Ecotoxicology and Environmental Safety 163 (2018) 594–603

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Algicidal properties of extracts from Cinnamomum camphora fresh leaves and T

their main compounds
Silan Chena,1, Tiefeng Zhenga,1, Chaolin Yea, Wulan Huannixia, Zumulati Yakefua, Yiyu Menga,
Xin Penga, Zhengfeng Tiana, Junhao Wangb, Yuandan Maa, Youyou Yangb, Zhongqing Mab,
⁎
Zhaojiang Zuoa,
a
School of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou 311300, China
b
Key Laboratory of Wood Science and Technology of Zhejiang Province, Zhejiang A & F University, Hangzhou 311300, China

A R T I C LE I N FO A B S T R A C T

Keywords: Plant allelochemicals are considered as the source of effective, economic and friendly-environmental algaecides.
Algaecide To uncover the anti-algal activities of Cinnamomum camphora fresh leaves and their main algicidal agents, we
Camphor investigated the inhibitory effects of water and methanol extracts from C. camphora fresh leaves on Microcystis
Cinnamomum camphora aeruginosa and Chlamydomonas reinhardtii cell growth, analyzed the composition of the water and methanol
Linalool
extracts, and determined the main compounds in extracts on the growth of the two algae and their anti-algal
α-terpineol
mechanism from photosynthetic abilities. Water and methanol extracts from C. camphora fresh leaves can inhibit
M. aeruginosa and C. reinhardtii cell growth, and methanol extracts showed stronger inhibitory effects, due to
their more compounds and higher molar concentration. There were 23 compounds in the water extracts, mainly
including terpenoids, esters, alcohols, and ketones. Compared to the water extracts, 9 new compounds were
detected in the methanol extracts, and the molar concentration of total compounds in methanol extracts in-
creased by 1.3 folds. Camphor, α-terpineol and linalool were 3 main compounds in the water and methanol
extracts. Their mixture (1: 3: 6) and individual compound showed remarkable inhibition on M. aeruginosa and C.
reinhardtii cell growth. The degradation of photosynthetic pigments and the reduction of maximum quantum
yield of photosystem II (PSII) photochemistry, coefficient of photochemical quenching as well as apparent
electron transport rate in C. reinhardtii cells aggravated gradually with increasing the concentration of the
mixture and individual compound, while the non-photochemical dissipation of absorbed light energy increased
gradually, which led to the decline of photosynthetic abilities. This indicated that camphor, α-terpineol and
linalool were 3 main algicidal agents in C. camphora fresh leaf extracts, and they inhibited algal growth by
inducing photosynthetic pigment degradation and declining PSII efficiency. Therefore, C. camphora fresh leaf
extracts and their main components have potential utilization values as algaecides.

1. Introduction
Eutrophication is a natural process concerning all habitats, and
becomes more serious with the increasing input of nutrients, mainly
nitrogen (N) and phosphorus (P), caused by human activities in the past
decades. Eutrophication promotes the excessive growth of phyto-
plankton and leads to algal blooms (Azevedo et al., 2002; Smayda,
2004). In recent decades, occurrences of algal blooms have increased
around the world. The main algae causing the blooms are cyano-
bacteria, and sometimes green algae (Azevedo et al., 2002; Hudnell and
Dortch, 2008; Kravtsova et al., 2014).
Algal blooms cause a series of ecological problems and lower the
water quality, due to the emission of secondary metabolites including
volatile organic compounds (VOCs) and algal toxins (Dodds et al., 2009;
Xu et al., 2017; Zuo et al., 2018a). Algae release a wide spectrum of
VOCs, mainly including sulfocompounds, alkanes, terpenoids, ben-
zenes, alcohols, aldehydes, ketones, and esters (Walsh et al., 1998; Zuo
et al., 2012a, 2012b; Ye et al., 2018), which have inhibitory effects on
the growth of other algae (Xu et al., 2017; Zuo et al., 2018b). Mean-
while, these VOCs cause unpleasant, earthy-musty odor in waters, and
geosmin and 2-methyl borneol are considered as the main odor com-
pounds (Huang et al., 2007; Fujise et al., 2010). The water odor

⁎
Corresponding author.
