ten capsulated/decapsulated cysts were introduced into each well, and each treatment was taken out in octuplicate. All plates were incubated under a continuous illumination with shaking at 28 °C. The hatchability was detected using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan) at 12, 18, 24 and 36 h. 2.4. Acute toxicity test The acute toxicity test was performed by adding 10 larvae (instar I, II and III) to each well of 24-well plates that contained 1 mL of α-Fe2O3NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or supernatants. The plates were incubated at 28 °C with shaking under a 16:8 h light/dark cycle. The larvae were not fed during the test. All of the tests were taken out in octuplicate. After 24 h, the numbers of dead larvae (completely motionless) were counted using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Larvae (instar I, II and III) were also randomly distributed into beakers (approximately 1000 larvae in each beaker) containing 100 mL of α-Fe2O3-NPs suspensions, and cultured as described above. After 24 h, the larvae were randomly sampled immediately prepared for
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morphological and behavioral analyses and ROS measurements. Samples were fixed in 2.5% glutaraldehyde for SEM and TEM analysis. Specimens for MDA content, total antioxidant capacity (T-AOC) and enzyme activity analysis were frozen in liquid nitrogen and stored at −80 °C.
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Fe by ICP-MS (Thermo Elemental X7, USA). The ferric contents were calculated according to the standard curve and then translated into corresponding α-Fe2O3-NPs contents. 2.10. Statistical analysis
2.5. Morphological and behavioral analysis After exposure for 24 h, 20 surviving larvae (instar I, II and III) were randomly selected for morphological and behavioral analysis. The swimming was recorded using a swimming behavior recorder that consisted of a video camera (Nikon, Japan) fixed on a microscope (Leica, Germany). The behavior recorder was placed in a dark room to exclude external sources of light. All larvae were dark-adapted for 5 min before the video recording to reach a steady speed, and then the swimming behavior was recorded. The result was shown as swimming inhibition, normalized to the average swimming speed of the control. Body length of A. salina was also recorded using the recorder. Individual dry weight of A. salina larvae was weighed following completely dried. In addition, a SEM (Hitachi S-4800, Japan) was used to investigate the attachment of NPs and the surface damage to A. salina. 2.6. ROS detection The fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) (Beyotime Biotech, Nantong, China) was used to determine the generation of ROS in larvae (instar I, II and III) following exposure to α-Fe2O3NPs for 24 h. In brief, approximately 500 larvae from each treatment were homogenized on ice in 500 μL ice-cold Tris–HCl buffer (100 mM, pH 7.4) using a homogenizer. The homogenates were then centrifuged at 12, 000 g for 15 min, after which the supernatants were collected for ROS detection. The detection was carried out in black 96-well plates and performed following the manufacturer's instructions. Fluorescence was measured on a microplate reader (Multiskan MK3, Thermo Labsystems Co., Beverly, MA) with an excitation and emission wavelength of 485 and 530 nm, respectively. All of the tests were carried out in triplicate. 2.7. MDA content and T-AOC and antioxidant enzymes activities After exposure for 24 h, approximately 500 larvae (instar I, II and III) were homogenized in 0.5 mL of ice-cold phosphate buffer for 5 min, and then centrifuged (12000g; 15 min) at 4 °C. The supernatants were collected for biochemical analysis. Total protein, MDA content, T-AOC and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] activities were measured using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. All of the tests were carried out in triplicate. 2.8. Uptake of α-Fe2O3-NPs The uptake of α-Fe2O3-NPs by A. salina was qualitatively examined using a microscope (Leica, Germany) at the end of the exposure. Images were captured by a digital camera (Nikon, Japan) from surviving larvae in petri dishes. At the same time, a TEM (JEOL, Tokyo, Japan) was also used to examine the uptake and distribution of α-Fe2O3-NPs in A. salina. 2.9. Uptake kinetics study To determine the uptake kinetics of α-Fe2O3-NPs in A. salina, 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. The cleaned samples were then dried using a freeze dryer (FD5-3, GOLD-SIM). To determine the ferric contents, 0.1 g of dry larvae were weighed and digested in 2 mL of trace metal grade nitric acid at 160 °C. The solutions were diluted to 5 mL with distilled water after completely digested and analyzed for
