Agricultural Biotechnology 2014, 3 ( 4 ) ; 45 - 48,52

Animal Science

Effects of I-Naphthol on Physiological Parameters of

Chlorella vulgaris
Liping QIU1, Yonghong LUO2, Jianhong QU1, Gengdong HU1, Jiazhang CHEN1'2*
I . Key Open Laboratory of Ecological Environment and Resources of Inland Fisheries, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sci
ences, Wuxi 214081, China; 2. Wuxi Fishery College, Nanjing Agricultural University, Wuxi 214081, China

Abstract [Objective] This study aimed to investigate the effects of 1-naphthol on primary productivity in aquatic ecosystems. [Method] The effects of 1-naphthol on biomass, chlorophyll a content, malondialdehyde (MDA) content and soluble protein content of Chlorella vulgaris were investigated. [Result] I-Naphthol
could significantly inhibit the growth of Chlorella vulgaris. On the 7 th d , the biomass of Chlorella vulgaris exposed to 1 .0 , 3 .0 , 6 .0 , 12.0 and 18.0 mg/L 1-naphthol was 60.26% , 48.15% , 41. 32% , 28.59% and 27.10% of that in control respectively, exhibiting a significant dose-response relationship. Chlorella vulgaris
was exposed to different concentrations of 1-naphthol ( 0 , 1 , 5 and 10 mg/L) for 7 d; with the increase of 1-naphthol concentration, chlorophyll a content of Chlorella vulgaris was reduced, MDA content increased gradually, soluble protein content increased first and then declined. [Conclusion] According to the experimen
tal results, 1-naphthol can significantly inhibit the growth of Chlorella vulgaris, thus reducing primary productivity in aquatic ecosystems.
Key words I -Naphthol; Chlorella vulgaris; Biomass; Physiological parameter

As a type of PAH ( polycyclic aromatic hydrocarbon) sub

stance, 1-naphthol is mainly used in organic synthesis and dye in
dustry as an important intermediate for the production and synthe
sis of pharmaceuticals, pesticides, dyes and cosmetics. I-Naphthol has low biodegradability and can influence physiological activ
ities of organisms111, with carcinogenic and teratogenic effects.
Due to the excessive use of 1-naphthol, it can enter the water
through various channels, thus posing a potential threat to the
aquatic environment121. Algae are the primary producers in water,
which play a very important role in aquatic ecosystems. The spe
cies diversity and primary production level of algae directly affect
the structure and function of aquatic ecosystems1341. Therefore,
algae are ideal indicator organisms of chemical substances151. In
vestigating the toxic effects of pollutants on algae is of scientific
significance to ecological risk assessment of pollutants.
Currently, most of studies on the toxic effects of 1-naphthol
are concentrated in animals1681 , while few studies have been car
ried out on the toxic effects of 1-naphthol on algae. In the early
1980s, the toxic effects of 1-naphthol on algae were analyzed for
the first time. Zeng et al. 191 investigated the toxic effects of
1-naphthol on Scenedesmus obliqnus Kutzing. using algal biomass
as an indicator and found that 1-naphthol could significantly inhib
it the growth of Scenedesmus obliqnus Kutzing. subsequently, Shen
et al. 1101 investigated the semi-effect concentration ( ECx ) of
1-naphthol to Chlorella vulgaris Beij. and effects of 1-naphthol on
chlorophyll a content; results showed that ECx of 1-naphthol to
Chlorella vulgaris Beij. was 11. 87 m g/L, 1-naphthol could pro
mote the biomass under low concentrations and inhibit the biomass
under high concentrations. Recently, Peng et al. 1111 investigated
the effects of 1-naphthol on physiological and biochemical indica
tors of two Chlorella species and found that 1-naphthol could in
Received: March 4 , 2014
Accepted; August 3 , 2014
Supported by Special Fund for the Construction of Modem Agricultural Industry
Technology System ( CARS-49 ) .
Liping QIU (1977 - ) , female, P. R. China, master, assistant professor,
devoted to research about fishery ecological environment protection.
* Corresponding author. E-mail; chenjz@ ffrc. cn