E-mail address: zuozhaojiang@126.com (Z. Zuo).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ecoenv.2018.07.115
Received 8 June 2018; Received in revised form 17 July 2018; Accepted 28 July 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
S. Chen et al.
Fig. 1. Effects of water (A and C) and methanol (B and D) extracts from C. camphora fresh leaves on M. aeruginosa (A and B) and C. reinhardtii (C and D) cell growth. *:
Compared to the control, significant difference at P < 0.05 level. **: Compared to the control, significant difference at P < 0.01 level. Data are means of four
independent experiments ± SE.
dramatically impacts water supplies and even induces drinking water
crisis (Zhang et al., 2010). Algal toxins are another kind of secondary
metabolites, including microcystin, neurotoxins, anatoxin-a, hepato-
toxins, neosaxitoxins, etc. (Codd, 2000; Frangópulos et al., 2004). These
toxins can poison other aquatic organisms, such as algae, zooplankton,
aquatic plants and fishes (Pflugmacher, 2002; Li and Li, 2012; Guzmán-
Guillén et al., 2013). In addition, they may threaten human health
when the waters polluted by the toxins are used for drinking and re-
creation and plants and fishes polluted are used as food (Hoeger et al.,
2007).
For the benefits of aquatic ecological system and human health, lots
of physical, chemical and biological methods have been developed to
control undesired algae. In physical method, the algae are artificially
salved during their blooms, but it cannot be practiced widely, due to the
high consumption of time and fees and hardly elimination. Some che-
micals, such as biquaternary ammonium salt (Liu et al., 2004), TiO2
(Kim and Lee, 2005), polyaluminium chloride (Lürling and van
Oosterhout, 2013), and copper sulfate (Costas and Lopez-Rodas, 2006;
Song and Wang, 2015), kill and/ or control the algae quickly, but they
can poison other aquatic organisms. Algal viruses (Garry et al., 1998)
and bacteria (Zhou et al., 2016) easily mutate, which leads to their
uncontrollability once released into the environment. These potential
defects limit the wide usage of the 3 methods in the wild. Therefore, it is
important to develop a new generation of algaecide that should be ef-
fective, economic and friendly-environmental in controlling undesired
algae.
Plant allelochemicals can effectively inhibit algal growth and be
degraded in the nature, which are considered as the potential source of
new algaecide. Extracts from 5 Chinese herbs such as Rhizoma coptidis,
Semen arecae, Isatis tinctoria, Sophora flavescens, and Houttuynia cordata
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Ecotoxicology and Environmental Safety 163 (2018) 594–603
can inhibit the growth of Alexandrium tamarense, and the extracts from
the front two species showed the strongest inhibitory effects with low
concentration and fast reaction (Zhou et al., 2007). Meanwhile, garlic
solution also inhibited the growth and activities of 3 species of Alex-
andrium (A. tamarense, A. satoanum and A. catenella), as well as
Scrippsiella trochoidea (Zhou et al., 2008). The extracts from barley and
rice straw (Choe and Jung, 2002; Su et al., 2014), Conyza canadensis
and Erigeron annuus (Ni et al., 2011), Artemisia annua (Ni et al., 2011,
2012), Iris wilsonii (Chen et al., 2012), Fructus ligustri Lucidi (Wu et al.,
2014) and Dracontomelon duperreanum (Wang et al., 2016) had re-
markable inhibitory effects on the growth of Microcystis aeruginosa, with
decreases in the membrane integrity and chlorophyll (Chl) fluorescence
in the treatment with rice straw, F. ligustri and D. duperreanum extracts.
In the extracts, phenolics and tannin were considered as the main anti-
algal compounds in I. wilsonii (Chen et al., 2012), and artemisinin was
in A. annua, with inhibitory effects on soluble protein content in M.
aeruginosa (Ni et al., 2012). In previous studies, we found that the ex-
tracts from grape leaves and stems can inhibit the growth of Chlamy-
domonas reinhardtii cells with decreases of photosynthetic pigments and
abilities, and the leaf extracts showed stronger inhibitory effects in
contrast to stem extracts (Zuo et al., 2015). In plant extracts, the anti-
algal compounds play inhibitory roles on algal growth and are the basic
functional compounds for developing new algaecides. Unfortunately,
the identified anti-algal compounds are limited and expensive for
practical application (Jančula and Maršálek, 2011). Therefore, rea-
sonably priced anti-algal compounds have practical significance in
controlling algal blooms and need to be identified.
Cinnamomum camphora (L.) Presl is an evergreen tree species and is
widely planted in the south of China for landscaping and forestation.
This species synthesizes an abundance of terpenoids to repel herbivore
S. Chen et al. Ecotoxicology and Environmental Safety 163 (2018) 594–603

Table 1
The main compounds in C. camphora fresh leaf extracts.
