Environmental Pollution 230 (2017) 683e691

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Developmental toxicity of Fe3O4 nanoparticles on cysts and three

larval stages of Artemia salina*
Song Zhu, Ming-Yang Xue, Fei Luo, Wei-Chao Chen, Bin Zhu, Gao-Xue Wang*
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China

a r t i c l e i n f o a b s t r a c t

Article history: Using Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) as experimental
Received 14 March 2017 models, the potential effects of Fe3O4 nanoparticles (Fe3O4-NPs) on marine ecosystems were investi-
Received in revised form gated. Hatchability, mortality and a number of ethological, morphological and biochemical parameters
14 June 2017
were selected as end-points to define the toxic responses. Data showed that the hatching rates of
Accepted 19 June 2017
capsulated and decapsulated cysts were significantly decreased (p < 0.01) following exposure to 600 mg/
Available online 14 July 2017
L for 24 and 36 h. The LC50 values for instar II and III were 482 and 561 mg/L (could not be measured for
instar I), and the EC50 values for swimming inhibition of instar I, II and III were 474, 365 and 421 mg/L,
Keywords:
Nanoparticle
respectively. Effects on hatchability, mortality and swimming were accounted for Fe3O4-NPs rather than
Developmental toxicity iron ion released from the NPs. Instar II larvae showed the greatest sensitivity to Fe3O4-NPs, and followed
Brine shrimp by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths of instar I, II and III larvae
Oxidative stress were decreased in dose-dependent manners. Fe3O4-NPs attached onto the gills and body surface,
Uptake resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant
capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities
were substantially increased following exposure, indicating that toxic effects were related to oxidative
stress. Mitochondrial malformation, cristae rupturing and membranous structure disruption were clearly
observed after Fe3O4-NPs exposure. Fe3O4-NPs were ingested and well distributed in the gut, yolk and
primary body cavity. Uptake kinetics data showed that the maximum Fe3O4-NPs content (16.4 mg/g) was
reached at 30 h. The combined results so far indicate that Fe3O4-NPs have the potential to affect aquatic
organisms when released into the marine ecosystems.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction biomedical application (Begum and Anantharaman, 2009; Linh

Nanotechnology is considered as one of the fastest-growing
fields today. With the development of nanotechnology, tremen-
dous interest has arisen in the application of nano-materials (Lin,
2015; Thorkelsson et al., 2015). As important parts of nano-
materials, nanoparticles are currently used in a wide range of
fields (Wolfbeis, 2015; Xiao et al., 2016). Nanoparticles possess
unique and enhanced physico-chemical properties, which give rise
to numerous multitask applications. As one of the most important
magnetic nanoparticles, Fe3O4 nanoparticles (Fe3O4-NPs) are being
used in many areas, such as pigment, sewage treatment and
*
This paper has been recommended for acceptance by Baoshan Xing.
* Corresponding author. Northwest A&F University, Xinong Road 22nd, Yangling,
Shaanxi, 712100, China.
E-mail address: wanggaoxue@126.com (G.-X. Wang).
et al., 2009; Ramírez, 2015). Moreover, the advances in Fe3O4-NPs
synthesis and chemical modification make Fe3O4-NPs suitable for
more commercial applications (Lv et al., 2008; Xuan et al., 2011).
However, along with the rapid increase of related products and
applications, Fe3O4-NPs are inevitably released into the environ-
ment during the processes of production, transportation, applica-
tion and disposal. Eventually, most of the Fe3O4-NPs will enter into
aquatic ecosystems, especially into marine ecosystems (Chen et al.,
2012; Scown et al., 2010). Therefore, aquatic organisms are partic-
ularly susceptible to the direct or indirect effects of Fe3O4-NPs. In
recent years, a few in vivo and in vitro studies have assessed the
potential toxicity of Fe3O4-NPs to human cells (Lin et al., 2012),
mice (Wang et al., 2013) and freshwater algae (Chen et al., 2012).
Nevertheless, there are few data available regarding the fate,
behavior and potential toxicity of Fe3O4-NPs to marine species
(Gambardella et al., 2014).
Artemia salina (A. salina) is an invertebrate zooplankton found in