duce significant changes of reduced glutathione ( GSH) content,

oxidized glutathione ( GSSG) content and glutathione reductase
(G R ) activity of Chlorella, suggesting that lipid peroxidation of
algal cells increases under 1-naphthol stress.
Chlorella vulgaris belongs to single-cell algae in Chlorophyta,
which is a natural food of aquatic animals. A large number of
studies have shown that inoculating Chlorella vulgaris in aquacul
ture water can regulate and optimize the structure of plankton com
munities, reduce nitrogen and phosphorus concentrations in water
and increase dissolved oxygen content, thereby improving aquatic
ecological environment112"131. Therefore, investigating the toxic
effects of 1-naphthol on Chlorella vulgaris is of important signifi
cance. In this study, physiological and biochemical responses of
Chlorella vulgaris to 1-naphthol stress were analyzed, which pro
vided scientific basis for rational evaluation, management and con
trol of pollution caused by 1-naphthol.

Materials and Methods

Experimental materials
Experimental algae species: Chlorella vulgaris was purchased
from Institute of Hydrobiology, Chinese Academy of Sciences,
which was incubated with BG-Il medium. After laboratory domes
tication incubation, healthy algae at the logarithmic growth stage
were collected for experiment.

Reagents and instruments

Reagents
I -Naphthol, AR, purity 5

9 9 .0 % , Sinopharm
Chemical Reagent Co. , Ltd. ; acetone, AR; trichloroacetic acid,
AR; thiobarbituric acid, AR; Coomassie brilliant blue G-250.
Instruments Constant shaking incubator ( Taicang Huamei Bio
chemical Instrument Factory); UV spectrophotometer UV-1200
( Shanghai Mapada Instruments Co. , Ltd. ) ; blood cell counting
plate, automatic electric-heating pressure steam sterilizer TOMY
AUTO CLAVE SS-523; automatic temperature-controlled high
speed centrifuge Z-16K (Sigma Corporation).

Methods
Domestication and incubation of algae species Firstly, 200 ml
of Chlorella vulgaris was incubated with BG-Il medium in a 500
ml flask at (25 2) cC under a light intensity of 2 800 - 3 000 Ix

46

Agricultural Biotechnology

for 12 h: 12 h (light: dark) and shaken three times everyday. The

transfer, domestication, incubation and determination were con
ducted under sterile conditions. Containers were sterilized with dry
heat sterilization method. Medium was sterilized at 120 tC for 30
min,
Concentrationdesign Six concentrations ( 0 , 1 .0 , 3 .0 , 6 .0 ,
12. 0 and 18.0 mg/L) were set to investigate the toxic effects of 1naphthol on ChloreUa vulgaris, with three replications. The growth
inhibitory rate of 1-naphthol on Chlorella vulgaris was 5% -7 5 % ,
and three concentrations were set ( 1 , 5 and 10 mg/L) , to investi
gate the effects of 1-naphthol stress on chlorophyll a content, malondialdehyde content and soluble protein content of Chlorella
vulgaris.
Determination of biomass Algal cells were counted under a mi
croscope using blood cell counting plates. The optical density of
algae liquid was measured at 680 nm, to establish a linear rela
tionship between the concentration and optical density of algal
cells. The biomass of algal cells was represented by optical densi
ty. The growth curve of Chlorella vulgaris was drawn to determine
the logarithmic growth stage of Chlorella vulgaris.
Determination of chlorophyll a content In accordance with
ITater and Wastewater Monitoring Analysis Method ( fourth edi
tion) , chlorophyll a content was determined, mg/m .
Determination of malondialdehyde content
In accordance
with Principle and Technology o f Plant Physiology and Biochemis
try Experiment^ , malondialdehyde content was determined with
thiobarbituric acid method, nmol/107 cells.
Determination of soluble protein content In accordance with
Principle and Technology o f Plant Physiology and Biochemistry Ex
periment^14] , soluble protein content was determined with Coomassie brilliant blue G-250 staining method, mg/g.