All experiments were repeated at least three times, and data were recorded as mean ± standard deviation (SD). The median effective concentration (EC50) for swimming speed alteration, median lethal concentration (LC50) and related 95% confidence limits were calculated using the Probit method. To perform statistical analysis, the SPSS Version 11.0 software package (SPSS Inc., Chicago, IL) was used. Data were analyzed for differences between the control and treatments using oneway ANOVA followed by Tukey's test, where p b 0.05 is considered significant and p b 0.01 extremely significant. 3. Results and discussion 3.1. α-Fe2O3-NPs characterization The physicochemical properties of nanoparticles, such as size and particulate state, have been proposed to be related to their uptake, bioaccumulation and toxicity (Ates et al., 2013b, 2015). In the study, αFe2O3-NPs were characterized by SEM, TEM, FTIR, XRD and DLS analysis (Fig. 1A–E). The SEM and TEM images of α-Fe2O3-NPs are shown in Fig. 1A and B, respectively, indicating that the α-Fe2O3-NPs are spherical with varying sizes. The FTIR spectra of α-Fe2O3-NPs are represented in Fig. 1C, and the characteristic absorption bands at 481 cm−1 and 574 cm−1 are assigned to α-Fe2O3 (Suresh et al., 2010). Fig. 1D shows the XRD spectrum of α-Fe2O3-NPs, all of the diffraction peaks are in accordance with the standard XRD card of rhombohedral α-Fe2O3 (JCPDS No. 87-1165). The peaks are sharp and no characteristic peak of impurities can be observed, indicating that the α-Fe2O3-NPs are wellcrystallized and high purity. Fe3+ released from α-Fe2O3-NPs was quantitatively measured using ICP-MS, and the data are shown in Table S2. Only a small fraction of the NPs was dissolved due to the high pH of the suspensions (pH = 8.6). The DLS data show that the hydrodynamic diameter of α-Fe2O3-NPs was ranged from 316 nm to 34.674 μm with a mean diameter of 8.134 μm (Fig. 1E). The hydrodynamic diameter is larger than the diameter which estimated by TEM (mean diameter: 44.56 nm; Fig. 1F). This result is due to the reduction of electrostatic repulsion in the FNSW and hydration of α-Fe2O3-NPs surfaces, and is consistent with previous findings (Ates et al., 2013a, 2015). Although aeration was provided to maintain homogeneity of the suspensions in the study, the agglomeration was inevitable. 3.2. Effects of α-Fe2O3-NPs and Fe3+ on hatchability Hatching rate of A. salina is a reliable and sensitive end-point, and has been used in many acute toxicity tests (Alyuruk and Cavas, 2013; Rotini et al., 2015). In this study, both capsulated and decapsulated cysts were conducted to evaluate the effects of α-Fe2O3-NPs on hatchability. As shown in Fig. 2, on the whole, for both capsulated and decapsulated cysts at 12, 18, 24 and 36 h, dose-dependent decreases in hatching rates are observed relative to the controls. Moreover, significant differences (p b 0.01) of hatching rates were found in 600 mg/L for capsulated and decapsulated cysts at 12, 18, 24 and 36 h compared to the controls. However, the data of dissolution test substantiate that αFe2O3-NPs did release a small amount of Fe3 + into the suspensions (Table S2), indicating that A. salina cysts and larvae were exposed to Fe3+ and aggregates of the NPs. Thus, the toxic effects may be attributed to Fe3+ from the dissolution of NPs. In order to elucidate the contribution of Fe3 + to the toxic effects, capsulated and decapsulated cysts were exposed to the supernatants. As shown in Fig. S1, there was no obvious influence of Fe3 + on the hatchability of capsulated and decapsulated cysts. Therefore, the results indicated that the toxic effects
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Fig. 1. The SEM image (A), TEM image (B), FTIR spectrum (C) and XRD pattern (D) of α-Fe2O3-NPs. Size distributions of α-Fe2O3-NPs detected by using DLS (E) and TEM (F).
