Journal of Environmental Protec tion, 2013, 4, 86-91
doi:10.4236/jep.2013.41b016 Published Online January 2013 (
Copyright © 2013 SciRes. JEP
Toxic Effect s o f Nan o -Cu O, Mi cro -CuO and Cu2+ on
Chlorella sp.
Liyan Wang, Mian Wang, Changsheng Peng*, Jinfen Pan
Key Lab of Marine Environmental Science and Ecology of the Ministry of Education,College of Environmental Science and Engi-
neering, Ocean University of China, Qingdao, China.
Email: *
Received 2013
The 96 h acute toxic effects of nano-CuO ( N-CuO), micro-CuO (M-CuO) and Cu2+ on Chlorella sp. were investigated
in this paper. The results showed that toxicities decreased in an order of Cu2+N-CuOM-CuO. The 96 h EC50 of
Cu2+ on Chlorella sp. was 1.06 mg /L, and of N-CuO it wa s 74.61 mg / L, while no p ronounced toxicity was observed
when the concentration of M-CuO was lowe r t han 160 mg/L. Further experi ments were carried out in order to study the
toxicity mechanis m of nano -CuO on Chlorella sp.. The r esults o f Cu2+ release from N-Cu O showed les s than 0 .2 mg/L
Cu2+ were released, so the release of Cu2+ was not responsible for the toxicity. Further exper iment s showed N-CuO in-
hibited formation of Chlorophyll A. Content of Chlorophyll A in the control group was 4.75 mg/108 cells, while it de-
clined to 2.89 mg/108 cells for 160 mg/L N-CuO after 96 h, which indicated that N-CuO c oul d inhib it p hoto syn the sis of
Chlorella sp.. Moreover, N-CuO condensed with algal cells. It affected the activity of SOD and POD, indic ating that
N-CuO could cause oxidant stress to Chlorella sp.. These may be the toxicity mechanism.
Keywords: Nano-CuO; Chlorella sp.; Toxic Effects; Photos ynt hesis; Oxidant Stress
1. Introduction
As a frontier technology, nano-science, as well as infor-
mation science and life science, are the top three pillar
sciences in contemporary society. It has been applied to
broad areas such as materials, information, environment,
life, national security and so on [1]. There are special
physical and chemical properties lying in nano-materials,
for example, small size effect, surface effect, macros-
copic quantum tunneling effect, etc [2]. Distinguishing
properties of nano-materials are observable in optics,
electricity, magnetism, calorifics, mechanic, and chemi-
cal properties [3]. Nano-CuO (N-CuO) has been applied
to catalysts, superconductors, thermoelectric materials,
sensor materials, glass, ceramics and other areas. Also, it
can be used as burning rate catalyzers. In a word, it is a
kind of widely applied nano-material.
Algae, the primary producers, which are the initiate
link of the food line of ecosystem and the origin of bio-
accumulation, play a very essential role in the balance of
the ecosystem. Highly sensitive, having short growth
cycle, easy to be cultivated separately and owning ob-
servable toxic effects directly from a cellular level, algae
are often used for risk assessment as the sensitive factors
to environmental toxic substances. With relatively high
resistance, Chlorella sp., which is one of the algae ap-
peared early on the earth, is a species commonly used for
assessment of toxic and harmful materials [4]. Nowadays,
the toxic studies on N-CuO are still limited, so we carry
on this study to investiga te the to xic effects of N-CuO on
Chlorella sp., aiming to assess the safety of nano-mate-
rial, and provide evidence for standard use of nano-ma-
2. Materials and Methods
2.1. Experimental Materi als and Apparatus
N-CuO, with the particular size of 40 ± 10~20 nm, spe-
cific surface area of 80 m2/g and purity of 99.9%, was
supplied by Beijing Nachen S&T Ltd., which was pro-
duced in chemical vapor deposition method. The TEM
analysis is s hown in Figure 1;
M-CuO, analytical reagent,was bought from Sino-
pharm Chemical Reagent CO., Ltd, with average partic-
ular size of 30 μm measured via Laser Particle Size
Analyzers in Figure 2;
CuCl2•H2O, analytical reagent, was bought from Tian-
jin Guan gchen g Chemic al Reage nt CO. , Ltd ;
Other reagents were all analytical;
Chlorella sp. was obtained from the Fisheries College
of Ocean Uni versity o f Chi na.