Retention time (min) Compounds Formula Concentration (μmol L−1)

Water extracts Methanol extracts

4.515 3-Methyl− 2-pentanone C6H12O 4.1 ± 0.3 38.5 ± 3.4

4.756 Isobutyl acetate C6H12O2 9.4 ± 0.6 91.6 ± 7.4
5.029 Mesityl oxide C6H10O –a 35.4 ± 1.1
5.237 Butyl acetate C6H12O2 2.6 ± 0.1 23.5 ± 3.5
7.218 α-Phellandrene C10H16 0.9 ± 0.1 5.5 ± 0.9
7.311 α-Terpinene C10H16 1.3 ± 0.4 2.4 ± 0.3
7.426 β-Cymene C10H14 1.4 ± 0.2 2.6 ± 0.4
7.530 D-Limonene C10H16 8.8 ± 0.4 52.4 ± 2.2
7.569 Eucalyptol C10H18O 31.2 ± 1.3 30.1 ± 4.2
7.799 γ-Terpinene C10H16 5.3 ± 0.2 30.3 ± 0.9
7.928 (S)-Linalool oxide C10H18O2 31.9 ± 4.5 25.1 ± 3.1
8.068 trans-Linalool oxide C10H18O2 80.3 ± 10.3 83.9 ± 10.0
8.162 Linalool C10H18O 961.9 ± 75.4 904.7 ± 93.9
8.589 Camphor C10H16O 151.4 ± 9.6 120.9 ± 9.6
8.746 δ-Terpineol C10H18O 16.8 ± 0.1 72.0 ± 4.8
8.854 Hotrienol C10H18O2 –a 1156.9 ± 13.4
8.922 α-Terpineol C10H18O 439.4 ± 22.5 312.7 ± 14.4
9.479 2,6-Dimethyl− 1,7-octadien− 3,6-diol C10H18O2 –a 381.8 ± 37.3
10.017 Ethyl linalool C12H22O 5.9 ± 0.9 33.4 ± 12.9
10.702 Coumaran C15H24 7.8 ± 0.1 38.2 ± 3.6
11.350 Epiglobulol C15H26O 0.8 ± 0.4 32.4 ± 7.8
11.850 E-Nerolidol C15H26O –a 75.2 ± 6.4
12.213 Spathulenol C15H24O 4.7 ± 0.4 129.8 ± 11.8
12.310 Caryophyllene oxide C15H24O 2.8 ± 0.4 22.4 ± 6.4
12.938 (-)-Spathulenol C15H24O 5.1 ± 0.7 26.2 ± 5.9
14.449 Isolongifolol C15H26O –a 80.1 ± 4.3
14.994 Palustrol C15H26O 9.7 ± 0.6 25.3 ± 0.7
15.705 Proximadiol C15H28O2 –a 51.7 ± 6.2
16.021 Isoaromadendrene epoxide C15H24O –a 21.6 ± 2.4
16.390 Platambin C15H26O2 7.5 ± 1.2 32.8 ± 4.5
19.459 Methyl linolenate C19H32O2 –a 82.4 ± 11.7
21.285 Octadecanol acetate C20H40O2 –a 42.1 ± 12.8

Data are means of three independent experiments ± SD.

a
No compound was found.

attack and resist fungal infection (Chen and Dai, 2012; Yang et al.,
2014; Zuo et al., 2017), indicating that the plants may have inhibitory
effects on algae. In our previous study, the inhibitory effects have been
detected using the extracts from the plant fallen leaves (Yakefu et al.,
2018). However, the plants fall less leaves in each year, which cannot
guarantee sufficient supply for algaecide preparation. To develop ef-
fective and reasonably priced algaecides using sufficient fresh leaves
and their main anti-algal compounds, we investigated the inhibitory
effects of the extracts from C. camphora fresh leaves on the growth of
typical species of cyanobacteria (M. aeruginosa) and green algae (C.
reinhardtii), analyzed the components of the extracts, and identified the
main anti-algal compounds in the extracts. Meanwhile, the variation of
photosynthetic pigments and photosystem II (PSII) efficiency in C. re-
inhardtii were investigated to uncover the anti-algal mechanism of the
main anti-algal compounds from photosynthesis.
2. Material and methods
2.1. Cell cultures
M. aeruginosa FACHB-912 was provided by Freshwater Algae
Culture Collection at the Institute of Hydrobiology, China, and grown in
BG-11 medium (Rippka et al., 1979). C. reinhardtii strain CC-125 wild
type mt + [137c] was provided by Dr E.H. Harris (Duke University,
Durham, NC, USA) and grown in TAP medium (Gorman and Levine,
1965). The growing condition was the regime of 16 h light
(30 μmol m−2 s−1) and 8 h dark, with temperature at 25 °C. When the
cell density reached to mid-logarithmic phase, they were used for the
experiments. The cell density was determined by using a 25 × 16 he-
mocytometer, with each value being the means of 6 repeats.