http://dx.doi.org/10.1016/j.envpol.2017.06.065
0269-7491/© 2017 Elsevier Ltd. All rights reserved.
684 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691
a variety of seawater systems from lakes to oceans. As one of the
most popular live foods for freshwater and marine fishes, A. salina
plays a key role in the energy flow of food chain (Nunes et al., 2006).
A. salina is a specie of nonselective filter feeder, and filters a large
amount of water per hour. Therefore, it faces a higher risk of
exposure to pollutants compared with other aquatic species (Ates
et al., 2013, 2015). Moreover, lots of common characteristics and
physiological features make A. salina as a suitable model organism
for ecotoxicity testing, such as ready availability, simple laboratory
culture and multiple life stages (Caldwell et al., 2003; Libralato,
2014; Nunes et al., 2006). A. salina capsulated/decapsulated cysts
are commercial availability and widely used in aquaculture. Be-
sides, capsulated and decapsulated cysts have been used as models
to assess the potential toxicity of pollutants (Arulvasu et al., 2014;
Caldwell et al., 2003; Zhu et al., 2017a). Different stages are
divided along the developmental process of A. salina. Published
studies showed that A. salina larvae exhibit discrepant sensitivity to
pollutants in relation to stages (Barahona and Sa
nchezfortún, 1996;
Caldwell et al., 2003). Recently, a number of studies have investi-
gated the potential toxicity of nanoparticles (e.g., CoO, ZnO, Zn and
Ag NPs) to A. salina (Arulvasu et al., 2014; Ates et al., 2013, 2016).
However, most studies generally focused on the effects of nano-
particles on a certain stage of A. salina. To get a systematic under-
standing of the toxicity, multiple stages of A. salina should be
conducted in the toxicity tests.
In the study, A. salina cysts and larvae were conducted as models
to elucidate the effects of Fe3O4-NPs on marine ecosystems.
Hatchability of capsulated and decapsulated cysts, and mortality of
instar I, II and III larvae were determined following Fe3O4-NPs
exposure. A number of ethological (swimming), morphological
(body length and surface damages) and biochemical parameters
[reactive oxygen species (ROS), malondialdehyde (MDA), total
antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GPx)] were efficiently assessed
to define the toxic responses. Uptake and distribution of Fe3O4-NPs
in A. salina were checked using microscope and transmission
electron microscope (TEM). In addition, the Fe3O4-NPs contents in
A. salina were measured to elucidate the accumulation and elimi-
nation patterns during the exposure for 72 h.
2. Materials and methods
2.1. Materials
Fe3O4-NPs were purchased from Beijing Dk Nano technology
Co., Ltd., (Beijing, China), and the structural parameters are listed in
Table S1. Trace metal grade nitric acid, glutaraldehyde (25%) and
TriseHCl were purchased from Sigma-Aldrich. Dichlorofluorescin
diacetate (DCFH-DA), total protein, MDA, T-AOC, SOD, CAT and GPx
kits were purchased from Biotechnology Development Co., Ltd.
(Nanjing, China). Solutions were prepared with deionized distilled
water.
2.2. Characterization of Fe3O4-NPs
Fe3O4-NPs were characterized by scanning electron microscope
(SEM; Hitachi S-4800, Japan) and TEM (JEM1200EX, Japan) with
accelerating voltages of 15 kV and 100 kV, respectively. Fourier
transform infrared spectrometer (FTIR; Bruker Vetex70, Germany)
was conducted to analyze the surface characteristics using KBr
pellet technique (Wang et al., 1998). The crystal characterization
was analyzed using X-ray diffractometer (XRD; Bruker D8 Advance)
with CuKa radiation (l ¼ 1.54 Å), and the Fe3O4-NPs were scanned
from 10 to 80 (2q) with a scanning rate of 1 min1. To prepare the
suspensions with nominal concentrations of 25, 50, 100, 200, 400
and 600 mg/L, Fe3O4-NPs were weighed on aluminum foil and
placed in 1 L beakers containing 900 mL of filtered natural seawater
(FNSW; 30‰ m/v; pH 8.6). The beakers were placed in an ice bath
and then sonicated with an ultrasonic processor (Scientz-IID,
China). The suspensions were sonicated for 1 h at 100 W using a
50% on/off cycle and left overnight at room temperature, and son-
icated again. Dissolution of Fe3O4-NPs was determined by ultra-
filtration as described by Ates et al. (2013). In brief, Fe3O4-NPs
suspensions were firstly centrifuged at 12,000 rpm for 30 min to
separate the suspending particulates, and then the supernatants
were passed through ultra-filtration filters with a molecular cut-off
3000 Da to separate the iron ion from the NPs. The iron ion con-
centrations were determined using inductively coupled plasma
mass spectrometry (ICP-MS, Thermo Elemental X7, USA). To esti-
mate the hydrodynamic size distribution of Fe3O4-NPs in FNSW, a