Fig. 2. Hatching percentages of capsulated (A) and decapsulated (B) cysts exposed to different α-Fe2O3-NPs concentrations. Values are presented as mean ± SD. Values that are significantly different from the control are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
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are accounted for α-Fe2O3-NPs rather than Fe3+ from the dissolution of NPs. 3.3. Mortality rate, behavioral and morphological analysis The end-point selection for acute toxicity tests is sometimes a vital factor to be considered, most frequently, mortality of larvae, behavioral and morphological parameters and biomarkers are selected as criterions (Nunes et al., 2006). In this study, instar I, II and III larvae of A. salina were conducted to elucidate the toxic effects of α-Fe2O3-NPs. As shown in Fig. 3A, dose-dependent increases in mortality rates of instar I, II and III larvae following exposure to α-Fe2O3-NPs suspensions. Mortality rates of all control groups were 2%–5%, indicating that absence of food did not induce any lethal effects on larvae even up to 96 h. Following treatments, the mortalities was significantly increased (p b 0.01) in 200, 400 and 600 mg/L, and the mean mortality rates in 600 mg/L treatment groups were 48.85%, 77.63% and 71.90% for instar I, II and III, respectively. The LC50 values for instar II and III are 177.424 and 235.495 mg/L, respectively (not calculable for instar I; Table S3). A. salina are relatively resistant to metal ions toxicity, and can tolerate a wide range of Fe3+ concentration (Gajbhiye, 1990; Kokkali et al., 2011). Gajbhiye (1990) systematically investigated the toxic effects of metal ions to A. salina and demonstrated that the LC50 values for Fe3+ were 18.2 and 13.9 mg/L in 24 and 48 h, respectively. Apparently, the concentrations of Fe3 + from the dissolution of NPs were all lower than the 24 h LC50 value, ranging from 0.47 to 1.98 mg/L (Table S2). Larvae (instar I, II and III) were exposed to the supernatants to elucidate the contribution of Fe3+ to the mortality rates. As shown in Fig. S2, all the
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controls and treatments showed about 2 to 5% mortality, and no statistically significant (p N 0.05) was found. Therefore, the results indicated that the toxic effects of α-Fe2O3-NPs on mortality are accounted for NPs rather than Fe3+ from the dissolution of NPs. Behavior and morphology are determinants that results from physiological and ecological aspects of toxicology. In the study, the swimming speed of instar I, II and III larvae showed concentration–dependent decreases following exposure to α-Fe2O3-NPs suspensions, and was significantly decreased (p b 0.01) in 100, 200, 400 and 600 mg/L. Likewise, Gambardella et al. (2014) demonstrated that swimming speed was significantly decreased in A. salina larvae following exposure to CeO2 NPs (Gambardella et al., 2014). The EC50 values for swimming speed inhibition of instar I, II and III were 259.956, 99.064 and 129.088 mg/L, respectively (Table S3). Moreover, instar II and III larvae showed significantly (p b 0.01) lower EC50 compared with instar I larvae. As shown in Fig. 3C, the body lengths were decreased with the rising of α-Fe2O3NPs concentrations, and significant reduction (p b 0.01) was observed at 400 and 600 mg/L for instar II and III larvae. For individual dry weight, significant reduction (p b 0.01) was only observed at 600 mg/L compared with the controls (Fig. 3D). A small amount of studies investigated the toxic effects of some materials on different stages of A. salina larvae (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003). Caldwell et al. (2003) assessed the toxicity of algal extracts and short chain aldehydes to cysts and different stages of A. salina, and demonstrated that hatching assay shows a lower sensitivity compared with the mortality assays. In addition, Barahona and Sánchezfortún (1996) compared the sensitivity of three stages of A. salina larvae to several compounds, and
Fig. 3. The mortality rate (A), swimming inhibition (SI; B), body length (C) and individual dry weight (D) for instar I, II and III larvae exposure to different concentrations of α-Fe2O3-NPs. Values are presented as mean ± SD. Values that are significantly different from the control are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
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demonstrated that 48-hours larvae (instar II) are more sensitive than the 24 (instar I) and 72-hours (instar III) larvae. In the study, the results of hatching assay, mortality assay and behavioral and morphological analysis revealed that the sensitivity of cysts and larvae to α-Fe2O3NPs is in the order of instar II N instar III N instar I N decapsulated cysts N capsulated cysts. The conclusion is somewhat similar to the results of previous studies (Barahona and Sánchezfortún, 1996; Caldwell et al., 2003). 3.4. Attachment of NPs and damages on the body surface Interactions of NPs with A. salina can be external, such as NPs attached onto the skin or exoskeleton, which cause direct damage to the lipid membranes. Mesarič et al. (2015) reported that carbon-based nanomaterials were extensively attached to the gills and caused the gill branches to fuse together, they also found the nanomaterials were attached onto the entire body surface of A. salina (Mesarič et al., 2015). In the study, the attachment of α-Fe2O3-NPs and damages on the body surface was checked by using a SEM, and the representative images are displayed in Fig. 4. Images of instar I, II and III larvae treated without α-Fe2O3-NPs are shown in Fig. 4A, B and C, respectively, indicating that the body surface was clean and undamaged. After exposure, α-Fe2O3-NPs were attached onto the gill (Fig. 4D) and body surface (Fig. 4E). In addition, some irreversible damages were observed, such as created “holes” (Fig. 4F–H) in the body surface and rupture of body surface (Fig. 4I).
