Marine Environmental Research 158 (2020) 105005

Contents lists available at ScienceDirect

Marine Environmental Research

journal homepage: http://www.elsevier.com/locate/marenvrev

Growth inhibition of the microalgae Skeletonema costatum under copper

nanoparticles with microplastic exposure
Xiaolin Zhu a, Weihong Zhao b, c, **, Xiaohua Chen a, Ting Zhao a, Liju Tan a, Jiangtao Wang a, *
a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao, 266100, China
b
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China
c
University of Chinese Academy of Sciences, Beijing, 100049, China

A R T I C L E I N F O A B S T R A C T

Keywords: In order to investigate the combined toxicities of copper nanoparticles (nano-Cu) with microplastic on micro­
Copper nanoparticles algae Skeletonema costatum, growth inhibition tests were carried out. The toxic effects of copper nanoparticles
Microplastic and microplastic on the microalgae under singleness and coexistence conditions were investigated. Both copper
Combined toxicities
nanoparticles and microplastic inhibited the growth of S. costatum. The growth inhibition ratio (IR) increased
Skeletonema costatum
with the increasing of particle concentrations and incubation time. The toxicity of copper nanoparticles was
reduced with the addition of microplastic. The concentrations of Cu2þ in the medium with or without addition of
microplastic were determined. It was found that adsorption of Cu2þ on microplastic and aggregation between
copper nanoparticles and microplastic are the main reasons for attenuation of toxicity of nano-Cu with adding
microplastic. The adhesion and aggregate interactions between microalgae and nanomaterial were also approved
by the observations through scanning electron microscopy.

1. Introduction 2013). Therefore, studies focusing on the combined toxicities of

Nanoparticles, defined as particles with a diameter between 1 and
100 nm in at least two dimensions, have many applications in the fields
of waterproof coatings, cosmetics, nano-medicine and paints due to their
unique capabilities such as small size effect, high surface effect and
quantum size effect (Georgantzopoulou et al., 2012; Rudramurthy and
Swamy, 2018). This expanding usage of nanoparticle increased the
amounts released into the environment through various paths, which
introduced new risks of exposure to ecosystems (Luo et al., 2018).
Microalgae, which lie at the lowest trophic level, are the main producer
and indispensable part of the marine food chain in aquatic environment
(Gilroy et al., 2014). Algae can be used as a model organism to inves­
tigate the toxicity and ecological risks of nanomaterial. The natural
environment is a complex system containing different types of nano­
particles, and the toxicity studies for only one type of nanoparticle may
not reflect its true impact. Nanomaterials undergo complex migration
changes in water environment and absorb many other substances to
form a complex compound. The exopolymeric substances from micro­
algae can also react with nanoparticle to alter its toxicity (Quigg et al.,
different nanoparticles are indispensable.
Currently, most researches about nano-metal on growth of marine
microalgae mainly focused on the effects of only single nanoparticle.
Boxall et al. (2007) suggested environmental concentrations of nano­
particles were approximately 1–100 g/L using a simplified box model
and known currents. Nano-metal in such concentration could inhibit the
growth and photosynthesis of algae and cause oxidative damage. Copper
nanoparticle (nano-Cu), a kind of conductive material, has widely
application in the world, however, there are no data available on the
concentration of nano-Cu in seawater now. Copper ion released by
nano-Cu is an indispensable micronutrient for plants and animals at low
concentration but becomes toxic when its concentration reaches high
quantity. The concentration of copper ion in aquatic environment can
exceed natural levels (>100 μg/L) due to anthropogenic activities
(Bellingeri et al., 2019; Hoang et al., 2009). Many studies reported that
copper ion can inhibit the growth of microalgae at a higher concentra­
tion that exceed natural levels (Barreto et al., 2019; Purbonegoro et al.,
2018). Nano-Cu could inhibit the growth of microalgae with the
maximum inhibition about 38% at the highest concentration of 3.3

* Corresponding author. Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao, 266100, PR
China.
** Corresponding author. Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, PR China.
E-mail addresses: whzhao@qdio.ac.cn (W. Zhao), jtwang@ouc.edu.cn (J. Wang).