dynamic light scattering (DLS, Brookhaven BI200SM, USA) was
used.
2.3. Model organism
A. salina dehydrated cysts (Tianjin, China) were used and kept at
4  C until required. Decapsulated cysts and instar I, II and III larvae
were acquired as described previously (Zhu et al., 2017a) (as
described in the supplementary file).
2.4. Hatching assay
Seven treatments (0, 25, 50, 100, 200, 400 and 600 mg/L) were
performed to examine the effect of Fe3O4-NPs on the hatchability of
capsulated and decapsulated cysts. Each treatment was taken out
eight times. The test concentrations were chosen on the basis of the
results of a preliminary screening test using hatching rate, mor-
tality and swimming inhibition as end-points. Fe3O4-NPs suspen-
sions were centrifuged and filtered as described above, and the
filtrates were collected. Capsulated and decapsulated cysts were
cultivated in the filtrates to evaluate the influence of iron ion
released from Fe3O4-NPs on the hatchability. Hatching tests were
performed in 24-well plates, and each well contained 10 capsu-
lated/decapsulated cysts and 1 mL test solution. All plates were
incubated at 28  C with a continuous illumination and shaking at
150 rpm. Hatching rates were determined by counting the number
of completely hatched larvae with the aid of a microscope
(Olympus Optical Co., Ltd., Tokyo, Japan) at 12, 18, 24 and 36 h.
2.5. Acute toxicity test
Acute toxicity test consisted of seven treatments (0, 25, 50, 100,
200, 400 and 600 mg/L), and each treatment was carried out eight
times. Larvae (instar I, II and III) were also cultivated in the filtrates
(obtained as described above) to evaluate the toxicity of iron ion
released from Fe3O4-NPs to A. salina. Test was performed according
to previous study (Zhu et al., 2017a).
For each treatment, instar I, II and III larvae were also randomly
added into beakers. Each beaker contained 100 mL FNSW and
approximately 1000 larvae, and cultured as described above. After
exposure for 24 h, larvae were randomly took and immediately
performed ethological and morphological analysis. Samples for
SEM and TEM analysis were fixed in 2.5% glutaraldehyde at 4  C.
Specimens for ROS, MDA content, T-AOC and enzyme activity
measurements were frozen in liquid nitrogen and stored at 80  C.
2.6. Ethological and morphological analysis
Swimming speed and body length were recorded using a video
camera (Nikon, Japan) fixed on a microscope (Leica, Germany). The
 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691
measurement and analysis were performed according to
Gambardella et al. (2014). Briefly, the equipment was firstly placed
in a dark room to exclude external light, and then larvae were
transferred into a petri dish and dark-adapted for 5 min to reach a
uniform spatial distribution and a steady speed. At last, the
swimming speed was recorded and normalized to the average
swimming speed of the controls. Body length was measured from
the images. Moreover, the attachment of Fe3O4-NPs and the
morphological damage to larvae were investigated using a SEM
(Hitachi S-4800, Japan).
2.7. Biochemical parameters
Measurements of ROS, MDA content, T-AOC and antioxidant
enzymes activities were performed according to previous studies
(Zhu et al., 2017a, 2017b) (as described in the supplementary file).
2.8. Uptake of Fe3O4-NPs
A microscope (Leica, Germany) was used to qualitative verify the
uptake of Fe3O4-NPs by A. salina larvae, and images were taken by a
digital camera (Nikon, Japan). Moreover, the uptake and distribu-
tion of Fe3O4-NPs in larvae were also observed by TEM (JEOL, Tokyo,
Japan).
2.9. Uptake kinetics
Larvae were collected at 1, 3, 5, 7, 9, 12, 15, 18, 24, 30, 36, 48, 60
and 72 h, and thoroughly washed with distilled water. At each
sampling time point, larvae treated without Fe3O4-NPs were also
collected as the control group. All samples were dried using a freeze
dryer (FD5-3, GOLD-SIM). Ferric contents in dried A. salina larvae
were determined using ICP-MS according to the protocol described
previously (Ates et al., 2015). In brief, the larvae (0.1 g) were
digested in 2 mL of trace metal grade nitric acid at 160  C. Once
completely digested, the solutions were diluted to 5 mL with
deionized water and analyzed for Fe by ICP-MS (Thermo Elemental
X7, USA). The Fe contents of the solutions were calculated according
to the standard curve, and then translated into corresponding Fe3O4
concentrations. Contents of Fe3O4 in the control group were sub-
tracted from that in the treatment groups to obtain the actual
content of Fe3O4.
2.10. Statistical analysis
All of the treatments were carried out at least three times, and
the data were expressed as mean ± standard deviation (SD). The
EC50, LC50 and related 95% confidence limits were calculated using