derived from lipid peroxidation, and has been widely used as an indicator of oxidative damages to membranes and oxidative stress (Ates et al., 2013a, 2013b, 2015). The production of ROS following NPs treatment seems to be a key event of the toxic effects, and the imbalance between ROS formation and the T-AOC result in oxidative stress occurs. SOD, CAT and GPx are antioxidants that catalyze the decomposition of ROS, and can prevent organisms from adverse effects of oxidative stress (Cazenave et al., 2006). In the study, on the whole, all of the MDA content, T-AOC, ROS and antioxidant enzymes (SOD, CAT and GPx) activities were increased in dose-dependent manners after exposure to α-Fe2O3-NPs suspensions (Fig. 5). Significant increases (p b 0.01) were observed at 400 and 600 mg/L for all of the metabolites, suggesting that the physical decline of larvae following exposure to α-Fe2O3-NPs was related to the oxidative damages. Increases of MDA contents confirm that α-Fe2O3-NPs induced oxidative stress and caused damages on the body surface of A. salina. The production of ROS causes an elevation of SOD, CAT and GPx activities as defense mechanisms against oxidative stress. Consistent with the result, induction of oxidative stress has also been reported in other studies (Ates et al., 2013b; Gambardella et al., 2014; Taze et al., 2016). For example, Taze et al. (2016) investigated the toxicity of iron oxide nanoparticles in Mytilus galloprovincialis. They demonstrated that the iron oxide nanoparticles induced significant increase in ROS production, lipid peroxidation, protein carbonylation, ubiquitin conjugates and DNA damage. Moreover, they concluded that iron oxide nanoparticles caused adverse effects on physiology by causing oxidative stress in hemocytes of exposed mussels.
3.5. MDA content, T-AOC, ROS and antioxidant enzymes activities 3.6. Uptake of α-Fe2O3-NPs Some studies reported that the toxic effects of NPs on A. salina were due to oxidative stress (Ates et al., 2013a, 2013b). Changes on the levels of certain metabolites, such as MDA, ROS and antioxidant enzymes activities have been described as biomarkers of oxidative stress (Ates et al., 2015; Mesarič et al., 2015; Zhu et al., 2016). MDA is a metabolite
Interactions of NPs with A. salina also can be internal, such as NPs intake. A. salina is non-selective filter feeder, and can ingest particles smaller than 50 μm (Ates et al., 2013a, 2013b). A. salina has a very primitively ingestive behavior compared with other crustaceans, it is a
Fig. 4. SEM images of instar I (A), II (B) and III larvae (C) treated without α-Fe2O3-NPs. Attachment of α-Fe2O3-NPs (red arrows) onto gills (D) and body surface (E) of A. salina. Created “holes” (F–H) in body surface and rupture of body surface (I) after being directly contacted with α-Fe2O3-NPs, the black arrows are pointed to the morphological damages. Scale bars in D and G are of 2 μm, and 10 μm for remaining images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Measurement of MDA content (A) and T-AOC (B), ROS (C) and the changes in SOD (D), CAT (E) and GPx (F) activities in A. salina larvae (instar I, II and III) following exposure to different concentrations of α-Fe2O3-NPs. Values are presented as mean ± SD. Values that are significantly different from the controls are indicated by asterisks (one-way ANOVA, *p b 0.05; **p b 0.01).