https://doi.org/10.1016/j.marenvres.2020.105005
Received 28 December 2019; Received in revised form 8 April 2020; Accepted 27 April 2020
Available online 4 May 2020
0141-1136/© 2020 Elsevier Ltd. All rights reserved.
X. Zhu et al.
mg/L at 24 h (Fu et al., 2007). Li et al. (2015) found that copper ions
released by nano-Cu induced generation of reactive oxygen species and
damaged cell membrane of microalgae S. costatum.
The invention of plastic provides great convenience for life. Plastic
was used in a variety of products such as toy, bags, plastic wares and so
on. About 10% of plastic products end up in the ocean through various
channels, which caused potential risks to environment. Due to weath­
ering, larger plastic objects fragment into smaller particles with diam­
eter less than 5 mm, which was called microplastic. Numerous studies
have shown that microplastic particles could be ingested by organisms
of different trophic levels, causing growth inhibition and lower survival
and reproductive capacity. Microplastic also made negative effect on
growth and photosynthesis of microalgae (Zhang et al., 2017; Zhao
et al., 2019).
According to a number of researches of combined toxicity, when
there exist simultaneously two or more nanoparticles, their toxicities
would present different patterns, such as synergistic, additive or
antagonistic effects (Tong et al., 2015; Ye et al., 2017; Zou et al., 2014).
The combined toxic effects of copper nanoparticles and microplastic on
microalgae were little studied. Both copper nanoparticles and micro­
plastic were toxic to microalgae. Copper ions released by nano-Cu were
the dominant factor for its toxic effect (Barreto et al., 2019; Müller et al.,
2016). Microplastic has a certain adsorption capacity for copper ions
due to high surface area and polarity (Brennecke et al., 2016). The
current work hypothesized that microplastic would affect nano-copper
toxicity through adsorbing Cu2þ released by nano-Cu. This was impor­
tant to mitigate the risk of environmental exposure for copper nano­
particles. Polyvinyl chloride (PVC) is one of the most typical plastics and
more degraded to micro- or nano-plastic than others in aquatic envi­
ronments (Andrady, 2011; Andrady and Neal, 2009; Wu et al., 2019).
Microsized polyvinyl chloride (mPVC) spheres combined with copper
nanoparticles were used in this work to testify their common effects on
the growth of S. costatum.
The objective of this study was to investigate the combined toxic
effects of copper nanoparticles with microplastic on microalgae
S. costatum. The growth inhibition tests were carried out. The adsorption
of Cu2þ on microplastic was analyzed to study the combined toxicity
mechanisms of copper nanoparticles and microplastic. The morphology
of algal cells and the interactions between nanoparticles and microalgae
were observed by SEM.
2. Materials and methods
2.1. Chemicals
Copper nanoparticles (99.9% purity, particle size distribution 10–30
nm, black powder) were purchased from Shanghai Aladdin Co., China.
Microplastic was the microsized polyvinyl chloride (mPVC) with
average particle sizes about 1 μm and was purchased from Shanghai
Youngling Electromechanical Technology Co. Ltd., China. Microsized
polyvinyl chloride was white spherical powder and obtained by a mill­
ing process. CuSO4⋅5H2O (99.9% purity) was purchased from Sino­
pharm Chemical Reagent Co., Ltd, China.
2.2. Algal cultures
The algal strain S. costatum was obtained from the Algal Center of
Key Laboratory of Marine Chemistry Theory and Technology, Ocean
University of China. All glassware was soaked with diluted HCl (10%)
and washed several times with Milli-Q water before and after use.
Seawater with salinity of 31.8 was filtered by 0.45 μm membrane to
remove impurities. The clean glassware and the filtered seawater were
autoclaved at 121 � C for 20 min before use. S. costatum was cultivated in
the f/2 medium. The culture temperature was 20 � 1 � C with a light
illumination of 72 μmol photons m 2 s 1 in a 12 h/12 h light/dark cycle.
The flasks were shaken twice a day to promote CO2 dissolution and
2
Marine Environmental Research 158 (2020) 105005
avoid the precipitation and adsorption to container walls. Samples were
collected at 0, 24, 48, 72 and 96 h to count algal cell density using he­
mocytometer under microscope (Leica, DM4000B).
2.3. Preparation of nanomaterial suspensions
Nanoparticle stock suspensions were prepared through dispersing
nano-Cu or mPVC into Milli-Q water to the final concentration of 100
mg/L and 3 � 103 mg/L, respectively. The suspensions were diluted to a
certain concentration with Milli-Q water, and then ultrasound bathed
30 min for dispersion before the toxicity assay. The size distribution of
nanomaterial suspensions and the zeta-potential in seawater of the f/2
medium were analyzed by a Malvern Mastersizer 3000 (England, Mal­
vern). The SEM characteristics of nano-Cu and miroplastic were showed
in the previous study at our laboratory (Zhang et al., 2017, 2018).
CuSO4⋅5H2O was dissolved in Milli-Q water to prepare a stock so­
lution of 5 mg/L Cu2þ then diluted to different concentration for sub­
sequent experiments.
2.4. Algal growth inhibition test
The toxic effects of nano-Cu and microplastic on microalgae
S. costatum were determined according to OECD Guidelines 201. 100 mL
pre-cultured algae cells in exponential growth phase were transferred
into 250 mL Erlenmeyer flask, and nanomaterials with different con­
centrations were added into the flask, respectively. The final concen­
trations of mPVC in algal cultures were 0, 3, 6, 10, 15, 20 and 30 mg/L
and the final concentrations of nano-Cu were 0, 0.1, 0.5, 1 and 2 mg/L,
respectively. In order to evaluate combined effects of nano-Cu and
mPVC on S. costatum, algae were exposed to a series of combinations of
mPVC and nano-Cu or Cu2þ as follows: nano-Cu plus increasing mPVC as
0.5 mg/L nano-Cu plus 0, 3, 6, 10, 15, 20 and 30 mg/L mPVC; mPVC
plus increasing Cu2þ as 6 mg/L mPVC plus 0, 0.1, 0.2, 0.3 and 0.5 mg/L
Cu2þ, and Cu2þ plus increasing mPVC as 0.2 mg/L Cu2þ plus 0, 3, 6, 10,
15 and 20 mg/L mPVC, respectively. Each experiment was repeated
three times.
2.5. Morphologic changes of the algae
The morphology of S. costatum and the interactions between nano­