2014

5
y = 65. 72b: - 0. 041 4
4

S2 = 0.999 4

I
0

0.02

0.04

0.06

Absorbance (/=680)

Fig. I

Rdationship between the number of algal cells and absor

bance

stage, and no significant increase was observed in the number of

algal cells. On the 3"1 - 8 d , Chlorella vulgaris was at the loga
rithmic growth stage with the maximum optical density; the absor
bance increased from 0. 044 to 2. 305 by 52. 39 times, and the
number of algal cells was 29.72 x IO6 cells / ml. On the 9th - 16th
d , Chlorella vulgaris was at the stable stage. Since the 17th d ,
the biomass of Chlorella vulgaris was reduced significantly and
entered the decline stage. The maximum biomass of Chlorella
vulgaris in this study was an order of magnitude higher than the
growth curve drawn by Xu et al. [15] , which might be due to the
different increase rates of Chlorella vulgaris biomass under differ
ent incubation conditions and different initial inoculation concen
tration.
2. 50

2.00

Data analysis
Experimental data were analyzed using SPSS 13.0 statistical
software with two significance levels ( a = 0. 0 5 , a = 0. 01 ).
* indicated significant differences at P < 0 . 05 level compared
with control group; * * indicated extremely significant differ
ences at P < 0 .0 1 level compared with control group.

I. 00
0.50

Results and Analysis

Relationship between the number and optical density of Chlorella vulgaris cells
By using blood cell counting plates and spectrophotometry,
Chlorella vulgaris at the logarithmic growth stage were determined.
The number of algal cells ( y ) and absorbance (A ) at 680 nm
were obtained to draw a fitting curve ( Fig. I ). Results indicated
that the number of algal cells and absorbance presented a good lin
ear relationship, and the fitting equation was:
y = 12. %91Am - 0.012 I ( P 2 =0.999 8)
To be specific, indicated the concentration of algal cells ( IO6
cells / m l ). According to verification results, the concentration
and optical density of algal cells were extremely significantly relat
ed ( P < 0 .0 1 ). Therefore, the absorbance was measured as an
indicator to determine the biomass of Chlorella.

Determination of growth curve of Chlorella vulgaris

The initial inoculation density of Chlorella vulgaris was ODm
= 0.044. The optical density of Chlorella vulgaris was determined
everyday, to draw the growth curve. As shown in Fig. 2 , within
the first two days, the biomass of Chlorella vulgaris was at the lag

0.04

0.06

0.08

Incubation d u ratio n Il d

Fig. 2

Growth curve of Chlorella vulgaris

Effects of 1-naphthol on biomass of Chlorella vulgaris

At the logarithmic growth stage of Chlorella vulgaris, the
effects of 1-naphthol on Chlorella vulgaris biomass were analyzed.
The results indicated that 1-naphthol had great toxic effects on
Chlorella vulgaris. As shown in Fig. 3 , within the first two days
after 1-naphthol addition, except the control, Chlorella vulgaris
biomass in various groups exhibited no significant increase, indi
cating that 1-naphthol extended the lad stage of Chlorella vulgaris.
On the 3rd d , the biomass of Chlorella vulgaris exposed to I mg/L
1-naphthol increased rapidly; the biomass of Chlorella vulgaris ex
posed to 3 and 6 mg/L 1-naphthol increased significantly on the
5th d; however, the biomass of Chlorella vulgaris exposed to 12
and 18 mg/L 1-naphthol exhibited no significant increase during
the entire growth period, and Chlorella vulgaris began to die; on
the 7th d , the biomass of Chlorella vulgaris exposed to different
concentrations of 1-naphthol was 60. 26% , 48. 15% , 41. 32% ,
28.59% and 27.10% of that in control, respectively.