Toxic Effect s of Nano-CuO, Micro-CuO and Cu2+ on Chlorella sp.
Copyright © 2013 SciRes. JEP
Figure 1 . TEM image of N-Cu O.
Figure 2 . Particle size analysis of M-CuO.
Table 1. The impr oved f / 2 formula of medi um.
Components Stock con c e ntr ation Medium
NaNO3 75g/L 75mg/L
NaH2PO4.H2O 5g/L 5mg/L
Na2SiO3.9H2O 20g/L 20mg/L
Na2ED TA 4.36g/L 4.36mg/L
FeCl3.6H2O 3.16g/L 3.16mg/L
CuSO4.5H2O 0.01g/L 0.01mg/L
ZnSO4.7H2O 0.023g/L 0.023mg/L
CoCl2.6H2O 0.012g/L 0.012mg/L
MnCl2.4H2O 0.18g/L 0.18mg/L
Na2MoO4.2H2O 0.07g/L 0.07mg/L
Vitamin B1 0.1mg/L 0.1µg/L
Vitamin B12 0.5mg/L 0.5µg/L
Biotin 0.5mg/L 0.5µg/L
Experimental apparatus: GZX illuminating incubator
(Ningbo Jiangnan Instrument Factory), KQ5200DE ul-
trasound distributor (Kunshan Ultrasound Instruments
Co., LTD.), CKX41 inverted fluorescence microscope
(Olympus), SCIENTZ-II D ultrasonic cell crusher (N ingbo
Scientz Biotechnology Co., LTD), UV2600 ultraviolet
and visible spectrophotometer (Shanghai Unico Instru-
ment Co., LTD), Rise 2000 Laser particle size analyzer
(Ji nan Ru nz hi T echno log y Co . , LT D.), TEM (JEM 1230,
2.2. Experimental Methods
2.2.1. Release of Cu2+ From N-CuO
250 mL suspensions of 50 and 100 mg/L N-CuO were
prepared with distilled water in 500 mL conical flasks.
The pH is adjusted to 7.5 by NaOH. T hen the flasks were
put in t he table of 20˚C, 150 r. After 24, 48, 72, 96, 120 ,
144 h, 10 mL of the shaken up solution was taken from
each flask, centrifuged 20 minutes at 4500 r and filtered
by 150 nm filter membrane. The content of Cu2+ was
measured by atomic absorption flame spectrometer .
2.2.2. Experiments of t he Growth of Chlo rella sp.
Influence d by N-CuO, M-CuO and C u2+
We used the improved f / 2 formula of medium (Guillard,
1962). Components of the culture medium are shown in
Table 2. One milliliter stock solution was added to one
liter seawater which had been sterilized by high temper-
ature and high pressure. After continuous training for
three gene rat ions, 20 mL alga e sus pe nsion o f lo gar ith mic
phase was added to 200 mL culture medium for 96 h in
500 mL conical flasks. Init ial algae density was about 6.0
× 105 cells/mL. Temperature of the illuminating incuba-
tor was 25˚C, illumination intensity was 5000 lux, and
light: dark wa s 12 h :12 h. Initial pH was about 8.0.There
were 3 samples in each group and the bottles were sha-
ken by hand 3 times a day. E xperimental equipment was
sterilized for 20 minutes, at 121˚C using pressure steam
sterilizer. Suspensions of 0 , 10 , 20 , 40 , 80 , 160 mg/L
N-CuO and M-CuO were prepared and dispersed by ul-
trasound for 60 min. Concentrations of Cu2+ were 0.10 ,
0.64 , 0.96 , 1.15 , 1.28 , 2.56 , 5.12 mg/L. Density of
algae cells was measured by blood counting chamber at
0 , 24 , 48 , 72 , 96 h dividedly.
2.2.3. Experiments of the Toxicity Mechanism of
N-CuO on C hlorella sp.
Cu2+ released from N-CuO: atomic absorption method;
Chlorophyll a: spectrophotometry; SOD: nitroblue tetra-
zolium method; POD: guaiacol method; CAT: perman-
ganate titration method; MDA: thiobarbituric acid method.
