Chemosphere 206 (2018) 802e808

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Ecotoxicity evaluation of natural suspended particles using the

microalga, Euglena gracilis
Yao Xiao a, Peng Zhao b, Yang Yang a, Mei Li a, *
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210023, China
b
Department of Environmental Engineering, School of Environmental Science and Engineering, Tianjin University, Tianjin, 300072, China

h i g h l i g h t s

Ecotoxicity of natural suspended particles (SPs) were investigated with microalgae.

Nanoscale SPs showed more toxic than common-scales ones in Gonghu Bay.

Ecological Restoration Area (ERA) showed significant influences on toxicities of SPs, especially nanoscale ones.

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

Article history: As vectors for pollutants, suspended particles (SPs) have been studied for many years. However, limited
Available online 15 May 2018 studies have focused on the ecotoxicity of natural SPs. This study examined ecotoxicity of natural SPs
Handling Editor: Jian-Ying Hu
isolated from Gonghu Bay and its Ecological Restoration Area (ERA) water samples by Tangential Flow
Filtration (TFF) using the microalga Euglena gracilis as a model organism. Effects of SPs on algae growth,
photosynthesis pigment contents, superoxide dismutase (SOD) activity and DNA damage were charac-
Keywords:
Suspended particles terized to determine the effects of ecological restoration. Additionally, SPs were separated into nanoscale
Nanoscale (<1 mm diameter) and common-scale (1 mm diameter) groups by size, to compare the differences in
Common-scale toxicity of SPs with different sizes. We found, in naturally occurring concentrations in Gonghu Bay,
Ecological restoration nanoscale SPs were more toxic than common-scale ones. However, no significant adverse effects were
Ecotoxicity detected in the nanoscale SPs from the ERA, which demonstrated that ecological restoration might
Euglena gracilis reduce the toxicity of nanoscale SPs. The results were supported by the inhibition of growth, SOD ac-
tivities and DNA damage, while no adverse influences were detected on pigment contents of E. gracilis in
all the treated groups. Our study provides new insights into the toxic effects of SPs.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction
Suspended particles (SPs) are important components of natural
aquatic environments. Naturally occurring SPs, including nano-
scale and common-scale ones, are commonly defined as particles
ranging from 1 nm to 62 mm that remain in suspension due to their
small sizes (Waters, 1995). Nanoscale SPs are defined as particles
ranging from 1 nm to 1 mm (Handy et al., 2008). Due to the different
size-based characteristics and the increasing emissions of engi-
neered nanoparticles and microplastic into the environment, both
nanoscale (<1 mm diameter) and common-scale (1 mm diameter)
SPs have caused extensive attention (Zhang et al., 2013; Sani-Kast
* Corresponding author.
E-mail address: meili@nju.edu.cn (M. Li).
et al., 2015). SPs serve as vectors for many pollutants of concern,
such as heavy metals and persistent organic pollutants (POPs)
(Zhang et al., 2007; Liu et al., 2017; Wang et al., 2017), which can
affect the biological, chemical, and physical status of aquatic eco-
systems (Schillinger and Gannon, 1985; Woelz et al., 2008; Rivetti
et al., 2015). Therefore, there is an increasing need for environ-
mental toxicologists to determine whether SPs have the potential
to threaten aquatic organisms.
Evidence has indicated that SPs can directly or indirectly, affect
aquatic organisms (Bilotta and Brazier, 2008). Physical abrasion and
interference with respiration and ingestion are the most common
ways in which SPs directly impact aquatic organisms (Kjelland
et al., 2015). Natural SPs are of various shapes, thus aquatic or-
ganisms are likely to be abraded by sharp particles which, under
extreme circumstances, may damage their body parts (Wilber and
Clarke, 2001). Zooplanktons, such as Daphnia magna, are known to