2.2. Preparation of C. camphora fresh leaf extracts
Fresh and health leaves were collected from C. camphora plants in
Zhejiang A & F University (30°15′ N, 119°43′ E). They were heated at
105 °C for 30 min for deactivation of enzymes, dried to constant weight
at 60 °C, and smashed with a pulverizer. The pulverized materials of
10 g were extracted with 100 ml distilled water and 50% methanol,
respectively. The extracted solution was centrifuged at 5000 r min−1
for 10 min to obtain the supernatant, with the concentration of
100 mg ml−1.
2.3. Treatments with C. camphora extracts
With addition of a certain amount of C. camphora extracts, M. aer-
uginosa (1 × 107 cells·ml−1) and C. reinhardtii (7 × 106 cells·ml−1)
cultures were treated with the extracts at 1, 5, 10 and 15 mg ml−1,
respectively. The algal cultures with addition of the same amount of
distilled water and 50% methanol were considered as the control of
water extract treatment and methanol extract treatment, respectively.
After 24 h and 48 h, the lived cell density of the two algae were de-
termined according to neutral red staining method (Wang et al., 2007).
2.4. Treatments with main compounds of C. camphora extracts
Camphor, α-terpineol and linalool solutions of 500 mM were pre-
pared using ethanol, and they were mixed with a ratio of 1: 3: 6 ac-
cording to their approximate ratio in C. camphora extracts. M. aerugi-
nosa (1 × 107 cells·ml−1) and C. reinhardtii (7 × 106 cells·ml−1)
cultures were treated with the single and mixed solution at 0.3, 0.6, 1.2
and 2.4 mM, respectively, according to the treated concentration of

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S. Chen et al.
Fig. 2. Effects of the mixture (A), camphor (B), α-terpineol (C) and linalool (D) on M. aeruginosa cell growth. *: Compared to the control, significant difference at
P < 0.05 level. **: Compared to the control, significant difference at P < 0.01 level. Data are means of four independent experiments ± SE.
total compounds in C. camphora extracts (about 0.27 mM in 15 mg ml−1
water extracts, and about 0.6 mM in 15 mg ml−1 methanol extracts).
The algal cultures that were added into the same amount of ethanol
were considered as the control. After 24 h and 48 h, the lived cell
density of the two algae was determined. The photosynthetic pigment
absorption spectra and Chl fluorescence parameters per 107 cells in C.
reinhardtii were measured after 48 h.
2.5. Measurement of photosynthetic pigment absorption spectra
The photosynthetic pigments in C. reinhardtii cells collected from
3 ml cultures were extracted with 3 ml 80% acetone, and their absor-
bance spectra were scanned from 400 to 700 nm using a TU-1900
UV–Vis spectrophotometer (Beijing Purkinje General Instrument Co.,
Ltd, Beijing, China). The cell number used for extracting photosynthetic
pigments was determined to calculate the absorbance spectra per 107
cells (Zuo et al., 2014).
2.6. Fourth derivative analysis of absorption spectra
Following our previous method (Zuo et al., 2014), the pigment
absorbance scans were performed 4th derivative analysis to obtain the
individual pigment spectrum from the overlapping peaks. The compo-
sition of pigments referred to the list of Rebeiz (2002).
2.7. Measurement of Chl fluorescence
C. reinhardtii cultures of 10 ml were harvested by centrifugation at
6000g for 5 min. The cells were resuspended in the same culture
medium of 10 μl, pipetted on a piece of filter paper to form a spot of
0.5 cm2, and kept in darkness for 15 min (Zuo et al., 2012a). The
measurement of their Chl fluorescence was performed using PAM-2500
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Ecotoxicology and Environmental Safety 163 (2018) 594–603
(Walz, Germany) according to our previous procedure (Zuo et al.,
2014). The maximum quantum yield of PSII photochemistry (Fv/Fm),
coefficient of photochemical quenching (qP) and non-photochemical
dissipation of absorbed light energy (NPQ) were calculated according to
the methods of Maxwell and Johnson (2000). The apparent electron
transport rate (ETR) through PSII was estimated as ETR = (F′m – Fs)/
F′m × PAR × 0.5 × 0.84 (Krall and Edwards, 1992).
2.8. Analysis of C. camphora extracts
Methanol was removed from C. camphora methanol extracts using a
rotary evaporator at 50 °C that was lower than the boiling point of
methanol at 64.5 °C. Twenty ml methanol extracts were distilled to
8 ml, and supplemented to 20 ml with addition of distilled water to
keep the extract concentration at 100 mg ml−1. Six ml removed me-
thanol extracts and water extracts were individually added in 1 ml ethyl
acetate for extracting the secondary metabolites. The extracted solution
of ethyl acetate was analyzed by gas chromatography-mass spectrum
(GC-MS) following our previous method (Zuo et al., 2015). The GC/MS
data were analyzed using NIST/ EPA/ NIH Mass Spectral Library (NIST
14) (National Institute of Standards and Technology, Gaithersburg,
USA) to obtain qualitative and quantitative results. Eucalyptol, D-li-
monene, terpinene, camphor, linalool and E-nerolidol were used as the
standard samples to calculate the concentration of the corresponding
compounds in the extracts. Sesquiterpenoid (C15) concentration was
calculated referring to E-nerolidol, while monoterpenoid (C10) and
other compound concentration referring to camphor.