3. Release of Cu2+
At pH 7.5, less than 0.17mg/L Cu2+ was detected in our
experiments. As illustrated in Figure 3, small amount of
Toxic Effect s of Nano-CuO, Micro-CuO and Cu2+ on Chlorella sp.
Copyright © 2013 SciRes. JEP
Cu2+ was released. It should be attributed to the aggrega-
tion of N-CuO and the slightly alkaline pH environment
of the susp ensi on.
4. Toxic Effects of N-CuO, M-CuO, and Cu2+
on Chlorella sp.
As illust rated by the growth curves in Figure 4-Figure 6,
the toxicity ranking of copper materials on Chlorella sp.
was: Cu2+>N-CuO>M-CuO. The toxicity of Cu2+ on
Chlorella sp. was pronounced. The linear interpolation
sugge sted that 96 h EC50 value was 1.06 mg /L. Th e toxic
effect of N-CuO was observed, and 96 h EC50 value was
74.61 mg /L . However, the toxic effect of M-CuO wi thi n
the concentration of 160 mg/ L was les s pronounc ed. T he
toxicity of N-CuO to the algae increased with the con-
centration. A dose-effect relationship between the con-
centration of N-CuO and the toxicity to Chlorella sp.
existed. Furthermore, studies showed that there was also
a dose-effect relationship of N-ZnO and N-T iO 2 on
Chlorella sp. [5].
Figure 3 . Cu2+ release fr om N- CuO.
Figure 4. Growing effect of N-CuO on Chlorella sp.
Figure 5. Growing eff f ect of M-CuO on Chlorella sp.
Figure 6. Growing eff f ect of Cu2+ on Chlorella sp.
5. The Mechanism of Toxicity of Nano-CuO
on Chlorella sp.
5.1. The Influence of Cu2+
Some studies attributed the toxicity of nanoparticles to
the release of metal ions [6-10]. Miao discovered that it
was the Ag+ released b y N-Ag that r esulte d in t he to xici-
ty to Thalassiosira, while no toxicity was detected after
the free Ag+ was infiltrated by membrane or was com-
plexed with mercaptan when most N-Ag formed non-
oxic aggregates which were larger than 0.22 µm in sea-
water[7]. However, there were studies indicating that the
Ag+ released by N-Ag could not totally explain t he toxic-
ity of N-Ag on Chlorella sp. [8]. Aruoja and Franklin
reported that M-ZnO and N-ZnO had similar toxicity to
Pseudokirchneriella subcapitata, which is primarily at-
tributed to the dissolved Zn2+ [9,10]. Aruoja discovered
that Cu2+ was the main cause of the toxic effect of N- uO
to Pseudokirchneriella [9]. But still some studies sug-
gested that N-CuO induced Chlamydomonas reinhardtii
to yield oxygen radicals ROS and the concentration of
Toxic Effect s of Nano-CuO, Micro-CuO and Cu2+ on Chlorella sp.
Copyright © 2013 SciRes. JEP
ions a lone ca nnot expl ain the toxic ity of N-CuO[11].
Solubility te sts of N-CuO sho wed tha t the a queo us so-
lubility of it was highly pH-dependent. At pH 7.5, 100
mg/ L N-CuO in distilled water released about 0.17 mg/L
Cu2+. Indicated by Figure 6, no pronounced toxic effect
of 0.17 mg/L Cu2+ was found on Chlorella sp.. Still, the
toxicity of N-CuO cannot be completely explained. In
conclusion, the toxicity of N-CuO to Chlorella sp. is not
caused by Cu2+ alone.
5.2. Condensation Effect
N-CuO aggregat ed with Chlorella sp. and precipitated, as
illustrated in Fig ure 7. In addition, the shading effect of
N-CuO on Chl orella sp. influenced the growth of it. Si-
milarly, studies showed that N-Ag could directly influ-
ence the surface of Chlorella sp. cells and form large
agglomeration. Though the 50nm Ag may not be able to
enter the cells, but it can perform as the bridge for cells
to connect with each other and accelerate the speed of
cell-aggregating [12]. The aggregations of N-ZnO and
anatase-TiO2 can trap, catch and enclose cells of Chlo-
rella sp. [5]. The toxicity of N-SiO2 to Scenedesmus is
probably caused by the absorption of nano-materials to
the surface of algae cells [13]. The surface of Chlorella
sp. and Scenedesmus absorb N-Al2O3 particles a nd lower
the utilization rate of light, which can be the cause for
inhibiting the gro wth [14]. Thus, the condensation effect
of N-CuO on Chlorella sp. may be one of the probable
explanations of i ts toxicit y.