https://doi.org/10.1016/j.chemosphere.2018.05.061
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
 Y. Xiao et al. / Chemosphere 206 (2018) 802e808
ingest SPs as they feed relatively non-selectively with respect to
particle size. This has been shown to decrease feeding efficiency,
therefore affecting growth, reproduction, or mortality (via me-
chanical interference with ingestion) (Kirk and Gilbert, 1990; Zhang
et al., 2013). Previous studies have also documented avoidance
behavior, decreased feeding, and reduced growth rates in a range of
aquatic species exposed to SPs (Kjelland et al., 2015; Suedel et al.,
2017). Moreover, a few studies have shown that SPs can cause
DNA damage in some aquatic organisms (Boettcher et al., 2010; Tse
et al., 2010). As reported in Tse et al. (2010), at the low concentra-
tion of SPs, a significant increase in tail length was detected in the
comet assay, which might due to the pollutants absorbed on the
SPs. However, studies on the genotoxicity of natural SPs remain
scarce. Previous studies have also shown that SPs can indirectly
affect aquatic organisms, commonly via changing the aquatic
environment (Redding et al., 1987). For example, SPs have been
reported to limit the amount of light penetrating the water column,
thus influencing photosynthesis in macrophytes and algae, then
affecting other higher-tier aquatic organisms through the food
chain. SPs can also reduce dissolved oxygen, which can cause a
reduction in the planktonic community (Kjelland et al., 2015).
However, a vast majority of previous studies chose materials like
kaolinite or montmorillonite, or collected natural sediment to
simulate natural SPs (Bilotta and Brazier, 2008; Zhang et al., 2013;
Kjelland et al., 2015). Although these materials may represent the
size of nanoparticles, they are different in chemical composition
and shape than natural SP. Furthermore, although natural sedi-
ments have a similar chemical composition to SPs, they still display
deviations, especially after some common pretreatments (e.g. air-
drying, grind etc.). Therefore, SPs directly isolated from the natu-
ral surface water are the best choice to investigate the effects of
these particles on aquatic organisms.
Tangential Flow Filtration (TFF) has been previously used to
separate and characterize SPs from natural waters. Compared to
classical filtration techniques, such as using 0.45 mm filters, the use
of TFF overcomes many analytical problems (Ran et al., 2000).
Moreover, this technique has also been widely employed to
concentrate cells, bacteria, particles or virus in medicinal and
biochemical fields (Cai et al., 2015). Indeed, TFF can be used to
collect SPs from water samples, which can then be used in the
experiment. As far as we know, limited analysis has focused on the
toxicity of natural SPs separated from water samples by TFF.
Euglena gracilis is a freshwater unicellular flagellated protozoa,
that generates energy through light-dependent photosynthesis,
and lives in many aquatic ecosystems (Sani-Kast et al., 2015).
Indeed, E. gracilis has been widely used as a reliable model organ-
ism for studying the biological effects of environmental contami-
nants, especially heavy metals and nanoparticles (Einicker-Lamas
et al., 2002; Miot et al., 2008; Azizullah et al., 2012). E. gracilis
was chosen to be used in this study due to the fact that it lacks cell
walls and thus may be sensitive to SPs.
Taihu Lake, the third largest freshwater lake in China, is an
important source of drinking water for the Yangtze River Delta
urban area (Ni et al., 2016). Gonghu Bay (GB) is an important part of
Taihu Lake, and serves as the main source of drinking water for
Wuxi City. However, in the past three decades, GB has experienced
serious eutrophication (Su et al., 2017). To improve the water
quality of GB, an Ecological Restoration Area (ERA), based on sub-
merged plants and aquatic animals, was established in 2013 (Gao
et al., 2017). However, the influence of the restoration on SPs and
the toxicological effects of these SPs remain unknown.
The objectives of this study are as follows: (1) to compare the
differences in toxicity between nanoscale and common-scale SPs
isolated from natural water samples and (2) to determine the ef-
fects of ecological restoration on the toxicity of SPs, by evaluating
803