2.9. Calculations and statistical analyses
For the algal growth and Chl fluorescence parameters, the response
index (RI) was calculated following the method proposed by
S. Chen et al.
Fig. 3. Effects of the mixture (A), camphor (B), α-terpineol (C) and linalool (D) on C. reinhardtii cell growth. *: Compared to the control, significant difference at
P < 0.05 level. **: Compared to the control, significant difference at P < 0.01 level. Data are means of four independent experiments ± SE.
Williamson and Richardson (1988).
C
⎧ 1− T When T ≥ C
RI =
⎨ T −1When T < C
⎩C
Where C is control response and T is treatment response. The positive
and negative values of RI indicate stimulation and inhibition by the
treatments, respectively, in contrast to the controls.
Statistical analyses of One-way ANOVA and drawing figures were
performed using Origin 8.0 (Origin Lab, USA).
3. Results
3.1. Effects of C. camphora fresh leaf extracts on algal growth
When M. aeruginosa cells were treated with C. camphora fresh leaf
water extracts at 1, 5, 10 and 15 mg ml−1, the cell growth was inhibited
markedly, with the RI of −0.05, −0.23 (P < 0.01), −0.49 (P < 0.01)
and −0.67 (P < 0.01), respectively, after 24 h. The inhibition en-
hanced after 48 h, with the RI of −0.12 (P < 0.01), −0.28
(P < 0.01), −0.55 (P < 0.01) and −0.71 (P < 0.01), respectively
(Fig. 1A). C. camphora fresh leaf methanol extracts also showed in-
hibitory effects on M. aeruginosa cells, and the inhibitory effect was
stronger than that of water extracts at the same concentration. The
whole of M. aeruginosa cells were killed by methanol extracts at
15 mg ml−1 after 48 h treatment (Fig. 1B).
Similar to M. aeruginosa, C. reinhardtii cell growth was also sig-
nificantly inhibited by C. camphora water and methanol extracts, and
methanol extracts showed stronger inhibitory effects compared to water
extracts at the same concentration. Meanwhile, the methanol extracts at
15 mg ml−1 killed the whole of C. reinhardtii cells after 48 h (Fig. 1C,
D).
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3.2. The main compounds in C. camphora fresh leaf extracts
Twenty-three compounds were detected in the water extracts
(100 mg ml−1) from C. camphora fresh leaves, mainly including terpenoids,
esters, alcohols, and ketones. Among these components, linalool, camphor
and α-terpineol were the main compounds, with the concentration of 961.9,
151.4 and 439.4 μmol L−1, respectively. Compared to the water extracts, 9
new compounds were detected in the methanol extracts, including mesityl
oxide (35.4 μmol L−1), hotrienol (1156.9 μmol L−1), 2,6-dimethyl-1,7-octa-
dien-3,6-diol (381.8 μmol L−1), E-nerolidol (75.2 μmol L−1), isolongifolol
(80.1 μmol L−1), proximadiol (51.7 μmol L−1), isoaromadendrene epoxide
(21.6 μmol L−1), methyl linolenate (82.4 μmol L−1), and octadecanol
acetate (42.1 μmol L−1) (Table 1).
In the water extracts, the concentration of 3-methyl-2-pentanone
was 4.1 μmol L−1, while its concentration in the methanol extracts in-
creased by 8.4 folds. Similarly, for most of other compounds, their
concentration in methanol extracts was higher than that in water ex-
tracts. Meanwhile, the concentration of total compounds in methanol
extracts was 2.3 folds of that in water extracts (Table 1).
3.3. Effects of camphor, α-terpineol, linalool and their mixture on algal
growth
In the treatments with the mixture of camphor, α-terpineol and li-
nalool at 0.3, 0.6, 1.2 and 2.4 mM, M. aeruginosa cell growth was sig-
nificantly inhibited, and the RI was −0.15 (P < 0.05), −0.25
(P < 0.01), −0.30 (P < 0.01) and −0.76 (P < 0.01), respectively,
after 24 h, and −0.22 (P < 0.01), −0.43 (P < 0.01), −0.58
(P < 0.01) and −0.96 (P < 0.01), respectively, after 48 h (Fig. 2A).