Liping QIU et al. Effects of I-Naphthol on Physiological Parameters of Chlorella vulgaris

47

0.5
Control
6 mg/L

OO

.
<0

I mg/L
12 mg/L

o J

Incubation d uration Il d

Fig. 3

Effects of 1-naphthol on the biomass of Chlorella vulgaris

Effects of 1-naphthol on chlorophyll a content of Chlorella

vulgaris
According to the effects of 1-naphthol on chlorophyll a content
Table I

of Chlorella vulgaris, with the increase of 1-naphthol concentration, chlorophyll a content of Chlorella vulgaris was reduced gradually, exhibiting a significant dose - response relationship.

Effects of different concentrations of 1-naphthol on chlorophyll a content of Chlorella vulgaris (7 days later)

I-Naphthol concentration //m g /L

Chi. a content //m g /m 3

10

2 086.726 + 89.940

I 713.192 195.204

I 183.699 60.245**

446.373 35.511***

.M

1.5

2. O

xn

Hl l l i
O

l-N afhthol concentration Il mg/L

Fig. 4

Effects of different concentrations of 1-naphthol on MDA

content of Chlorella vulgaris (7 days later)

Effects of different concentrations of 1-naphthol on MDA con

tent of Chlorella vulgaris
Effects of different concentrations of 1-naphthol on MDA con
tent of Chlorella vulgaris were shown in Fig. 4. MDA content of
Chlorella vulgaris exposed to 1 , 5 and 10 mg/L 1-naphthol was
0. 668 , 0.820 and I. 041 nmol/107 cells, which was 111. 89% ,
137.35% and 173.37% of that in control, respectively. MDA
content of Chlorella vulgaris was improved significantly, which in
dicated that I -naphthol increased the level of lipid peroxidation in
algal cells, suggesting that there was a significant dose-response
relationship between MDA content and 1-naphthol concentration.

Effects of different concentrations of 1-naphthol on soluble

protein content of Chlorella vulgaris
Effects of 1-naphthol on soluble protein content of Chlorella
vulgaris were shown in Fig. 5. Soluble protein content of Chlorella
vulgaris exposed to I mg/L 1-naphthol was improved, exhibiting
significant differences from that in control ( P < 0 .0 5 ) . With the
increase of 1-naphthol concentration, soluble protein content of
Chlorella vulgaris was reduced gradually. To be specific, soluble
protein content of Chlorella vulgaris exposed to 10 mg/L 1-naph
thol was lower than that in control ( 12.19% ) , exhibiting signifi
cant differences ( P < 0.05 ).

I-Naihtho I concent ra t i a t

Fig. 5

10

I! mg/L

Effects of different concentrations of 1-naphthol on solu

ble protein content of Chlorella vulgaris (7 days later)

Discussion
According to the effects of 1-naphthol on the biomass of Chlo
rella vulgaris, the growth lag stage of Chlorella vulgaris exposed to
different concentrations of 1-naphthol was extended; since the 3
d , the biomass of Chlorella vulgaris exposed to I mg/L 1-naphthol
increased, but that of Chlorella vulgaris exposed to 12 and 18
mg/L 1-naphthol exhibited no significant increase during the en
tire growth stage, which indicates that 1-naphthol can inhibit the
growth of Chlorella vulgaris, and the inhibitory effects are posi
tively correlated with the concentration of 1-naphthol. Previous
studies have shown that low concentrations of 1-naphthol can pro
mote the increase of the biomass of Chlorella vulgaris ^161 , which is
in consistent with the conclusion of this study, and that may be
due to the different initial inoculation densities of Chlorella vulgar
is or different actuation durations of drugs on algae.
Studies have shown that the toxic effects of 1-naphthol are
mainly induced by photoinduction, which can produce active oxy
gen free radicals or be decomposed into active intermediate metab
olites by light-catalyzed reaction, thus destructing the structure
and function of cell membrane and other intracellular macromole
cules1171 . Photosynthetic organs are one of the major sources of re
active oxygen species in plant cells. Photosynthetic pigments and