the Probit method. The SPSS Version 11.0 software package (SPSS
Inc., Chicago, IL) was used to perform statistical analysis. Significant
differences between the controls and treatments were examined
using one-way ANOVA followed by Tukey's test. Significance was
accepted at p < 0.05 and extremely significant at p < 0.01.
3. Results and discussion
3.1. Characterization of Fe3O4-NPs
The intrinsic properties of nanoparticles (such as size, shape and
particulate state) have been proposed to be related to their uptake,
bioaccumulation and toxicity (Gil et al., 2010). In the study, SEM,
TEM, FTIR, XRD and DLS analysis were performed to characterize
the Fe3O4-NPs (Fig. 1AeE). As shown in Fig. 1A and B, the Fe3O4-NPs
are rhombic with varying sizes. Fig. 1C shows the FTIR spectra of
Fe3O4-NPs, the absorption peak at 581 cm1 is attributed to the
685
stretching vibration mode of FeeO bond in Fe3O4 (Arsalani et al.,
2010). XRD spectrum of Fe3O4-NPs is represented in Fig. 1D, the
diffraction peaks are in accordance with the standard XRD card of
cubic Fe3O4 (JCPDS No. 01e1111). No characteristic peaks of impu-
rities are identified, and the sharp and high intense peaks indicate
the Fe3O4-NPs are well-crystallized. The actual size distribution of
Fe3O4-NPs in FNSW was determined by DLS, and the result shows a
wide distribution of particle size (Fig. 1E). The hydrodynamic
diameter ranges from 186 nm to 19.9 mm with a mean diameter of
5.52 mm, and is larger than that estimated by TEM (mean diameter:
170 nm; Fig. 1F). Aggregation of NPs in aqueous solution is inevi-
table due to the hydration and reduction of electrostatic repulsion
in the solution (Blinova et al., 2010). The result indicated that
A. salina were actually exposed to aggregates of Fe3O4-NPs rather
than the NPs, and similar phenomena were reported by other
studies (Ates et al., 2013, 2015). Dissolution of Fe3O4-NPs was
quantitatively measured, and the result is shown in Table S2. The
iron ion contents are ranged from 0.72 to 3.88 mg/L, and a large
fraction of the NPs is still undissolved due to the high pH of FNSW
(pH ¼ 8.6).
3.2. Effects of Fe3O4-NPs and iron ion on hatchability
As shown in Fig. 2A and B, on the whole, the hatching rates
decreased with the Fe3O4-NPs concentrations increased from 0 to
600 mg/L for both capsulated and decapsulated cysts at 12, 18, 24
and 36 h. Significant decreases (p < 0.01) were found in 600 mg/L at
24 and 36 h for capsulated cysts and in 600 mg/L at 12, 18, 24 and
36 h for decapsulated cysts compared with the controls. The result
indicates that decapsulated cysts showed a higher sensibility to
Fe3O4-NPs than capsulated cysts. In addition, decapsulated cysts
also showed a higher hatchability compared with capsulated cysts.
A. salina cysts have a relatively low energy content, the hatchability
is improved by decapsulation due to a lower energy requirement to
break out of decapsulated cysts (Sorgeloos et al., 1986).
Several studies reported that toxic effects of NPs were related to
the metal ions released from the NPs into the solution (Ates et al.,
2013; Heinlaan et al., 2008). In this study, the influence of iron
ion released from Fe3O4-NPs on the hatchability was evaluated. As
shown in Fig. S1, the hatching rates were substantially decreased
following exposure to different concentrations of iron ion. How-
ever, there was no statistically significant (p > 0.05) compared with
the controls, indicating that the toxic effects on hatchability are
accounted for Fe3O4-NPs rather than iron ion from the dissolution
of NPs.
3.3. Acute toxicity
As shown in Fig. 3A, mortality rates in the controls ranged be-
tween 2% and 5% for instar I, II and III larvae. As mentioned above,
larvae were not fed during the exposure. Therefore, the result
indicated that absence of food did not induce any lethal effects on
larvae even up to 72 h. Similar phenomenon has been reported by
other studies (Ates et al., 2013, 2015). After treated with Fe3O4-NPs
for 24 h, mortality rates of instar I, II and III larvae were increased
with the Fe3O4-NPs concentrations increased from 0 to 600 mg/L.
The mean mortality rates in 600 mg/L were 37.1, 59.3 and 51.8% for
instar I, II and III, and the LC50 values for instar II and III were 482
and 561 mg/L, respectively (could not be measured for instar I;
Table S3). Gambardella et al. (2014) investigated the effects of
Fe3O4-NPs on A. salina after exposure for 48 h. They demonstrated
that no significant lethal effect was observed following exposure to
Fe3O4-NPs up to a concentration of 1.0 mg/mL. The obvious
disagreement may be induced by many factors, such as size, shape
and abiotic factors, which have been verified to have profound
686 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691