Similar inhibitory effects were also detected in the treatments with the
individual compound, camphor, α-terpineol and linalool (Fig. 2B, C, D).
Camphor, α-terpineol, linalool and their mixture also inhibited the
S. Chen et al.
Fig. 4. Effects of the mixture (A), camphor (B), α-terpineol (C) and linalool (D) on the absorbance spectra of photosynthetic pigments in C. reinhardtii. The cells were
killed by the mixture, camphor, α-terpineol and linalool at 2.4 mM, so the absorbance spectra were not determined. Data are means of four independent experiments.
growth of C. reinhardtii cells, and the inhibition enhanced with in-
creasing the compound concentration and prolonging the treatment
time. The whole of the cells were killed by the mixture, α-terpineol and
linalool at 2.4 mM after 24 h, and camphor at 2.4 mM after 48 h (Fig. 3).
3.4. Changes in absorbance spectra of photosynthetic pigments in C.
reinhardtii
When C. reinhardtii cells were treated with the mixture, camphor, α-
terpineol and linalool for 48 h, the absorbance peak of photosynthetic
pigments at wavelength of 413, 433, 457 and 663 nm gradually de-
clined with increasing the compound concentration, indicating that the
photosynthetic pigments had been degraded (Fig. 4).
3.5. Variations in 4th derivative spectra in C. reinhardtii
In the treatment with the mixture of camphor, α-terpineol and li-
nalool, the degradation of divinyl-pheophytin a (419 nm), divinyl-
chlorophyll a (438 nm), divinyl-chlorophyll b (468 nm), xanthophyll
(475 nm), monovinyl-chlorophyll b (645 nm) and monovinyl-chlor-
ophyll a (663 nm) was found, and the degradation enhanced gradually
with increasing the mixture concentration (Fig. 5A). Similar degrada-
tion was also found in the treatments with camphor, α-terpineol and
linalool, and xanthophyll was nearly completely degraded in the
treatments with camphor and linalool at 1.2 mM (Fig. 5B, C, D).
3.6. Changes of PSII efficiency in C. reinhardtii
When C. reinhardtii cells were treated with the mixture, camphor, α-
terpineol and linalool, the Fv/Fm gradually declined with increasing
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Ecotoxicology and Environmental Safety 163 (2018) 594–603
the compound concentration, and reached the lowest at 1.2 mM, with
the RI of −0.38 (P < 0.01), −0.61 (P < 0.01), −0.49 (P < 0.01)
and −0.43 (P < 0.01), respectively (Fig. 6A). Similar decreases were
also detected in the qP and ETR (Fig. 6B, D). However, NPQ increased
in the treatments with the mixture, camphor, α-terpineol and linalool,
and the maximum increase was detected at 1.2 mM, with the RI of 0.50
(P < 0.01), 0.55 (P < 0.01), 0.64 (P < 0.01) and 0.49 (P < 0.01),
respectively (Fig. 6C).
4. Discussion
Plant allelochemicals are considered as the effective, economic and
friendly-environmental algaecides, due to their effective inhibition on
algae, convenient preparation and easy degradation in the nature.
Previous studies have reported that some plant extracts had algicidal
properties, such as, extracts from R. coptidis, S. arecae, I. tinctoria, S.
flavescens, H. cordata and garlic on Alexandrium (Zhou et al., 2007,
2008), extracts from grape leaves and stems on C. reinhardtii (Zuo et al.,
2015), extracts from rice straw, C. canadensis, E. annuus, A. annua, I.
wilsonii, F. ligustri, D. duperreanum, Ailanthus altissima and Sagittaria
trifolia on M. aeruginosa (Ni et al., 2011; Chen et al., 2012; Su et al.,
2014; Wu et al., 2014; Meng et al., 2015; Li et al., 2016; Wang et al.,
2016), and extracts from Arundo donax on Prymnesium parvum (Patiño
et al., 2018). In this study, the extracts from C. camphora fresh leaves
showed significant inhibitory effects on M. aeruginosa and C. reinhardtii
(Fig. 1), which were similar with our previous studies using C. camphora
fallen leaves (Yakefu et al., 2018). Compared to the fallen leaves, water
extracts from C. camphora fresh leaves showed stronger inhibitory ef-
fects on M. aeruginosa (Yakefu et al., 2018). These results indicated that
both fresh and fallen leaves of the plants have potential values for
S. Chen et al.
Fig. 5. Fourth derivative analysis of absorption spectra in C. reinhardtii cells in the treatment with the mixture (A), camphor (B), α-terpineol (C) and linalool (D). 419:
Divinyl-pheophytin a, 438: Divinyl-chlorophyll a, 468: Divinyl-chlorophyll b, 475: Xanthophyll, 623: Monovinyl-protochlorophyllide a, 645: Monovinyl-chlorophyll
b, 663: Monovinyl-chlorophyll a. Data are means of four independent experiments.
developing as algaecides, and fresh leaves are more suitable for con-
trolling M. aeruginosa using water as the extracting agent.