materials and microalgae were examined by Scanning Electron Micro­
scope (SEM). After 24 h exposure under 0.5 mg/L nano-Cu treatment,
algal cells were collected by centrifugation (3000 rpm, 5 min), and the
supernatants were removed. The cells were fixed by 2.5% glutaralde­
hyde at 4 � C overnight (12 h). After fixation, the samples were washed
three times for 30 min with 0.1 M phosphate buffer solution (PBS, pH ¼
7.4) and then centrifugated (3000 rpm, 5 min). The samples were
dehydrated with increasing concentration of ethanol solution (30%,
50%, 70%, 80%, 90%, 95% and 100%) for 20 min at room temperature.
After dehydration, samples were fixed with tert-butyl alcohol and
freeze-dried for final observation. The effects of 20 mg/L mPVC with
nano-Cu and without nano-Cu on microalgae were also observed with
the similar method.
2.6. Adsorption of Cu2þ on mPVC
To investigate the mechanism of the combined toxicity of mPVC and
nano-Cu, the adsorption of Cu2þ on mPVC was investigated. Cu2þ so­
lution at concentrations of 0, 0.1, 0.15, 0.2, 0.4 and 0.6 mg/L was pre­
pared and then the suspensions of mPVC was added to make the final
concentration of 6 mg/L. The suspensions were shaken for 24 h and
centrifuged at 4000 rpm for 15 min. The concentrations of Cu2þ in the
supernatant were measured by ICP-OES (Thermo Fisher Scientific, USA).
X. Zhu et al.
2.7. Data analysis
The growth inhibition ratio (IR) was calculated as follows: IR (%) ¼
(1-T/C) � 100%, where T and C were cell density in the experimental
group and control group, respectively.
The result data obtained were expressed as average � standard de­
viation (n ¼ 3). Data were determined in a one-way analysis of variance
with SPSS 16 software. Difference were considered statistically signifi­
cant when p < 0.05.
3. Results
3.1. Particle characterization
The hydrodynamic diameter of copper nanoparticles ranged from
180.3 nm to 388.8 nm in Milli-Q water as shown in Fig. 1A, while the
original average size was about 30 nm. However, the particles were
agglomerated in seawater with an average size of 1215 nm. The primary
average size of mPVC was 1 μm, and the particles size in Milli-Q water
ranged from 6.1 μm to 14.9 μm as shown in Fig. 1B. Microplastic had an
average hydrodynamic diameter of 9.1 μm in seawater. The zeta po­
tential indicated the stability of colloidal system. Both nano-Cu and
mPVC had negative surface charge with zeta potentials about 12.3 and
19.2 mV in seawater, respectively.
3.2. Acute toxicity assay
The addition of nanomaterials to incubation cultures led to inhibi­
tion of cell growth as shown in Fig. 2. The inhibition rate (IR) increased
gradually with the increasing nanoparticles concentration and exposure
time. The IR of nano-Cu on S. costatum reached up to 6.8% at 24 h and
25.2% at 96 h under 0.1 mg/L concentration of nano-Cu. The inhibition
rate reached up maximum at the highest concentration of nano-Cu. IR
was 71.7% under the concentration of 2 mg/L with 96 h exposure as
shown in Fig. 2A.
Microplastic also had negative effects on the growth of S. costatum.
Under low concentration (<6 mg/L) of mPVC, there had no significant
difference between treatments and control (p > 0.05). The IR was below
zero within a short time under low concentration treatments. Then the
inhibition rate (IR) of mPVC on microalgae increased with the concen­
tration and cultivation time that showed the same trend with the toxic
effects of nano-Cu. IR reached up to 14.9% at 72 h and 18.1% with 96 h
exposure under the concentration of 10 mg/L. The maximum IR was
30.5% under 30 mg/L mPVC treatment at 96 h.
Fig. 1. Size distribution of particles in Milli-Q water (A: nano-Cu; B: mPVC).
3
Marine Environmental Research 158 (2020) 105005
3.3. The interaction between particles and S. costatum
The change of morphological feature of S. costatum under different
treatments was observed through scanning electron microscope. The
interaction between particles and microalgae was shown in Fig. 3. SEM
showed that nano-Cu or mPVC wrapped on the surface of microalgae
cells, result in the cell membrane disruption or roughened.
3.4. Combined toxicity of Cu2þ and microplastic to algae
Since 6 mg/L of microplastic had no obvious influence on algae, the
concentration of 6 mg/L was chosen to study the combined toxic effects
of copper ion in the presence of mPVC. The introduction of mPVC
appeared to alleviate the adverse effects of Cu2þ on microalgae
(Fig. 4A). The relative inhibition rate of 0.2 mg/L Cu2þ on the algae was
28.2%, and decreased to 17.8% as mPVC was added with 72 h exposure.
The IR was 57.3% under 0.5 mg/L Cu2þ treatment while IR decreased to
51.5% as the co-exposure of Cu2þ and mPVC. The IR increased with the
concentration of copper ions, however, the average difference between
the IR was reduced gradually and then kept constant about 8%.
To investigated the influence of Cu2þ on mPVC, different concen­
trations of microplastic were added into the cultures that contained
fixed amount of copper ions (0.2 mg/L) to study the inhibition on
S. costatum (Fig. 4B). The IR increased with the increasing concentration
of mPVC, while the change of inhibition rate was complicated under the
co-exposure treatments. The inhibition rate of 0.2 mg/L Cu2þ with
addition of 6 mg/L mPVC on the algae was 16.2% that was higher than
that of mPVC alone, and the enhancement was light compared to 3 mg/L
mPVC added. When the added concentration of mPVC increased to 10
mg/L, the IR reduced drastically about 8.5%–13.5% at 72 h. As the
added concentration of mPVC continuously increased over 10 mg/L, the
inhibition rate enhanced and a dose-response relationship was present.
The inhibition rate was maximum about 30.8% under 20 mg/L mPVC
with of 0.2 mg/L Cu2þ treatment at 72 h.
3.5. Cu-ion adsorption by microplastic
The adsorption of Cu2þ by mPVC was shown in Fig. 5. The amount of
2þ
Cu adsorbed onto microplastic (adsorption capacity, mg/g) was
calculated based on the concentration of non-adsorbed Cu2þ (Cu2þ
equilibrium concentration, mg/L). A significant quantity of Cu2þ was
adsorbed by mPVC of 6 mg/L. The adsorption amounts increased
gradually and reached saturated extent of adsorption as Cu2þ concen­
tration was over 0.2 mg/L.
X. Zhu et al. Marine Environmental Research 158 (2020) 105005