48

Agricultural Biotechnology

the thylakoid membrane pigments combined with have unsaturated

polyene structures and are vulnerable to the attack of reactive
oxygen species1'81. Under exposure to different concentrations of
1-naphthol, the content of photosynthetic pigments in Chlorella
vulgaris also varies significantly. To be specific, with the increase
of 1-naphthol concentration, the content of photosynthetic
pigments in Chlorella vulgaris is reduced greatly, and the content
of photosynthetic pigments in Chlorella vulgaris exposed to 5 and
10 mg/L 1-naphthol varies significantly compared with control
( P < 0. 0 1 ) , which may be due to that the accumulation of intra
cellular reactive oxygen species under stress of moderate and high
concentrations of 1-naphthol destructs chloroplast structure and
inhibits chloroplast synthesis '91. Alberte et al. 1201 believed that
the reduction of chloroplast content under stress may be due to that
the synthesis of light-harvesting chlorophyll a/b-protein complexes
in chloroplast lamellae is inhibited.
Malondialdehyde (MDA) is an important indicator of cellular
oxidative damage and its content reflects the degree of damage1211.
In this study, MDA content in Chlorella vulgaris increases with the
increase of 1-naphthol concentration, suggesting that the active
oxygen scavenging system in algal cells is inactivated under
1-naphthol stress and can not effectively establish a new balance
with active oxygen, leading to membrane lipid peroxidation and
rapid increase of MDA content in algal cells, thusing seriously
damaging Chlorella vulgaris cells. Higher concentrations of
1-naphthol cause greater oxidative damage to Chlorella vulgaris,
which is basically consistent with the conclusion of Peng et a l1'61.
Protein is the material basis of life. Wang et al. 1221 believed
that changes of plant cell proteins are mainly reflected in the chan
ges of soluble proteins and enzymes. Most soluble proteins in plant
cells are metabolism-regulating enzymes with specific function,
and some soluble proteins may play the role in dehydration protec
tion and provide a combination matrix for intracellular bound water
to increase the content of bound water in plant tissues, thus pre
venting the cell structure from greater damage in dehydration1231.
The present study show that I mg/L 1-naphthol promotes the in
crease of soluble protein content in Chlorella vulgaris, which may
be due to that normal protein synthesis in plants under stress is
generally inhibited, but the stress tends to induce the production
of some new proteins or significantly increase the content of origi
nal proteins. Plants can be direcdy involved in the adaptation
process to stress by improving the synthesis of soluble proteins,
which is of positive significance to the adaptation to adverse envi
ronmental conditions1241. With the increase of 1-naphthol concen
tration, the content of soluble proteins is reduced gradually, indi
cating that high concentrations of 1-naphthol has undermined the
adaptation process of Chlorella vulgaris to stress, caused metabolic
disorders and led to irreversible damage to algal cells, thereby in
hibiting the formation and accumulation of soluble proteins in
Chlorella vulgaris.

Conclusion
l-Naphthol significantly inhibited the growth of Chlorella vul
garis. On the 7 th d , the biomass of Chlorella vulgaris exposed to
1. 0, 3 . 0 , 6. 0, 12. 0 and 18. 0 mg/L 1-naphthol was 60. 26% ,
48.15% , 41.32% , 28. 59% and 27. 10% of that in control re
spectively, exhibiting a significant dose - response relationship.
Determination of various biochemical indicators showed that

2014
MDA
Chlorella vulgaris. To be

1-naphthol significantly in flu en ced chlorophyll a c on ten t,

content and soluble protein content o f

sp e c ific , w ith the increase o f 1-naphthol concen tration, chloro

phyll a content of

Chlorella vulgaris was reduced and MDA content

increased gradually, exhibiting a significant dose - response rela

tionship. H ow ever, soluble protein content o f

Chlorella vulgaris

increased first and then d eclin ed w ith the increase o f 1-naphthol

concentration.

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Agricultural Biotechnology

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Editor; Xiaohui FAN

P ro o fread er; X iaoyan WU

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E ditori Xiaohui FAN

P ro o fread er; X iaoyan WU

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