Fig. 6. Ingestion of α-Fe2O3-NPs (red arrows) by A. salina larvae. (A) The gut is empty in the control. (B) A. salina larvae start to ingest α-Fe2O3-NPs. (C) α-Fe2O3-NPs is visible as a dark line inside the gut of treatment. (D–F) TEM characterization of intracorporal localization of α-Fe2O3-NPs in A. salina larvae. Scale bars in A–C are of 300 μm. nd, nephridial duct; pbc, primary body cavity; inc, intestinal cell; in, intestine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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continuous non-selective, obligate phagotrophic filter-feeder (Provasoli and Shiraishi, 1959). Suspended particles (no matter what their nature is) with suitable size are continuously ingested by A. salina (Reeve, 1963). In the study, uptake of α-Fe2O3-NPs was checked under a microscope, and representative images are shown in Fig. 6. The gut for the control was empty (Fig. 6A), and larvae started to ingest α-Fe2O3-NPs (Fig. 6B) following exposure to the NPs. Gradually, the gut was almost entirely filled with α-Fe2O3-NPs, verified by a dark line inside the gut (Fig. 6C) of the larvae. Several studies also reported the uptake of NPs by A. salina (Ates et al., 2013a; Gambardella et al., 2014; Ozkan et al., 2015). TEM was used to check the distribution of NPs in A. salina. αFe2O3-NPs were visible within the nephridial duct (nd; Fig. 6D), primary body cavity (pbc; Fig. 6E) and intestine (in; Fig. 6F). 3.7. Uptake kinetics As shown above, α-Fe2O3-NPs were ingested and well distributed in A. salina. In order to quantitatively assess the uptake profile, the iron contents in A. salina were measured using ICP-MS. The contents were based on the dry weight of A. salina, reflecting the total body burden from 1 to 72 h. As shown in Fig. 7, result revealed a general increase during the first 36 h followed by a decrease from 36 to 60 h, and a steady state was reached after 60 h. Average NPs contents were ranged from 0.178 to 8.818 mg/g. Uptake of NPs was slowly increased during the first 12 h (instar I), probably due to the mouth and anus of larvae are not yet completely opened, and the digestive tract is not fully formed (yolk sac consumption period). Moreover, mouth size is generally correlated with body size, thus instar I larvae have a smaller mouth size compared with other stages. The small mouth size restricts the size of particles which can be ingested (Sorgeloos et al., 1986). After molt into the instar II, tissues and organs of A. salina are fully formed, and the mouth size are larger. Therefore, a fast accumulation of NPs was occurred. The maximum content was reached at 36 h (instar II). As mentioned before, instar II larvae show the greatest sensitivity to α-Fe2O3NPs. The high content may be responsible for the strong toxic responses. There is a decrease from 36 to 60 h, probably because of redistribution and discharge of α-Fe2O3-NPs. As shown above, NPs were visible within the nephridial duct, indicating that a portion of NPs may be eliminated by A. salina. A steady state was achieved after 60 h may be due to the accumulation and elimination was finally balanced. 4. Conclusion In this study, we evaluated the toxic effects of α-Fe2O3-NPs on A. salina to elucidate the impacts to marine ecosystems. The results so far pointed to the fact that the acute exposure of cysts (capsulated and
Fig. 7. The contents of α-Fe2O3 uptake by A. salina at different time points.
