Science of the Total Environment 598 (2017) 847–855

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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, mor-

tality, and other end-points
• Effects are accounted for α-Fe2O3-NPs
and mediated by oxidative stress.
• Instar II larvae show the greatest sensi-
tivity 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 a b s t r a c t

Article history: Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) were exposed to α-Fe2O3 nano-
Received 25 February 2017 particles (α-Fe2O3-NPs) to evaluate the effects on marine ecosystems. Hatchability, mortality and a number of
Received in revised form 14 April 2017 ethological, morphological and biochemical parameters were selected as end-points to define the toxic re-
Accepted 24 April 2017
sponses. Results indicate that the hatchability of capsulated and decapsulated cysts was significantly decreased
Available online 27 April 2017
(p b 0.01) following exposure to 600 mg/L at 12, 18, 24 and 36 h. Both increases of mortality and decreases of
Editor: D. Barcelo 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
Keywords: 259.956, 99.064 and 129.088 mg/L, respectively. Instar II larvae show the greatest sensitive to α-Fe2O3-NPs,
Toxicity and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths and individual dry
Brine shrimp weight of instar I, II and III larvae were decreased following exposure. α-Fe2O3-NPs attached onto the gills and
Iron oxide nanoparticles body surface of larvae, resulting in irreversible damages. All of malondialdehyde content, total antioxidant capac-
Oxidative stress ity, reactive oxygen species and antioxidant enzymes activities were substantially increased in dose-dependent
Uptake
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 authors. With the rapid development of nanotechnology, tremendous inter-

E-mail addresses: wangcjzz@163.com (C. Wang), jiahualizk@163.com (H. Jia). est has arisen in the field of nano-materials. Due to their distinctive

http://dx.doi.org/10.1016/j.scitotenv.2017.04.183
0048-9697/© 2017 Elsevier B.V. All rights reserved.
848 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855
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 ap-
plications (Alagiri and Hamid, 2014; Gao et al., 2009; Predescu and
Nicolae, 2012; Wu et al., 2008). According to a previous report, the glob-
al market for magnetic nanoparticles in electronic, magnetic, and opto-
electronic 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-Fe2O3-
NPs worldwide, they will be likely released into the environment at sig-
nificant 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-Fe2O3-
NPs could be introduced into the environment with the discharge of
wastewater from the production processes. Eventually, most of the re-
leased nanoparticles will enter into the aquatic environment, especially
into marine environment (Chen et al., 2012; Scown et al., 2010). There-
fore, the subsequent impacts of α-Fe2O3-NPs to marine ecosystem have
drawn significant attentions.
In recent years, using aquatic invertebrates as models to assess toxico-
logical 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 feed-
er, and filters a lot of water per hour. Therefore, it has significant interac-
tions 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 characteris-
tics, 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 hatch-
ability, 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 an-
tioxidant 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 α-
Fe2O3-NPs on both cysts (capsulated and decapsulated) and larvae (in-
star 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 transmis-
sion 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 accelerat-
ing 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 tech-
nique (Wang et al., 1998). X-ray diffraction (XRD) analysis was per-
formed 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 com-
pared 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 centri-
fuged at 12,000 rpm for 30 min to pellet α-Fe2O3-NPs. The Fe3+ content
in supernatants were then determined using inductively coupled plas-
ma mass spectrometry (ICP-MS, Jarrell-Ash, MA). A dynamic light scat-
tering (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 an-
other 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-Fe2O3-
NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) to study the ef-
fects of α-Fe2O3-NPs on the hatchability. In order to evaluate the influ-
ence of Fe3 + released from α-Fe2O3-NPs on the hatchability, the α-
Fe2O3-NPs suspensions were centrifuged at 12,000 rpm for 30 min
and the capsulated and decapsulated cysts were cultivated in the super-
natants. 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-Fe2O3-
NPs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or superna-
tants. 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 lar-
vae (completely motionless) were counted using a microscope (Olym-
pus Optical Co., Ltd., Tokyo, Japan).
Larvae (instar I, II and III) were also randomly distributed into bea-
kers (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
 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855
morphological and behavioral analyses and ROS measurements. Sam-
ples were fixed in 2.5% glutaraldehyde for SEM and TEM analysis. Spec-
imens for MDA content, total antioxidant capacity (T-AOC) and enzyme
activity analysis were frozen in liquid nitrogen and stored at −80 °C.
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 swim-
ming inhibition, normalized to the average swimming speed of the con-
trol. 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 genera-
tion of ROS in larvae (instar I, II and III) following exposure to α-Fe2O3-
NPs 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 wave-
length 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 col-
lected 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
849
Fe by ICP-MS (Thermo Elemental X7, USA). The ferric contents were cal-
culated according to the standard curve and then translated into corre-
sponding α-Fe2O3-NPs contents.
2.10. Statistical analysis
All experiments were repeated at least three times, and data were
recorded as mean ± standard deviation (SD). The median effective con-
centration (EC50) for swimming speed alteration, median lethal concen-
tration (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 ana-
lyzed for differences between the control and treatments using one-
way ANOVA followed by Tukey's test, where p b 0.05 is considered sig-
nificant 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, bio-
accumulation and toxicity (Ates et al., 2013b, 2015). In the study, α-
Fe2O3-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 ac-
cordance with the standard XRD card of rhombohedral α-Fe2O3 (JCPDS
No. 87-1165). The peaks are sharp and no characteristic peak of impuri-
ties can be observed, indicating that the α-Fe2O3-NPs are well-
crystallized and high purity. Fe3+ released from α-Fe2O3-NPs was quan-
titatively 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 re-
pulsion in the FNSW and hydration of α-Fe2O3-NPs surfaces, and is con-
sistent 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 hatch-
ability. 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, signif-
icant 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 α-
Fe2O3-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 contribu-
tion of Fe3 + to the toxic effects, capsulated and decapsulated cysts
were exposed to the supernatants. As shown in Fig. S1, there was no ob-
vious influence of Fe3 + on the hatchability of capsulated and
decapsulated cysts. Therefore, the results indicated that the toxic effects
850 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855