Terpenoids are considered as the first largest class of plant sec-
ondary metabolic compounds, and many studies revealed their anti-
algal activities. In the extracts from A. annua, C. canadensis and E. an-
nuus, terpenoids may be the main active ingredients in inhibiting M.
aeruginosa growth (Ni et al., 2011). The volatile oil from A. ordosica can
inhibit the growth of desert soil microalgae Palmellococcus miniatus by
inducing reactive oxygen species (ROS) production and inhibiting Fv/
Fm, and terpenoids were considered as the main anti-algal compounds
(Yang et al., 2012). In grape extracts, terpenoids and phenolic com-
pounds were the main anti-algal compounds in inhibiting C. reinhardtii
cell growth and photosynthesis (Zuo et al., 2015). Artemisinin identi-
fied from A. annua is a terpenoid, which can inhibit M. aeruginosa cell
growth with decreasing soluble protein content and increasing ROS
levels (Ni et al., 2012). Meanwhile, abundant VOCs including lots of
terpenoids released from M. flos-aquae and M. aeruginosa cells under
non-N or non-P condition can inhibit Chlorella vulgaris and C. reinhardtii
cell growth, and two terpenoids eucalyptol and limonene had been
identified as the main functional compounds in the VOCs (Zhao et al.,
2016; Xu et al., 2017; Zuo et al., 2018b).
In Cinnamomum genus, an abundance of terpenoids were detected to
perform antimicrobial activities (Ragasa et al., 2013; Monteiro et al.,
2017; Taha and Eldahshan, 2017; Wei et al., 2017; Yuan et al., 2017).
For C. camphora, the main compounds in the fresh and fallen leaf ex-
tracts were terpenoids, especially oxygenated monoterpenes (Yang
et al., 2014; Yakefu et al., 2018). Moreover, monoterpenoids were the
main VOCs released from the plants, and the emission amount of oxy-
genated monoterpenes was higher than that of monoterpenes (Zuo
et al., 2017). In the present study, amounts of terpenoids were detected
from C. camphora fresh leaf extracts, mainly including oxygenated
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Ecotoxicology and Environmental Safety 163 (2018) 594–603
monoterpenes and sesquiterpenes (Table 1), which was consistent with
the results in previous studies. These terpenoids should play inhibitory
roles on M. aeruginosa and C. reinhardtii cell growth. Among them,
eucalyptol and limonene had been proved to have anti-algal activities
(Zhao et al., 2016; Xu et al., 2017; Zuo et al., 2018b), but there was
lower content in C. camphora extracts (Table 1). Previous studies have
reported that camphor, α-terpineol and linalool can inhibit the growth
of several microorganisms, such as Candida albicans (Zore et al., 2011;
Pinto et al., 2014), Geotrichum citriaurantii (Zhou et al., 2014), Schis-
tosoma japonicum (Yang et al., 2014), Microsporum canis and M. gypseum
(Silva et al., 2017), Campylobacter spp. (Duarte et al., 2016), Escherichia
coli and Staphylococcus aureus (Cutillas et al., 2017), by affecting
membrane integrity, cell cycle and hyphae formation (Zore et al., 2011;
Pinto et al., 2014). In the present study, camphor, α-terpineol and li-
nalool were 3 main terpenoids in C. camphora fresh leaf extracts, which
can inhibit M. aeruginosa and C. reinhardtii cell growth, induce photo-
synthetic pigment degradation, and decline PSII efficiency (Figs. 2–6),
indicating that they should be 3 main anti-algal compounds.
Compared to the same molar concentration of total compounds in C.
camphora water (about 0.27 mM in 15 mg ml−1 extracts) and methanol
(about 0.6 mM in 15 mg ml−1 extracts) extracts, the inhibitory effects of
camphor, α-terpineol, linalool and their mixture on M. aeruginosa and
C. reinhardtii cell growth were lower (Figs. 1–3), demonstrating that
other compounds also contributed to the algal inhibition and the
combined effect of more compounds was stronger than that of the three
ones (camphor, α-terpineol and linalool) and the single effect of each
one.