decapsulated) and larvae (instar I, II and III) to α-Fe2O3-NPs causes significant changes in hatchability, mortality, and ethological, morphological and biochemical parameters. The toxic effects were mediated by oxidative stress. Instar II larvae show the greatest sensitivity to αFe2O3-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts, indicating that instar II would be a suitable candidate for toxicological test. Although α-Fe2O3-NPs rapidly aggregate in seawater to form large particles, there is no effect on the uptake of the NPs. α-Fe2O3-NPs were accumulated in the gut and well distributed in nephridial duct and primary body cavity of A. salina. Moreover, the uptake kinetics data show that the accumulation of α-Fe2O3-NPs in A. salina was firstly increased and decreased then, and a steady state was reached in the end. The results revealed short-term effects (24 h) of α-Fe2O3-NPs on A. salina, for safe and commercial purposes, longterm treatments and other complementary studies must be undertaken. Acknowledgements This work is supported by the Natural Science Foundation of Henan Province (Program No. 162300410197), the Doctoral Scientific Research Foundation of Zhoukou Normal University (Program No. ZKNUB2013001) and the Scientific Research Innovation Foundation of Zhoukou Normal University (Program No. ZKNUA201701). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.04.183. References Alagiri, M., Hamid, S.B.A., 2014. Green synthesis of α-Fe2O3 nanoparticles for photocatalytic application. J. Mater. Sci. Mater. Electron. 25, 3572–3577. Alyuruk, H., Cavas, L., 2013. Toxicities of diuron and irgarol on the hatchability and early stage development of Artemia salina. Turk. J. Biol. 37, 151–157. Aragay, G., Merkoçi, A., 2012. Nanomaterials application in electrochemical detection of heavy metals. Electrochim. Acta 84, 49–61. Ates, M., Daniels, J., Arslan, Z., Farah, I.O., 2013a. Effects of aqueous suspensions of titanium dioxide nanoparticles on Artemia salina: assessment of nanoparticle aggregation, accumulation, and toxicity. Environ. Monit. Assess. 185, 3339–3348. Ates, M., Daniels, J., Arslan, Z., Farah, I.O., Rivera, H.F., 2013b. Comparative evaluation of impact of Zn and ZnO nanoparticles on brine shrimp (Artemia salina) larvae: effects of particle size and solubility on toxicity. Evnviron. Sci. Process. Impacts 2013, 225–233. Ates, M., Demir, V., Arslan, Z., Daniels, J., Farah, I.O., Bogatu, C., 2015. Evaluation of alpha and gamma aluminum oxide nanoparticle accumulation, toxicity, and depuration in Artemia salina larvae. Environ. Toxicol. 30, 109–118. Ates, M., Demir, V., Arslan, Z., Camas, M., Celik, F., 2016. Toxicity of engineered nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater. Water Air Soil Pollut. 227, 1–8. Barahona, M.V., Sánchezfortún, S., 1996. Comparative sensitivity of three age classes of Artemia salina larvae to several phenolic compounds. Bull. Environ. Contam. Toxicol. 56, 271–278. Basilevsky, M.V., Shamov, A.G., 2003. Topical review: functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D. Appl. Phys. 36, R198–R206(199). Caldwell, G.S., Bentley, M.G., Olive, P.J.W., 2003. The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes. Toxicon 42, 301–306. Cazenave, J., Bistoni, M.L., Pesce, S.F., Wunderlin, D.A., 2006. Differential detoxification and antioxidant response in diverse organs of Corydoras paleatus experimentally exposed to microcystin-RR. Aquat. Toxicol. 76, 1–12. Chen, X., Zhu, X., Li, R., Yao, H., Lu, Z., Yang, X., 2012. Photosynthetic toxicity and oxidative damage induced by nano-Fe3O4 on in aquatic environment. Open J. Ecol. 02, 21–28. Gajbhiye, S.N., 1990. Toxicity of heavy metals to brine shrimp Artemia. J. Indian Fish. Assoc. 20. Gambardella, C., Mesarič, T., Milivojević, T., Sepčić, K., Gallus, L., Carbone, S., Ferrando, S., Faimali, M., 2014. Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses. Environ. Monit. Assess. 186, 4249–4259. Gao, J., Gu, H., Xu, B., 2009. Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc. Chem. Res. 42, 1097–1107. Giersig, M., Khomutov, G.B., 2008. Nanomaterials for Application in Medicine and Biology. Hu, J., Wang, D., Wang, J., Wang, J., 2012. Bioaccumulation of Fe2O3(magnetic) nanoparticles in Ceriodaphnia dubia. Environ. Pollut. 162, 216–222. Khoshakhlagh, A., Nazari, A., Khalaj, G., 2012. Effects of Fe2O3 nanoparticles on water permeability and strength assessments of high strength self-compacting concrete. J. Mater. Sci. Technol. 28, 73–82.