the effects of nanoscale and common-scale SPs isolated from GB
and its ERA on E. gracilis. Growth of E. gracilis was analyzed to
evaluate acute toxicity and the comet assay was employed to
evaluate genotoxicity. Photosynthesis pigment and superoxide
dismutase (SOD) activity were analyzed to determine the mecha-
nism of the toxic effects. To our knowledge, this study established,
for the first time, the effects of natural SPs directly isolated from
surface water on freshwater microalga and determined whether
ecological restoration will affect the toxicity of SPs.
2. Materials and methods
2.1. Materials
E. gracilis was obtained from the Freshwater Algae Culture
Collection at the Institute of Hydrobiology (FACHB-collection).
Cultures were grown and maintained in 250 ml Erlenmeyer flasks
containing Checcucci culture medium at 25 ± 1  C. Concentration of
nutrients in the medium was based on the formula given by Hu
et al., (Hu et al., 2015). The light/dark cycle was 12 h/12 h with a
photon irradiance of 85e90 mmol m2s1, provided by cool white
fluorescence lights. These culture conditions were also used for all
toxicity experiments. Algae inoculums were prepared three days
before the toxicity tests to obtain exponential growth.
2.2. Study sites and sample collection
Water samples were collected from GB and its ERA in December
2016 (Fig. 1). Four sampling sites were set at GB (S1eS4) and
another four were set at the ERA (S5eS8). These sites were chosen
due to their proximity to cities and distance from discharge points,
as they may be affected by anthropogenic activities. December was
chosen as the month to collect water samples for two reasons.
Firstly, the water quality index we monitored steadily met the re-
quirements of the Gonghu Bay Ecological Restoration project in
December. Secondly, winter is a dry season, thus the chance of
rainfall (interrupting sample collection) is limited. All the samples
were collected at 0.5 m beneath the water-surface. Final samples
were obtained by mixing equal volume of water from all four sites
at GB and ERA respectively. Water samples need hypothermic
preservation before filtered with coarse gauze, and the processed
samples were stored at 4  C until used in experiments. Inductively
Fig. 1. Sampling sites in Gonghu Bay (S1eS4) and the Ecological Restoration Area
(ERA) (S5eS8).
804 Y. Xiao et al. / Chemosphere 206 (2018) 802e808
Coupled Plasma Mass Spectrometry (ICP-MS) (NexION 300, USA)
was used to detect essential ion concentration in all water samples.
As lake water was different in space, a formulated water aimed to
prevent the experiment results from being affected by dissolved
substances was set. The formulated water here is a kind of artificial
water consisting of pure water and average concentration of
essential ions in these two areas. The size distribution of SPs were
detected using Laser Particle Size Analyzer (Mastersizer 3000,
England).
2.3. Fractionation of particles
Particles were isolated from water samples by Tangential Flow
Filtration (TFF, SYR2-U20-0LN, USA). Meanwhile, natural SPs were
separated into nanoscale (<1 mm diameter) and common-scale
(1 mm diameter) groups and dissolved in the formulated water.
SPs isolated from 100 mL of original water samples were dissolved
in the same volume of formulated water to maintain the environ-
mental concentration. To verify the separation, Laser Nanometer
Size Analyzer (Malvern Zetasizer Nano-S, England) was used to
detect the size distribution of the nanoscale SPs. After pretreat-
ment, all the water samples were stored at 4  C until the toxicity
tests were conducted.
2.4. Microalga growth curve
E. gracilis was exposed to different sizes of SPs in a 96 h toxicity
bioassay. 100 ml of algae cell suspension was centrifuged at 3500 g
for 15 min, removing the supernatant and then re-suspended in
5 ml phosphate buffer solution (PBS). 200 ml of re-suspended algal
culture was added to 20 ml glass bottle preloaded with 10 ml
formulated water containing different sizes of SPs. The initial
cellular concentration was approximately 1  105 cells mL1. Algal
growth (as cell density) was measured spectrophotometrically at a
wavelength of 680 nm by Multiscan Spectrum (INFINITE M200,
China) every 24 h for 96 h. As our previous study showed a linear
relation between cell number and OD680, the linear fitting equation
was as follows (Li et al., 2014):
 