Fig. 2. The growth inhibition rate (IR) of Cu nanoparticles (A) and microplastic (B) on S. costatum, respectively. Vertical bars represent averages � standard de­
viation (n ¼ 3).

Fig. 3. Interactions between particles and microalgae (A: control; B: nano-Cu; C: mPVC). Substances inside circle and rectangle outlines were recognized as nano-Cu
and mPVC aggregates, respectively.

Fig. 4. Change in the inhibition rate (IR) of S. costatum under copper ion and microplastic treatment at 72 h. Vertical bars represent averages � standard deviation (n
¼ 3).

3.6. Combined toxicity of nano-Cu with microplastic shown in Fig. 6. The IR on S. costatum was 32.8% under 0.5 mg/L copper
nanoparticles treatments at 72 h exposure, and IR was 4.9% with the
The growth inhibition of mPVC with 0.5 mg/L Cu nanoparticles on presence of 6 mg/L mPVC alone (Fig. 2). However, the combined toxic
S. costatum was carried out to investigate the combined toxic effects as effects of them were less than the sum. The IR was 31.8% under 0.5 mg/

4
X. Zhu et al. Marine Environmental Research 158 (2020) 105005

L nano-Cu with 6 mg/L mPVC treatments with 72 h exposure (Fig. 6),

and then the combined toxicity increased with the increasing concen­
tration of microplastic. IR reached up to 45.2% under the co-exposure
treatment, while the IR was 22.3% in presence of 15 mg/L mPVC
alone. The highest growth inhibition was 56.7% at the concentration of
30 mg/L mPVC with 0.5 mg/L nano-Cu. The difference of IR was
gradually decreased to a certain level and kept constant over 15 mg/L
mPVC.
The SEM images also provided useful information on the interaction
of nano-Cu with mPVC and algal cells. The algal cell was damaged and
changed its original shape. Heterogeneous aggregates, including nano-
Cu adsorbed onto microplastic and the adhesion of nanoparticles on
the surface of algal cells, were found in Fig. 7.