C. Wang et al. / Science of the Total Environment 598 (2017) 847–855 Kokkali, V., Katramados, I., Newman, J.D., 2011. Monitoring the effect of metal ions on the mobility of Artemia salina nauplii. Bios 1, 36–45. Mesarič, T., Gambardella, C., Milivojević, T., Faimali, M., Drobne, D., Falugi, C., Makovec, D., Jemec, A., Sepčić, K., 2015. High surface adsorption properties of carbon-based nanomaterials are responsible for mortality, swimming inhibition, and biochemical responses in Artemia salina larvae. Aquat. Toxicol. 163, 121–129. Nunes, B.S., Carvalho, F.D., Guilhermino, L.M., Van, S.G., 2006. Use of the genus Artemia in ecotoxicity testing. Environ. Pollut. 144, 453–462. Ozkan, Y., Altinok, I., Ilhan, H., Sokmen, M., 2015. Determination of TiO2 and AgTiO2 nanoparticles in Artemia salina: toxicity, morphological changes, uptake and depuration. Bull. Environ. Contam. Toxicol. 125, 1–7. Predescu, A., Nicolae, A., 2012. Adsorption of Zn, Cu and Cd from waste waters by means of maghemite nanoparticles. Upb Sci. Bull. 74. Provasoli, L., Shiraishi, K., 1959. Axenic cultivation of the brine shrimp Artemia salina. Biol. Bull. 117, 347–355. Reeve, M.R., 1963. The Filter-feeding of Artemia: II. In Suspensions of Various Particles. 40 p. 207. Rotini, A., Manfra, L., Canepa, S., Tornambè, A., Migliore, L., 2015. Can Artemia hatching assay be a (sensitive) alternative tool to acute toxicity test? Bull. Environ. Contam. Toxicol. 95, 745–751. Scown, T.M., Van, A.R., Tyler, C.R., 2010. Review: do engineered nanoparticles pose a significant threat to the aquatic environment. Crit. Rev. Toxicol. 40, 653–670.
855
Sorgeloos, P., Wielen, R.V.D., Persoone, G., 1979. The use of Artemia nauplii for toxicity tests—a critical anaysis. Ecotoxicol. Environ. Saf. 2, 249–255. Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W., Versichele, D., 1986. Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Suresh, R., Vijayalakshmi, L., Stephen, A., Narayanan, V., 2010. Hydrothermal Synthesis and Characterization of Cobalt Doped α-Fe2O3. 1276 pp. 362–367. Taze, C., Panetas, I., Kalogiannis, S., Feidantsis, K., Gallios, G.P., Kastrinaki, G., Konstandopoulos, A.G., Václavíková, M., Ivanicova, L., Kaloyianni, M., 2016. Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis. Aquat. Toxicol. 172, 9–20. Wang, Y., Muramatsu, A., Sugimoto, T., 1998. FTIR analysis of well-defined α-Fe2O3 particles. Colloids Surf. A Physicochem. Eng. Asp. 134, 281–297. Wu, X.L., Guo, Y.G., Wan, L.J., Hu, C.W., 2008. α-Fe2O3 nanostructures: inorganic salt-controlled synthesis and their electrochemical performance toward lithium storage. J. Phys. Chem. C 112, 16824–16829. Xu, P., Zeng, G.M., Huang, D.L., Feng, C.L., Hu, S., Zhao, M.H., Lai, C., Wei, Z., Huang, C., Xie, G.X., 2012. Use of iron oxide nanomaterials in wastewater treatment: a review. Sci. Total Environ. 424, 1–10. Zhu, S., Zhu, B., Huang, A., Hu, Y., Wang, G., Ling, F., 2016. Toxicological effects of multiwalled carbon nanotubes on Saccharomyces cerevisiae: the uptake kinetics and mechanisms and the toxic responses. J. Hazard. Mater. 318, 650–662.