y ¼ ð94:31  D680 þ 0:91Þ  104 R2 ¼ 0:999
where, y indicated algal cell concentration and D680 indicated
absorbance at 680 nm.
2.5. Photosynthetic pigment analysis
After 96 h of exposure, 1 ml of algal cell suspension was
centrifuged for 15 min at 3500 g, and then extracted with 80%
acetone for 24 h. Extracts were maintained at 4  C until analysis and
all experiments were performed in dim green safe light to avoid
photo-degradation. Absorbance was determined via Multiscan
Spectrum Analysis (INFINITE M200, China). For chlorophyll a (Chl
a), chlorophyll b (Chl b) and carotenoid (CAR) determinations, ex-
tracts were measured at 663 nm, 645 nm, and 470 nm as described
in Lichtenthaler (1987). The calculation formula was as follows:
Chl a ¼ 12:21A663  2:81A645
Chl b ¼ 20:13A645  5:03A663
CAR ¼ ð1000A470  3:27Chl a  104Chl bÞ=229
2.6. Superoxide dismutase (SOD) activity
For enzyme assays, 5 ml of algal cell suspension was cultured for
96 h and then centrifuged at 3500 g for 15 min, removing the su-
pernatant. One ml of pre-cooled sodium phosphate buffer (PBS)
was subsequently added to re-suspend the algae cells. After soni-
cation (JY88-II, China; 50 Hz, 3 s pulse, and 5 s interval) for 5 min in
an ice bath, the cell debris was removed via centrifugation at 5000g
for 15 min at 4  C. The supernatant was used for the biochemical
assays. SOD activity was measured using a commercial SOD-kit
(Jiancheng Co. Ltd., Nanjing, China). Enzyme activities were stan-
dardized by protein content. Each experiment was performed in
triplicate.
2.7. Comet assay
After 48 h of exposure, 1 ml of algal cell suspension was
centrifuged at 3500 g for 15 min. Nuclear DNA damage was deter-
mined using Single Cell Gel Electrophoresis (SCGE), which is
commonly called the comet assay. Experimental steps were based
on the methods described in Dou et al. (2015). Slides were exam-
ined with a Fluorescent Microscope (BX41, Olympus, Japan). Images
were analyzed by CASP software. Tail DNA%, Tail Moment (TM) and
Olive Tail Moment (OTM) were chosen as endpoints to analyze the
genotoxicity of SPs.
2.8. Statistical analysis
Each experiment was in triplicate. Results were analyzed using
SPSS 23.0 software (SPSS Inc.). One-way ANOVA tests were used to
evaluate the statistic differences between both SP-treated groups
and the control group for biological parameters. Results were
presented as mean ± SD of three replicates. A probability
value < 0.05 (p < 0.05) was accepted as significance. Comet assay
results were analyzed using CASP software.
3. Results and discussion
3.1. Characteristics of original water samples
In the present study, seven kinds of essential ions were detected,
including three kinds of positive ions (Ca2þ, Mg2þ, Naþ) and four
kinds of negative ions (Cl, SO2  
4 , NO3 , HCO3 ) (Table 1). Ion con-
centrations were significantly different between GB and ERA
(p < 0.05), especially the NO 3 . We hypothesized that these were
due to the nitrification and anti-nitrification reactions associated
with eutrophication.
As indicated in Fig. 2, SPs size distribution of GB and ERA ranged
from 1 nm to 22.9 mm. This agrees with previous studies that have
reported that the sizes of SPs are commonly ranged from 1 nm to
62 mm (Waters, 1995). SPs isolated from GB (SPs-GB) consisted of
78.8% nanoscale particles and 21.2% common-scale ones. The size
distribution of SPs isolated from ERA (SPs-ERA) was similar to SPs-
GB, including 74.1% nanoscale particles and 25.9% common-scale
particles. No significant differences in size distribution of the SPs
were observed between GB and ERA. Results indicated that there
was a diversity in concentrations between nanoscale and common-
scale SPs in natural water samples. However, compared to
common-scale SPs, nanoscale SPs, which cannot freely settle in
static situations, were barely removed during drinking water
treatment (Xu et al., 2015). As concentration is an important factor
in ecotoxicity, this result may evidence supporting that nanoscale
SPs may be larger health threats than common-scale SPs.
 Y. Xiao et al. / Chemosphere 206 (2018) 802e808 805