5.3. Inhibit ed Pho tosynthesis
Photosynthesis is an important influential factor of the
growth of algae. Chlorophyll A is the material basis of
photosynthesis, the content of which represents the grow-
ing condition of algae. Figure 8 illustrates how the con-
centration of N-CuO influenced the Chlorophyll A con-
tent at 96 h.
As the concentration of N-CuO increased, the Chloro-
phyll A content in Chlorella sp. decreased. In control
group, the Chlorophyll A content is 4.75 mg/108 cells
and decreased to 2.89 mg/108 cells after being influenced
by the 160 mg/ L N -CuO for 96 h, indicatin g that N-CuO
could influence the synthesis of Chlorophyll A in
Chlorella sp. and inhibit the photosynthesis of Chlorella
5.4. Oxidative Stress
Nanomaterials can cause oxidative stress to cells, yi elding
superoxide anions, hydroxyl radicals, hydrogen peroxides
etc. Superoxide dismutase SOD is a kind of enzyme which
can remove superoxide anion free radicals; peroxidase
POD is a kind of enzyme which relies on hydrogen
peroxides as the electron acceptor to catalyze and oxidize
substra t es. Thus, by measuring the activity of SOD and
POD of algae cells, the extent that cells are being
oxidative stressed can be indirectly measured.
Figure 7. Light microscopic images of algal cells: control group (a), 80 mg/L (b),sediment of the control (c), sedi ment of 80
mg/L (d).
Toxic Effect s of Nano-CuO, Micro-CuO and Cu2+ on Chlorella sp.
Copyright © 2013 SciRes. JEP
Figure 9 demonstrated that the impact of N-CuO on
antioxidase activities of Chlorella sp. varied with the
concentration of N-CuO. The activity of SOD was
gradually enhanced as the concentration of N-CuO
increased,. In the control group the SOD concentration
was only 0.49 U/108 cells, and increased to 7.88 U/108
cells under the impact of 160 mg / L N-CuO for 96 h. Thi s
figure is 16 times higher than the one before, indicating
that under the oxidative stress of N-CuO, the concentration
of O2- increased, stimulatin g the activity of SOD to resist
the stress caused by N-CuO. When the concentration of
N-CuO was between 0 mg/L and 80 mg/L, the POD
concentration increased from 0.48 U/(108 cells*min)to
Figure 8. The influen ce of N-CuO o n Chlorophyll A content
in Chlorella sp.
Figure 9. The impact of N-CuO on SOD and POD of Chlo-
rella sp.
1.09 U/(108 cells*min), indica ting the i ncreased abilit y of
cells to resist peroxide. When the concentration of N-
CuO reached 160mg/L, the activity of POD declined to
0.39 U/(108 cells*min), one probable explanation of
which might be that under the oxidative stress of high
concentration of N-CuO, POD was destroyed, leading to
the declined an ti peroxidative ability.
6. Conclusion
The experiment demonstrates that N-CuO inhibits the
growth of Chlorella sp. and the toxicity cannot be
completely attributed to the released Cu2+. N-CuO can
absorb cells of Chlorella sp. and cause them to subside.
It also inhibits the composition of Chlorophyll A in
Chlorella sp. and thus influences the p hotosynthesis. T he
concentration of cells of Chlorella sp. was lower
compared to control and the number of cells was smaller.
The antioxidant enzyme system of algal cells is affected
by N-CuO, indicating that N-CuO can cause oxidative
stress. The results provide certain theory basis for
evaluati ng t he sa fety of N -CuO.
7. Acknowledgement s
This work was supported by the Cultivation Fund of the
Key Scientific and Technical Innovation Project, Ministry
of Education of China (NO. 708060), Special Funds for
Environmental Nonprofit Research Project (200809095)
and Public science and technology research funds
projects of ocean, China (201105020).
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