4. Discussion

4.1. The growth inhibition of nanomaterials on S. costatum

The usual tendency of nanomaterial aggregating was enhanced in

Fig. 5. Adsorption isotherm of Cu2þ on microplastic (6 mg/L) at 25 � C.
Fig. 6. Inhibition rate (IR) of S. costatum under the combination exposure of Cu
nanoparticles and mPVC at 72 h. Vertical bars represent averages � standard
deviation (n ¼ 3).
seawater, where the increasing ionic strength will decrease the repulsive
forces between nanoparticles, and thus favoring agglomeration (Bian
et al., 2011). Müller et al. (2016) found that nano-Cu with an initial
particle size of 6–7 nm agglomerated in freshwater and the average size
of the agglomeration was 140–200 nm. The agglomerates were smaller
than the size of S. costatum. Zeta potential indicated the stability of
colloidal system. The dispersion of copper nanoparticles with a lower
potential was more labile than that of microplastic in seawater. The
suspensions should be always freshly prepared and mixed prior to each
experiment in order to minimize the effects of particle aggregation.
Cu nanoparticles have high reactivity and bactericidal properties
which make them popular in many fields and increased the amount
released into ecosystems and the potential ecological risk. Toxicity of
nano-Cu has been reported for many different types of algae. Nano-Cu
was transferred from the culture medium to the cells affecting
biochemical equilibrium. The growth inhibition of nano-Cu on micro­
algae was increased by the added concentration and the exposure time.
The IR on S. costatum was 72.5% and 87.2% at 96 h under 0.15 and 0.3
mg/L nano-Cu treatments, respectively, which was similar to the inhi­
bition rate of 79.2% in presence of 0.3 mg/L nano-Cu at 72 h (Li et al.,
2015). The IR of nano-Cu on S. costatum reached up to 86% under 1.0
mg/L nano-Cu at 96 h and was related to the concentration of copper
ions released by nano-Cu (Zhang et al., 2018).
The ecological risk of microplastic has drawn attention recently.
Microplastic was harmful to many phytoplankton species at high con­
centration. Microplastic also inhibited microalgal photosynthesis and

Fig. 7. SEM images of S. costatum treated with the mPVC/nano-Cu system. B is enlarged from the square frame of figure A. The circle outlines showed the particles of
nano-Cu and mPVC.