Table 1
Ion concentrations (mg/L) in water samples from GB and ERAa.

Ca2þ Mg2þ Naþ Cl SO2-

4 NO
3 HCO
3

GB 35.45 ± 3.21 9.56 ± 0.20 27.02 ± 0.67 38.58 ± 0.78 70.44 ± 1.28 5.32 ± 0.11 132.08 ± 0.30
ERA 37.45 ± 2.35 11.45 ± 0.41 21.65 ± 0.40 24.81 ± 0.26 76.65 ± 1.37 0.19 ± 0.09 124.27 ± 0.36
a
GB means Gonghu Bay, ERA means Ecological Restoration Area.

Fig. 2. Size distributions of SPs in natural water samples from Gonghu Bay (A) and Ecological Restoration Area (B). Results shown are mean of three replicates.

3.2. Growth inhibition of E. gracilis
As shown in Fig. 3, no significant changes in the growth were
detected when E. gracilis was exposed to total SPs, no matter
whether isolated from GB or ERA. This was in agreement with
previous results reported by Zhang et al. (2013) i.e. that no adverse
impact of natural suspended sediment on Daphnia magna was
observed. However, when particle size was considered, SPs-GB
showed significant (p < 0.05) inhibition of E. gracilis growth, espe-
cially the nanoscale particles (p < 0.01). After 96 h of exposure, the
growth inhibition rates of E. gracilis exposed to nanoscale and
common-scale SPs of GB were 53.1% and 22.5%, respectively.
Common-scale SPs-ERA also significantly (p < 0.05) inhibited
growth rate of E. gracilis, at an inhibition rate of 18.2%.
For SPs-ERA, though the percentage of nanoscale SPs were
significantly higher than common-scale ones (p < 0.05), the
growth inhibition rate of E. gracilis was significantly lower
(p < 0.05). This was consistent with a previous study that indi-
cated that at the same concentration, the nanoscale particles were
less toxic than common-scale ones (Adams et al., 2006; Zhang