Fig. 1. The SEM image (A), TEM image (B), FTIR spectrum (C) and XRD pattern (D) of Fe3O4-NPs. Size distributions of Fe3O4-NPs measured using DLS (E) and TEM (F).

Fig. 2. Hatching rates of capsulated (A) and decapsulated (B) cysts exposed to different Fe3O4-NPs concentrations. Values are presented as mean ± SD.

impacts on toxicity (Gil et al., 2010; Nunes et al., 2006).
Effect of iron ion released from Fe3O4-NPs on A. salina was
checked and the result is presented in Fig. S2A. Mortality rates of
instar I, II and III larvae were slightly increased and ranged between
2% and 5%, while no significant difference (p > 0.05) was observed
compared with the controls. The result indicated that the toxic
effects on mortality are accounted for Fe3O4-NPs rather than iron
ion. A. salina is relatively resistant to heavy metal ion and can
tolerate wide ranges of metal ion concentration (Gajbhiye, 1990;
Kokkali et al., 2011). Gajbhiye (1990) investigated the toxic effects
of several heavy metal ions to A. salina and demonstrated that the
24 h LC50 values for Fe3þ were 18.2 mg/L. In this study, Fe3O4-NPs
were slightly dissolved into the FNSW, the highest concentration of
iron ion was 3.88 mg/L, which apparently lower than the 24 h LC50
value.
 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691 687

Fig. 3. Mortality rate (A), swimming inhibition (SI; B) and body length (C) for instar I, II and III larvae exposure to different concentrations of Fe3O4-NPs. Values are presented as
mean ± SD.