5
X. Zhu et al.
influenced the growth of algae. The inhibit rate of mPVC on dinofla­
gellate Karenia mikimotoi was 45.8% under 100 mg/L of mPVC and
depended on the exposure concentration and cultivation time (Zhao
et al., 2019). Sjollema et al. (2016) reported that Dunaliella tertiolecta
growth rate was inhibited by 57% at 250 mg/L of polystyrene (PS)
concentration relative to controls.
Microalgae can adsorb nano-Cu on its surface and cause mechanical
damage or oxidative damage (Bellingeri et al., 2019; Sabatina et al.,
2009). The interaction between microplastic and S. costatum showed
that microplastic also wrapped on the surface of microalgae cells
(Fig. 3). Physical adsorption of nano-Cu and microplastic on algal cell
prevented energy and substance transfer between the cell and environ­
ment and caused the decrease of nutrient uptake and the availability of
light, which therefore led to the adverse effects on the growth of algae.
The adsorption of particle agglomerates on algae cells resulted in
co-precipitation between mPVC, nano-Cu and algae cells, which also
influenced the cell growth (Bhattacharya et al., 2010; Chen et al., 2018;
Zhang et al., 2017).
4.2. The joint effects of copper ion and microplastic on S. costatum
Ions released by nanometals were the dominant factor causing
toxicity. The algal toxicity of Cu nanoparticles was mainly explained by
bioavailable Cu ions leaching from the particles. The ions could inhibit
the photosynthesis process and induce oxidative stress response,
resulting in the growth inhibition of algae (Barreto et al., 2019; Li et al.,
2015). The inhibitory effects of Cu ion on S. costatum were obvious, and
increased with the concentration of Cu ion (Fig. 4A). Müller et al. (2016)
found that only 1–2% of Cu2þ released by nano-Cu had an inhibitory
action on microalgae Chlamydomonas reinhardtii for growth and photo­
synthesis. Minocha and Mumper (2012) considered that the content of
Cu2þ released by copper nanoparticles were reduced by carbon coating,
which caused lower toxicity of nano-Cu with carbon coating than
without carbon coating.
The toxicity of Cu2þ on algae was apparently higher than that of
mPVC as shown in Fig. 2, and the toxic effects of mPVC less than 6 mg/L
were almost negligible. Under the co-exposure of Cu2þ and 6 mg/L
mPVC, the inhibition rate was lower compared to the group only added
Cu2þ. The IR of 0.2 mg/L Cu2þ in presence of 6 mg/L mPVC was higher
than that of only mPVC added but lower than that of only Cu2þ added
(Fig. 4). The content of Cu2þ in culture media decreased through
adsorption by microplastic, resulting in the decrease of Cu2þ toxic ef­
fects. When the concentration of Cu ions increased over 0.2 mg/L, the
difference of IR between only Cu2þ added and co-exposure treatments of
Cu2þ plus 6 mg/L microplastic was almost constant as shown in Fig. 4A,
possibly due to the amounts of adsorbed Cu2þ onto 6 mg/L mPVC
reached saturation, which was confirmed by the conclusion of Fig. 5.
Then the inhibition rate increased mainly caused by the increased Cu2þ
concentration. When the concentration of microplastic increased over 6
mg/L, the IR of Cu2þ plus mPVC decreased until the mPVC concentra­
tion of 10 mg/L as shown in Fig. 4B, which suggested amounts of Cu2þ in
culture reduced since more Cu2þ was adsorbed onto microplastic with
increasing mPVC concentration. After copper ions were almost adsorbed
as the concentration of microplastic of 10 mg/L, the combined toxicity
was mainly caused by the microplastic and increased gradually with the
increased mPVC concentration.
Many previous studies have shown that hypotoxic nanomaterials
could decrease the toxic effects of heavy metals through adsorption, for
example, Tong et al. (2015) found that TiO2 NPs could alleviate the
toxicity of nano-ZnO due to their adsorption of Zn ions. Nano-TiO2 also
decreased the toxicity of Cu2þ on microalgae by absorption processes
(Chen et al., 2015; Fan et al., 2011). Hartmann et al. (2010) found that
TiO2 attenuated the toxicity of Cr3þ toward Scendesmus obliquus by
surface adsorption of Cr ions. Hu et al. (2016) considered that graphene
oxide decreased the inhibitory effect of copper ions on microalgae
Scendesmus obliquus. Microplastic had the same role as above
6
Marine Environmental Research 158 (2020) 105005
nanomaterials. The adsorption of Cu2þ by microplastic reduced the
bioavailability of Cu2þ for S. costatum, which alleviated the toxic effects
of Cu ions on algae. The accumulation of Cu2þ on mPVC increased until
reaching saturation adsorption (Fig. 5), then the increased toxicity
depended on the increasing concentration of Cu ions or mPVC (Fig. 4).
4.3. The combined toxicity of nano-Cu and mPVC on S. costatum
The IR of Cu2þ was higher than that of nano-Cu at the same con­
centration of 0.5 mg/L as shown in Figs. 2 and 4. Nano-metal partially
dissolved into ions and adsorbed each other to form a larger aggrega­
tion. The aggregates in media could wrap and entrap the algal cells and
contribute to the algal growth inhibition (Aruoja et al., 2015; Ji et al.,
2011). Nanoparticles (such as nano-ZnO, nano-CuO and nano-Co) had
other effects on algae compared to ions. Nanoparticles wrapped onto cell
membrane and affected the photosynthesis of algae. The shading assay
proved that the nanoparticle decreased the light availability, which led
to the growth inhibition (Aruoja et al., 2009; Chen et al., 2018; Zhang
et al., 2016).
Adsorption of Cu ions and copper nanoparticles on microplastic
helped to alleviate the toxicity of nano-Cu on S. costatum. It was sug­
gested that there were two mechanisms for the combined toxic effects of
nano-Cu with mPVC on S. costatum. One was that adsorption of Cu ions
released by copper nanoparticles on microplastic which helped to alle­
viate the toxicity of nano-Cu on S. costatum. Microplastic was hypotox­
icity at low concentration and mainly as an adsorbent that adsorbed an
amount of copper ions and decreased the bioavailability of Cu2þ for
algae, resulting in the decreased toxic effects of nano-Cu. Another was
heterogeneous agglomeration between microplastic and nano-Cu. Nano-
Cu and mPVC adsorbed each other to form aggregation (Fig. 7), which
may hinder the interaction between the algal cells and nano-Cu parti­
cles, resulting in the decreased toxic effects of nano-Cu.
5. Conclusion
The toxicity of nano-Cu with and without mPVC on S. costatum was
investigated and algal growth inhibition tests were carried out. Micro­
plastic and copper nanoparticles had toxic effects on S. costatum and the
toxicity of nano-Cu on algae was higher than that of mPVC. The growth
inhibition rates increased with the concentration of nanomaterial and
the incubation time. Both nano-Cu and microplastic were adsorbed by
S. costatum, resulting in the cells membrane damaged and growth inhi­
bition. Adsorption of Cu2þ on mPVC and the formation of aggregates
between nano-Cu and mPVC were the main reasons for alleviating the
toxicity of nano-Cu with adding mPVC.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Acknowledgements
The study was financially supported by the National Natural Science
Foundation of China [grant number 41876078]; Shandong Provincial
Natural Science Foundation of China [grant number ZR2018MD016];
National Oceanic Administration Public Welfare Project [grant number
201505034] and the Special Fund for Basic Scientific Research in Cen­
tral Colleges and Universities [grant number 201713059].
X. Zhu et al. Marine Environmental Research 158 (2020) 105005