Fig. 3. Growth curve of E. gracilis after 96 h of exposure to SPs isolated from Gonghu Bay (A) and Ecological Restoration Area (B). Water means formulated water, D means diameter
(D < 1 ¼ nanoscale SPs; D  1 ¼ common-scale SPs). Data were presented as mean ± SD of three replicates.
806 Y. Xiao et al. / Chemosphere 206 (2018) 802e808
et al., 2013). However, the growth curve of E. gracilis exposed to
SPs-GB showed that nanoscale SPs were much more toxic than
common-scale ones. These differences in toxicity may be due to
the difference in concentration of particles between GB and the
ERA. As nanoscale SPs have larger specific surface areas, they can
absorb large number of pollutants (Bengtsson and Picado, 2008).
Therefore, the larger concentration of nanoparticles in the GB
(with pollutants absorbed onto them) is likely responsible for the
increased toxicity observed. In addition, Wheatland et al. (2017)
found that microorganisms are important for SPs, therefore the
differences in microorganisms present on nanoscale and
common-scale SPs might also effect the toxicity. However, limited
studies have focused on the distribution of microorganisms on
natural SPs of different sizes. SPs-GB were shown to be much
more toxic to the growth of E. gracilis than the SPs-ERA, especially
the nanoscale ones, which demonstrated that ecological restora-
tion affected the acute toxicity of nanoscale SPs.
3.3. Photosynthetic pigment content
Keeping photosynthetic pigment content in balance is essential
for the microalga to maintain efficient utilization of carbon sources
and to meet their energy demands. Therefore, photosynthetic
pigment content is an important indicator of photosynthetic effi-
ciency of microalgae and macrophytes (Li et al., 2015). Indeed, it is
widely used in environmental toxicity assessments to indicate
contaminant stress on photosynthesis.
Fig. 4 shows the photosynthetic pigment contents of E. gracilis,
determined via spectrophotometric analysis after 96 h of exposure.
Photosynthetic pigment contents of E. gracilis, including chloro-
phyll a, chlorophyll b and carotinoid, in all the SPs-treated groups
showed a significant (p < 0.05) increase compared to the control,
especially the chlorophyll a and b levels, indicating that SPs can
influence photosynthesis. When exposed to nanoscale SPs-GB, the
maximum increase rate of algae chlorophyll a and carotinoid
reached 60.3% and 53.9% respectively. Similarly, the concentration
of chlorophyll b reached 0.18 mg/105 cells in the group treated by
nanoscale SPs-GB.
Most previous studies have reported a decrease of photosyn-
thetic pigment content after exposure of algae to aquatic pollutants
(Azizullah et al., 2011; Sani-Kast et al., 2015). On the contrary, an
Fig. 4. Photosynthetic pigment contents of E. gracilis after 96 h of exposure to SPs isolated from Gonghu Bay (A) and Ecological Restoration Area (B). Water means formulated water,
D means diameter (D < 1 ¼ nanoscale SPs; D  1 ¼ common-scale SPs). Data were presented as mean ± SD of three replicates. *p < 0.05, **p < 0.01.
increase of photosynthetic pigment content was detected in this
study, which was close to that reported in Deng et al. (2017). Ac-
cording to this result, SPs had no adverse impact on photosynthetic
pigment contents. We conjectured that this might be due to the
high concentration of nitrogen and phosphorus in GB, resulting
from strong eutrophication that was reported to have occurred in
June 2016. Nitrogen is an essential element of chloroplast, therefore
the increased elemental concentration of N and P in GB could have
induced an increase of chlorophyll content in the microalga
(Dahmen-Ben Moussa et al., 2017; Zhang et al., 2017). Furthermore,
restricted ability to maintain photosynthetic function, stemming
from a decrease in chlorophyll content, in response to N and S
starvation has previously been reported (Cakmak et al., 2012).
However, as photosynthetic pigment content does not completely
represent the photosynthetic efficiency, further studies need to be
conducted to determine the effects of SPs on photosynthesis of
microalga.
3.4. Superoxide dismutase activity
As a key antioxidant defense enzyme, SOD plays important roles
in normal cellular metabolism. SOD eliminates redundant ROS and
protects exposed organisms from the potential toxic effects of
pollutants. Therefore, SOD activity is an important biomarker for
the estimation of oxidative stress (Deng et al., 2017).
As shown in Fig. 5, exposure to total SPs caused a significant
(p < 0.05) increase in SOD activity, especially SPs-GB (p < 0.01).
Nanoscale SPs-GB also induced an increase of SOD activity. This
observed increase in the enzymatic activity in E. gracilis was
attributed to stress responses induced by excessive intracellular
ROS generation, indicating that oxidative stress was induced after
96 h of exposure. Although SOD activity was significantly increased
in E. gracilis after exposure to total SPs isolated from GB and the
ERA, no significant inhibitions of growth were detected. However,
previous study concluded that SOD is the first line of defense
against oxidative stress in plants, and may be more sensitive than
the growth inhibition test (Li et al., 2009; Dahmen-Ben Moussa