Fig. 4. SEM images of instar I (A), II (B) and III larvae (C) treated without Fe3O4-NPs. Attachment of Fe3O4-NPs (red arrows) onto body surface (D) and gills (E) of A. salina. Rupture of
body surface (F) and created “holes” (GeI) in body surface after being directly contacted with Fe3O4-NPs, the black arrows are pointed to the surface damages. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Ethological and morphological analysis
Ethological and morphological changes are resulted from mo-
lecular, physiological and ecological aspects of toxicology
(Gambardella et al., 2014; Little et al., 1990). Therefore, ethological
and morphological analysis can provide insights into various levels
of body tissues. As important ethological and morphological pa-
rameters, swimming speed and body length have been widely used
in many studies (Gambardella et al., 2014; Ozkan et al., 2015).
In the study, swimming speed and body length were measured
after exposure for 24 h, and the results are shown in Fig. 3B and C,
respectively. Swimming speed showed concentrationedependent
inhibition, and decreased as much as 58.9, 74.9 and 64.3% in
600 mg/L for instar I, II and III, respectively. The EC50 values for
swimming inhibition of instar I, II and III were 474, 365 and 421 mg/
L, respectively (Table S3). Instar II and III larvae showed signifi-
cantly lower (p < 0.01) EC50 values compared with instar I larvae.
Body length (Fig. 3C) was shown concentrationedependent
decrease. However, no significant difference (p > 0.05) was
observed compared with the controls.
688 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691
Fig. 5. ROS (A), MDA content (B), T-AOC (C) and the changes in CAT (D), SOD (E) and GPx (F) activities in A. salina larvae (instar I, II and III) following exposure to different
concentrations of Fe3O4-NPs. Values are presented as mean ± SD. Values that are significantly different from the controls are indicated by asterisks (one-way ANOVA, *p < 0.05;
**p < 0.01).
Effect of iron ion on the swimming is shown in Fig. S2B.
Swimming was slightly inhibited and no significant difference
(p > 0.05) was observed compared with the controls. Kokkali et al.
(2011) studied the effect of Fe2þ on A. salina, and demonstrated that
the 24 h EC50 value for swimming inhibition was 66.3 mg/L.
Apparently, the concentrations of iron ion released from Fe3O4-NPs
were much lower than the 24 h EC50 value.
Several studies have investigated the toxic effects of pollutants
on different stages of A. salina, and demonstrated that it exhibits
discrepant sensitivity in relation to stages (Barahona and

nchezfortún, 1996; Caldwell et al., 2003). Caldwell et al. (2003)
Sa
reported that hatching assay showed a lower sensitivity to algal
extracts and short chain aldehydes compared with the mortality
assay. Besides, Barahona and S
anchezfortún (1996) studied the
sensitivity of three stages of A. salina larvae to several compounds,
and demonstrated that 48-h larvae were more sensitive than the 24
and 72-h larvae. In the study, based on the hatching assay, mortality
assay and ethological and morphological analysis, it can be
concluded that the sensitivity of A. salina to Fe3O4-NPs is in the
order of instar II > instar III > instar I > decapsulated
cysts > capsulated cysts. Larvae show a higher sensitivity than cysts
may be due to the cortical layer of cysts can provide an effective
protective barrier to Fe3O4-NPs. In some ways, the result is similar

nchezfortún,
to the reports of previous studies (Barahona and Sa
1996; Caldwell et al., 2003; Zhu et al., 2017a).
3.5. Fe3O4-NPs attachment and surface damages
Interactions of nanomaterials with organisms can be external,
such as nanomaterials attached onto the skin or exoskeleton, which
cause direct damages to the body surface. Nanomaterials possess a
smaller size and a higher specific surface area, therefore, they are
more easily to attach onto the surface of organisms than bulk
materials. Mesari c et al. (2015) investigated the effects of carbon-
based nanomaterials on A. salina. They demonstrated that the
nanomaterials were extensively attached onto the gills and entire
body surface, causing gill branches to fuse together. Moreover, they
also reported that high surface adsorption properties of nano-
materials were responsible for mortality, swimming inhibition, and
biochemical responses in A. salina (Mesari c et al., 2015).
In this study, the attachment of Fe3O4-NPs and surface damages
of larvae were checked using a SEM, and the representative images
are shown in Fig. 4. Body surfaces of instar I (Fig. 4A), II (Fig. 4B) and
III (Fig. 4C) larvae treated without Fe3O4-NPs were clean and un-
damaged. After exposure for 24 h, Fe3O4-NPs attached onto the
body surface (Fig. 4D) and gill (Fig. 4E) of A. salina. The attachment
 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691 689