References enriched agriculture soils: implications in everglades restoration. Water Air Soil
Pollut. 199, 79–93.
Hu, C., Hu, N., Li, X., Zhao, Y., 2016. Graphene oxide alleviates the ecotoxicity of copper
Andrady, A.L., 2011. Microplastic in the marine environment. Mar. Pollut. Bull. 62,
on the freshwater microalga Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 132,
1596–1605.
360–365.
Andrady, A.L., Neal, M.A., 2009. Applications and societal benefits of plastics. Philos.
Ji, J., Long, Z., Lin, D., 2011. Toxicity of oxide nanoparticles to the green algae Chlorella
Trans. R. Soc. B-Biol. Sci. 364, 1977–1984.
sp. Chem. Eng. J. 170, 525–530.
Aruoja, V., Dubourguier, H.-C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles
Li, F.F., Pan, R., Zhang, C., Wang, J.T., 2015. Inhibition effects of copper nanoparticles
of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total
on the growth of Skeletonema costatum. China Environ. Sci. 35, 2874–2880.
Environ. 407, 1461–1468.
Luo, Z., Wang, Z., Yan, Y., Li, J., Yan, C., Xing, B., 2018. Titanium dioxide nanoparticles
Aruoja, V., Pokhrel, S., Sihtm€ae, M., Mortimer, M., M€ adler, L., Kahru, A., 2015. Toxicity
enhance inorganic arsenic bioavailability and methylation in two freshwater algae
of 12 metal-based nanoparticles to algae, bacteria and protozoa. Environ. Sci. Nano.
species. Environ. Pollut. 238, 631–637.
2, 630–644.
Minocha, S., Mumper, R.J., 2012. Effect of carbon coating on the physic-chemical
Barreto, D.M., Tonietto, A.E., Amaral, C.D.B., Pulgrossi, R.C., Polpo, A., N� obrega, J.A.,
properties and toxicity of copper and nickel nanoparticles. Small 8, 3289–3299.
Lombardi, A.T., 2019. Physiological responses of Chlorella sorokiniana to copper
Müller, E., Behra, R., Sigg, L., 2016. Toxicity of engineered copper (CuO) nanoparticles to
nanoparticles. Environ. Toxicol. Chem. 38, 387–395.
the green alga Chlamydomonas reinhardtii. Environ. Chem. 13, 457–463.
Bellingeri, A., Bergami, E., Grassi, G., Faleri, C., Redondo-Hasselerharm, P., Koelmans, A.
Purbonegoro, T., Suratno Puspitasari, R., Husna, N.A., 2018. Toxicity of copper on the
A., Corsi, I., 2019. Combined effects of nanoplastics and copper on the freshwater
growth of marine microalgae Pavlova sp. and its chlorophyll-a. IOP Conf. Ser. Earth
alga Raphidocelis subcapitata. Aquat. Toxicol. 210, 179–187.
Environ. Sci. 118, 012060.
Bhattacharya, P., Lin, S., Turner, J.P., Ke, P.C., 2010. Physical adsorption of charged
Quigg, A., Chin, W.C., Chen, C.S., Zhang, S., Jiang, Y., Miao, A.J., Santschi, P.H., 2013.
plastic nanoparticles affects algal photosynthesis. J. Phys. Chem. C 114,
Direct and indirect toxic effects of engineered nanoparticles on algae: role of natural
16556–16561.
organic matter. ACS Sustain. Chem. Eng. 1, 686–702.
Bian, S.W., Mudunkotuwa, I.A., Rupasinghe, T., Grassian, V.H., 2011. Aggregation and
Rudramurthy, G.R., Swamy, M.K., 2018. Potential applications of engineered
dissolution of 4 nm ZnO nanoparticles in aqueous environments: influence of pH,
nanoparticles in medicine and biology: an update. J. Biol. Inorg. Chem. 23,
ionic strength, size, and adsorption of humic acid. Langmuir 27, 6059–6068.
1185–1204.
Boxall, A.B.A., Chaudhry, Q., Sinclair, C., Jones, A., Aitken, R., Jefferson, B., Watts, C.,
Sabatini, S.E., Juarez, A.B., Eppis, M.R., Bianchi, L., Luquet, C.M., de Molina, M.D.C.R.,
2007. Current and Future Predicted Environmental Exposure to Engineered
2009. Oxidative stress and antioxidant defenses in two green microalgae exposed to
Nanoparticles. Central Science Laboratory, Department of the Environment and