In contrast to the water extracts from C. camphora fresh leaves,
methanol extracts showed stronger inhibition on M. aeruginosa and C.
reinhardtii cell growth (Fig. 1), which was consistent with the results of
the extracts from C. camphora fallen leaves (Yakefu et al., 2018),
S. Chen et al.
Fig. 6. PSII efficiency in C. reinhardtii cells treated with the mixture, camphor, α-terpineol and linalool. A: Fv/Fm, B: qP, C: NPQ, D: ETR. *: Compared to the control,
significant difference at P < 0.05 level. **: Compared to the control, significant difference at P < 0.01 level. Data are means of four independent experiments ± SE.
fenugreek seeds (Belguith-Hadriche et al., 2013) and grape leaves and
stems (Zuo et al., 2015). The concentration of camphor, α-terpineol and
linalool was similar in both methanol extracts and water extracts from
C. camphora fresh leaves, while there were more compounds with
higher concentration in the methanol extracts (Table 1), which may
contribute to the stronger inhibition of methanol extracts (Fig. 1). In-
terestingly, when M. aeruginosa and C. reinhardtii cells were treated with
C. camphora fresh leaf water extracts at 33.3 mg ml−1 (the molar con-
centration of total compounds equaled to that in methanol extracts at
15 mg ml−1, about 0.6 mM), about 93% M. aeruginosa cells and 96% C.
reinhardtii cells were killed after 48 h (data not shown), which were
slightly lower than algicidal effects of methanol extracts at 15 mg ml−1
(the two algal cells were killed completely). These results suggest that
methanol should be better for preparing the plant original algaecide.
Chl are essential photosynthetic pigments and serve as capturing
light and transducing it to biochemical energy. In addition to capturing
light, carotenoids function as preventing photo-oxidative damage and
protecting Chl and photosynthetic apparatus (Pérez-Pérez et al., 2012).
When C. reinhardtii cells were treated with grape leaf and stem extracts,
their Chl content declined significantly (Zuo et al., 2015). In the
treatment with D. duperreanum defoliation extracts, a remarkable de-
crease was found in M. aeruginosa Chl content (Wang et al., 2016).
When C. vulgaris cells were exposed to eucalyptol and limonene, re-
markable degradation was detected in carotenoids and Chl, including
xanthophyll, divinyl-pheophytin a, divinyl-Chl a, divinyl-Chl b, mono-
vinyl-protochlorophyllide a, monovinyl-Chl b, and monovinyl-Chl a,
which enhanced with increasing the compound concentration (Zhao
et al., 2016). Similar degradation was also detected in C. reinhardtii cells
in exposure to camphor, α-terpineol, linalool and their mixture (Fig. 5),
which led to the decline of light absorption by the alga. Meanwhile, the
degradation of xanthophyll may reduce the protection to Chl and
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Ecotoxicology and Environmental Safety 163 (2018) 594–603
photosynthetic apparatus and aggravate their oxidative damage.
Photosynthesis is the most fundamental biosynthetic process sup-
porting algal multiplication and growth. Chl fluorescence can rapidly
investigate PSII photochemical efficiency and status of photosynthetic
apparatus without invasiveness (Maxwell and Johnson, 2000). When C.
reinhardtii cells exposed to camphor, α-terpineol, linalool and their
mixture, their photosynthetic abilities declined significantly, due to the
degradation of photosynthetic pigments (Fig. 5), reduction of PSII
quantum yield (Fv/Fm), photochemical quenching (qP) and electron
transport (ETR), as well as the increase of heat dissipation of absorbed
light energy (NPQ) through xanthophyll cycle (Ivanov et al., 2008).
These results were consistent with previous studies about plant extracts
and terpenoids (eucalyptol and limonene) on algal photosynthetic
abilities (Yang et al., 2012; Zuo et al., 2015, 2018b; Zhao et al., 2016;
Xu et al., 2017). The reduction of photosynthetic abilities can decrease
the accumulation of carbohydrates, which may slow down C. reinhardtii
cell growth.
C. camphora is an evergreen tree species and is widely planted in the
south of China, which guarantees the sufficiency of leaf materials for
extract preparation. The 3 anti-algal compounds (camphor, α-terpineol
and linalool) widely exist in lots of plants and are easily extracted and
artificially synthesized. Meanwhile, the applied solution can be con-
veniently prepared with the leaves and main anti-algal compounds and
perform effective inhibition on algae. These advantages suggest that C.
camphora fresh leaf extracts and their main components have potential
values for developing as reasonably priced algaecides.
Acknowledgements
This research was supported by the Natural Science Foundation of
Zhejiang Province (No. LY17C160004), the National Students’ Innovation
S. Chen et al.
and Entrepreneurship Training Program (No. 102–2013200053), the
Student Research Training Program in Zhejiang A & F University (No.
2013200038), the National Natural Science Foundation of China (No.
31300364), and the Personnel Startup Project of the Scientific Research
and Development Foundation of Zhejiang A & F University (No.
2013FR069).
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