Fig. 6. Ingestion of Fe3O4-NPs (red arrows) by A. salina larvae. (A) The gut is empty in the control. (B) A. salina larvae start to ingest Fe3O4-NPs. (C) Fe3O4-NPs are visible as a dark line
inside the gut of treatment. (D) Fe3O4-NPs are excreted by A. salina larvae. TEM characterization of intracorporal localization of Fe3O4-NPs in yolk (y; E), primary body cavity (pbc; F)
and intestine (in; G). (H) TEM image of mitochondria in the control. Mitochondrial malformation (I), cristae rupturing (J and K) and membranous structure disruption (L and M) after
Fe3O4-NPs exposure. The abnormal mitochondria are marked with black asterisks. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

resulted in some irreversible damages to the lipid membranes, such
as ruptured (Fig. 4F) and created “holes” (Fig. 4GeI) in the body
surface, causing the surface to wither.
3.6. Biochemical responses
Several studies showed that the toxic effects of metal oxide
nanomaterials on A. salina were mediated by the oxidative stress
(Ates et al., 2013, 2016; Gambardella et al., 2014). ROS formation
following nanomaterials treatment seems to be a key event of the
toxic responses, and the imbalance between ROS production and
the T-AOC results in oxidative stress. MDA is a by-product derived
from lipid peroxidation which has been widely used as an indicator
of oxidative stress and oxidative damages to membranes (Ates
et al., 2013, 2016). Antioxidant enzymes catalyze the decomposi-
tion of ROS and prevent organisms from adverse effects of oxidative
stress (Cazenave et al., 2006).
As shown in Fig. 5, on the whole, ROS, MDA content, T-AOC and
antioxidant enzymes (CAT, SOD and GPx) activities were increased
after exposure to Fe3O4-NPs, indicating that the toxic effects were
related to oxidative stress. The result is consistent with previous
studies (Ates et al., 2013; Gambardella et al., 2014). For example,
Ates et al. (2013) demonstrated that the MDA contents of A. salina
were increased following exposure to Zn and ZnO NPs, and the toxic
effects were related to oxidative stress. For instar I and II larvae, the
SOD activity showed a gradual increase and followed by a decrease.
Increase of SOD activity may be due to a response to the superoxide,
high SOD activity can efficiently degrade superoxide. SOD activity
690 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691
may be inhibited by the elevated ROS level, high concentrations of
ROS are able to inhibit the antioxidant enzymes activities.
3.7. Uptake of Fe3O4-NPs
Interactions of nanomaterials with organisms also can be in-
ternal, such as uptake of nanomaterials. Accumulation of nano-
materials in A. salina may cause damage to body tissues. A. salina is
a non-selective filter feeder, and can ingest particles smaller than
50 mm. Suspended particles (no matter what their nature is) with
suitable size are continuously ingested by A. salina (Ates et al., 2013;
Reeve, 1963).
In this study, the uptake of Fe3O4-NPs by A. salina was observed
using a microscope and representative images are shown in
Fig. 6AeD. Fig. 6A shows that the gut for the control was empty.
After exposure, larvae started to ingest Fe3O4-NPs (Fig. 6B). Grad-
ually, the gut was almost entirely filled with Fe3O4-NPs, manifested
by a dark line inside the gut (Fig. 6C). Finally, the accumulated
Fe3O4-NPs was excreted by A. salina (Fig. 6D). The result is consis-
tent with previous study (Ates et al., 2015). Ates et al. (2015)
evaluated the accumulation and depuration of Al2O3-NPs by
A. salina. They demonstrated that Al2O3-NPs deposited inside the
guts and the ingested NPs can be excreted by A. salina. Moreover,
the uptake and distribution of Fe3O4-NPs in A. salina were also
checked using a TEM. Fe3O4-NPs were clearly visible within the
yolk (y; Fig. 6E), primary body cavity (pbc; Fig. 6F) and intestine (in;
Fig. 6G), indicating Fe3O4-NPs can pass through the membrane into
body tissues.
Mitochondrion plays an important role in cell metabolism, and
is one of the most sensitive organelles to xenobiotics (Sun et al.,
2011). There is a close relationship between ROS production and
mitochondrial damage. Disruption of ROS balance can result in the
mitochondrial structure injury. In addition, damage to the mito-
chondria can lead to increased ROS production (Li et al., 2012; Yu
et al., 2013). Mitochondria are considered to be the major organ-