copper. Ecotoxicol. Environ. Saf. 72, 1200–1206.
Rural Affairs, London, UK.
Sjollema, S.B., Redondo-Hasselerharm, P., Leslie, H.A., Kraak, M.H., Vethaak, A.D.,
Brennecke, D., Duarte, B., Paiva, F., Cacador, I., Canning-Clode, J., 2016. Microplastic as
2016. Do plastic particles affect microalgal photosynthesis and growth? Aquat.
vector for heavy metal contamination from the marine environment. Estuar. Coast
Toxicol. 170, 259–261.
Shelf Sci. 178, 189–195.
Tong, T., Wilke, C.M., Wu, J., Binh, C.T.T., Kelly, J.J., Gaillard, J.F., Gray, K.A., 2015.
Chen, J., Qian, Y., Li, H., Cheng, Y., Zhao, M., 2015. The reduced bioavailability of
Combined toxicity of nano-ZnO and nano-TiO2: from single- to multi-nanomaterial
copper by nano-TiO2 attenuates the toxicity to Microcystis aeruginosa. Environ. Sci.
systems. Environ. Sci. Technol. 49, 8113–8123.
Pollut. Res. 22, 12407–12414.
Wu, P., Cai, Z., Jin, H., Tang, Y., 2019. Adsorption mechanisms of five bisphenol
Chen, X., Zhang, C., Tan, L., Wang, J., 2018. Toxicity of Co nanoparticles on three species
analogues on PVC microplastic. Sci. Total Environ. 650, 671–678.
of marine microalgae. Environ. Pollut. 236, 454–461.
Ye, N., Wang, Z., Fang, H., Wang, S., Zhang, F., 2017. Combined ecotoxicity of binary
Fan, W., Cui, M., Liu, H., Wang, C., Shi, Z., Tan, C., Yang, X., 2011. Nano-TiO2 enhances
zinc oxide and copper oxide nanoparticles to Scenedesmus obliquus. J. Environ. SCI.
the toxicity of copper in natural water to Daphnia magna. Environ. Pollut. 159,
Health Part A-Toxic. 52, 555–560.
729–734.
Zhang, C., Wang, J., Tan, L., Chen, X., 2016. Toxic effects of nano-ZnO on marine
Fu, F., Liu, Z.Q., Chen, C.H., 2007. Pilot study about effects of copper nanoparticle on
microalgae Skeletonema costatum: attention to the accumulation of intracellular Zn.
phytoplankton growth. Ecol. Sci. 2, 126–130.
Aquat. Toxicol. 178, 158–164.
Georgantzopoulou, A., Balachandran, Y.L., Rosenkranz, P., Dusinska, M., Lankoff, A.,
Zhang, C., Chen, X., Wang, J., Tan, L., 2017. Toxic effects of microplastic on marine
Wojewodzka, M., Hoffmann, L., 2012. Ag nanoparticles: size-and surface-dependent
microalgae Skeletonema costatum: interactions between microplastic and algae.
effects on model aquatic organisms and uptake evaluation with NanoSIMS.
Environ. Pollut. 220, 1282–1288.
Nanotoxicology 7, 1168–1178.
Zhang, C., Chen, X., Tan, L., Wang, J., 2018. Combined toxicities of copper nanoparticles
Gilroy, K.D., Neretina, S., Sanders, R.W., 2014. Behavior of gold nanoparticles in an
with carbon nanotubes on marine microalgae Skeletonema costatum. Environ. Sci.
experimental algal–zooplankton food chain. J. Nanoparticle Res. 16, 2414.
Pollut. Res. 25, 13127–13133.
Hartmann, N.B., Von der Kammer, F., Hofmann, T., Baalousha, M., Ottofuelling, S.,
Zhao, T., Tan, L., Huang, W., Wang, J., 2019. The interactions between micro polyvinyl
Baun, A., 2010. Algal testing of titanium dioxide nanoparticles—testing
chloride (mPVC) and marine dinoflagellate Karenia mikimotoi: the inhibition of
considerations, inhibitory effects and modification of cadmium bioavailability.
growth, chlorophyll and photosynthetic efficiency. Environ. Pollut. 247, 883–889.
Toxicology 269, 190–197.
Zou, X., Shi, J., Zhang, H., 2014. Coexistence of silver and titanium dioxide
Hoang, T.C., Schuler, L.J., Rogevich, E.C., Bachman, P.M., Rand, G.M., Frakes, R.A.,
nanoparticles: enhancing or reducing environment risks? Aquat. Toxicol. 154,
2009. Copper release, speciation, and toxicity following multiple floodings of copper
168–175.