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).
 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855
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 criteri-
ons (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 in-
star 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. Fol-
lowing 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 toler-
ate 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). Lar-
vae (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
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).
851
controls and treatments showed about 2 to 5% mortality, and no statis-
tically 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 physi-
ological and ecological aspects of toxicology. In the study, the swimming
speed of instar I, II and III larvae showed concentration–dependent de-
creases following exposure to α-Fe2O3-NPs suspensions, and was signif-
icantly decreased (p b 0.01) in 100, 200, 400 and 600 mg/L. Likewise,
Gambardella et al. (2014) demonstrated that swimming speed was sig-
nificantly decreased in A. salina larvae following exposure to CeO2 NPs
(Gambardella et al., 2014). The EC50 values for swimming speed inhibi-
tion of instar I, II and III were 259.956, 99.064 and 129.088 mg/L, respec-
tively (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-Fe2O3-
NPs 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 com-
pared 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
852 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855
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-Fe2O3-
NPs 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 re-
sults 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 at-
tached 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, indi-
cating 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).
3.5. MDA content, T-AOC, ROS and antioxidant enzymes activities
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 ac-
tivities have been described as biomarkers of oxidative stress (Ates
et al., 2015; Mesarič et al., 2015; Zhu et al., 2016). MDA is a metabolite
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.
derived from lipid peroxidation, and has been widely used as an indica-
tor 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 oxida-
tive damages. Increases of MDA contents confirm that α-Fe2O3-NPs in-
duced 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. Consis-
tent 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 conju-
gates and DNA damage. Moreover, they concluded that iron oxide nano-
particles caused adverse effects on physiology by causing oxidative
stress in hemocytes of exposed mussels.
3.6. Uptake of α-Fe2O3-NPs
Interactions of NPs with A. salina also can be internal, such as NPs in-
take. 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 prim-
itively ingestive behavior compared with other crustaceans, it is a
 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855 853

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.
854 C. Wang et al. / Science of the Total Environment 598 (2017) 847–855
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 micro-
scope, 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. α-
Fe2O3-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 dur-
ing 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 corre-
lated with body size, thus instar I larvae have a smaller mouth size com-
pared 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 oc-
curred. The maximum content was reached at 36 h (instar II). As men-
tioned before, instar II larvae show the greatest sensitivity to α-Fe2O3-
NPs. 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 sig-
nificant changes in hatchability, mortality, and ethological, morpholog-
ical and biochemical parameters. The toxic effects were mediated by
oxidative stress. Instar II larvae show the greatest sensitivity to α-
Fe2O3-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 sea-
water 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 up-
take 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, long-
term 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.
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