elle that can be seriously affected by NPs toxicity (Yu et al., 2013;
Zhu et al., 2016, 2017b).
According to the TEM images, disruptions of mitochondrial
morphology were clearly visible. In the control, mitochondria
maintained the structural integrity with a well-defined outer
membrane, a narrow inter membrane space and rich cristae
(Fig. 6H). After exposure, mitochondria were slightly swollen with
some vacuoles (Fig. 6I). Mitochondrial cristae was ruptured and
disappeared (Fig. 6J and K). Moreover, mitochondrial membranous
structure disruption was also clearly observed (Fig. 6L and M).
Mitochondrial damages may be likely caused by oxidative stress
and the direct contact of Fe3O4-NPs, and similar results have been
reported in other studies (Park et al., 2014; Sun et al., 2011). Sun
et al. (2011) investigated the mitochondrial damage of HepG2 cell
caused by silica nanoparticles, and demonstrated that the oxidative
stress induced by silica nanoparticles plays an important role in
mitochondrial damage. They also showed that mitochondrial
damage was related to the direct injurious effect of nanoparticles.
To investigate the exact mechanism of mitochondrial damage,
further study should be performed.
3.8. Uptake kinetics
In order to elucidate the accumulation and elimination patterns,
Fe3O4-NPs contents in A. salina were quantificationally measured
by ICP-MS. The content was based on the dry weight of A. salina,
reflecting the total body burden across the exposure from 1 to 72 h.
As shown in Fig. 7, the average Fe3O4-NPs contents were ranged
from 0.38 to 16 mg/g. The result shows an increase during the first
30 h followed by a decrease from 30 to 48 h, and a steady state was
Fig. 7. Fe3O4-NPs contents in A. salina at different time points.
reached after 48 h. The content was slowly increased during the
first 12 h, and rapidly increased from 15 to 30 h. It is the early stage
of instar I during the first 12 h, the mouth and anus of larvae are not
yet completely opened, and the digestive tract is not fully formed
(Sorgeloos et al., 1979). Therefore, a slow increase was observed.
After A. salina molt into instar II, the tissues and organs are fully
formed, thus a fast accumulation of Fe3O4-NPs was occurred. The
decrease from 30 to 48 h is probably due to the discharge of Fe3O4-
NPs. As shown above, the accumulated Fe3O4-NPs can be excreted
by A. salina. Finally, a balance was achieved between accumulation
and elimination, and a steady state was reached. The maximum
content was reached at 30 h (instar II). As mentioned before, instar
II larvae showed the greatest sensitivity to Fe3O4-NPs. Therefore,
the high content may be responsible for the strong toxic responses.
4. Conclusion
In the study, effects of Fe3O4-NPs on A. salina were evaluated to
elucidate the impacts to marine ecosystems. The data so far showed
that acute exposure of cysts (capsulated and decapsulated) and
larvae (instar I, II and III) to Fe3O4-NPs results in significant effects
on hatchability, mortality, and ethological, morphological and
biochemical parameters. The toxic effects were accounted for
Fe3O4-NPs rather than iron ion from the dissolution of NPs, and
were related to oxidative stress. Mitochondrial morphology was
disrupted, and the disruption may be likely caused by oxidative
stress and the direct contact of Fe3O4-NPs. Instar II larvae show the
greatest sensitivity to Fe3O4-NPs, indicating that instar II would be a
suitable candidate for nanotoxicological test. Fe3O4-NPs were
accumulated in the gut and well distributed in yolk and primary
body cavity. In addition, the uptake kinetics data showed that the
Fe3O4 contents in A. salina were firstly increased and then
decreased, and a steady state was finally reached. Results revealed
that Fe3O4-NPs have the potential to affect aquatic organisms when
released into the marine ecosystems. The study mainly focused on
the laboratory evaluation, but chronic exposure at environmentally
feasible levels is necessary for safe and commercial purposes.
Acknowledgements
This work is supported by the Special Funds for Talents in
Northwest A&F University to B. Zhu (Program No. Z111021510) and
China Postdoctoral Science Foundation (Program No.
2015M580888).
 S. Zhu et al. / Environmental Pollution 230 (2017) 683e691
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2017.06.065.
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