et al., 2017).
On the contrary, common-scale SPs-GB and both nanoscale and
common-scale SPs-ERA had no significant impacts on SOD activity
in E. gracilis. Therefore, other effect mechanisms such as abrasion or
 Y. Xiao et al. / Chemosphere 206 (2018) 802e808
Fig. 5. SOD activity in E. gracilis after 96 h of exposure to SPs isolated from Gonghu Bay (A) and Ecological Restoration Area (B). Water means formulated water, D means diameter
(D < 1 ¼ nanoscale SPs; D  1 ¼ common-scale SPs). Data were presented as mean ± SD of three replicates.
ingestion may be more critical in the toxic mechanism of action of
SPs on E. gracilis.
3.5. DNA damage
As a simple, convenient, rapid and sensitive technique, the
comet assay has been widely used to determine DNA damage and
evaluate genotoxicity of environmental pollutants in recent years
(Rothschild and Pearson, 1998; Aoyama et al., 2003; Santiago
Martinez et al., 2015).
Percentage of tail DNA (% tDNA), Tail Moment (TM) and Olive
Tail Moment (OTM) are widely used parameters in the comet assay
to determine DNA damage. These parameters positively correlate
with genotoxicity (Li et al., 2014). After 96 h of exposure, significant
differences were detected between the control and all SP treated
Fig. 6. Induction of DNA damage (represented as % tDNA, Tail Moment and Olive Tail
Moment) in E. gracilis after 96 h of exposure to SPs isolated from Gonghu Bay (A) and
Ecological Restoration Area (B). Water means formulated water, D means diameter
(D < 1 ¼ nanoscale SPs; D  1 ¼ common-scale SPs). Data were presented as mean ± SD
of three replicates. Typical images from comet assays of E. gracilis cells in formulated
water (no obvious damage) (C) and exposed to nanoscale SPs isolated from Gonghu
Bay (significant damage) (D).
807
groups (Fig. 6). The maximum increase of % tDNA, TM and OTM was
observed in the group exposed to nanoscale SPs-GB (Fig. 6A).
Photographs of nuclear DNA in comet assays show that the
formulated water caused no DNA damage (Fig. 6C) and the nano-
scale SPs-GB caused significant DNA damage (Fig. 6D). The results
of the comet assay presented here showed that the SPs isolated
from GB and the ERA could both induce DNA damage in E. gracilis,
which may affect the survival of this microalga after long-term
exposure to SPs. Furthermore, nanoscale SPs-GB showed higher
genotoxicity than common-scale ones, which was in agreement
with the results of the acute toxicity tests. We also concluded that
the comet assay was more sensitive than other experiments, which
has been determined in a previous study (Dou et al., 2015).
3.6. Effects of ecological restoration
We found that both acute toxicity and genotoxicity of natural
nanoscale SPs were reduced after ecological restoration. However,
ecological restoration had no significant effects on common-scale
SPs. These results demonstrated that ecological restoration is
likely to affect the toxicity of nanoscale SPs. We hypothesized that
this occurred for several reasons. First, ecological restoration might
have affected the location of the SPs promoting their flocculation
and subsidence, which was supported by a previous study (Gao
et al., 2017). Indeed, a significant difference in the concentration
of total suspended sediment was detected between GB and ERA.
Second, there was a similar size distribution of SPs in both GB and
its ERA (Fig. 1). Although the concentration of common-scale SPs
was reduced, the toxicity was not changed. Therefore, we specu-
lated that flocculation or subsidence was not the only reason for the
toxicological differences between the ecologically restored area and
GB. The influences of ecological restoration on microorganisms and
contaminants absorbed to SPs might be another reason for these
differences. Indeed, a previous study reported that pollutants are
more inclined to be absorbed on particles with a smaller size
(Changcheng et al., 2006; Zhang et al., 2007). Nonetheless, the in-
fluences of ecological restoration on toxicity of SPs are still unclear
and needs to be further studied.
4. Conclusions
In conclusion, our study showed SPs-GB significantly inhibited
808 Y. Xiao et al. / Chemosphere 206 (2018) 802e808
the growth of E. gracilis and induced an increase in SOD activity.
Furthermore, nanoscale SPs-GB were more toxic than common-
scale ones both in the acute toxicity experiments and comet
assay. Ecological restoration was found to reduce the toxicity of SPs,
especially the toxicity of nanoscale ones. However, the differences
between toxicological mechanisms of action for nanoscale and
common-scale SPs and the effects of ecological restoration on the
toxicity of SPs are still unclear, thus need to be further studied. This
study provides new insights into the toxic effects of SPs.
Conflicts of interest
The authors have declared that no competing interests exist.
Acknowledgments
This research was supported by National Major Project of Sci-
ence & Technology Ministry of China (Grant No. 2014ZX07204-
005), Science and Technology Support Program of Jiangsu Province
(No. BE2016736) and National Natural Science Foundation of China
(Grant No. 41773115, 41571468).
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