Bioaccumulation and ROS generation in Coontail Ceratophyllumdemersum L. exposed to phenanthrene

Ying Yin • Xiaorong Wang • Liuyan Yang •

Yuanyuan Sun • Hongyan Guo

Accepted: 31 March 2010 / Published online: 14 April 2010

� Springer Science+Business Media, LLC 2010

Abstract Phenanthrene bioaccumulation, induction free

radicals and their consequent biochemical responses in

coontail (Ceratophyllum demersum L.) were examined.

Plants were exposed to different levels (0.01, 0.02, 0.05,

0.07 and 0.1 mg/l) of phenanthrene for 10 days. Results

showed that the phenanthrene concentration in the plants

was exponentially correlated to exposure concentration

(R2 = 0.958) and phenanthrene exposure significantly

increased the total free radicals and superoxide anion in the

plants. The activities of antioxidant enzymes and the con-

tents of glutathione were determined. The superoxide dis-

mutase (SOD) activity and reduced glutathione (GSH)

content were inhibited, while the catalase (CAT), peroxi-

dase (POD), glutathione-s-transferase (GST) activities and

oxidized glutathione (GSSG) content were significantly

induced. Changes in the contents of chlorophyll and mal-

ondialdehyde (MDA) indicated that the MDA content was

enhanced after phenanthrene exposure and the contents of

chlorophyll were significantly increased in the 0.01 mg/l

group. These experimental data demonstrated that the

bioaccumulation of phenanthrene induced the production

of free radicals and ROS, and changed the antioxidant

defense system, ultimately resulting in oxidative damage in

C. demersum.

Keywords Bioaccumulation � Ceratophyllum demersum �Electron paramagnetic resonance (EPR) � Free radical �Phenanthrene

Introduction

Polynuclear aromatic hydrocarbons (PAHs) are a ubiqui-

tous wide spread class of environmental chemical pollu-

tants which are known to exert acutely toxic effects as

well as have mutagenic and carcinogenic properties. There

is an increasing concern about the deleterious effects of

PAHs on estuarine and coastal ecosystems (Aas et al.

2000; Krang 2007). However, the consequences and

mechanisms of the toxic effect of PAHs on aquatic

organisms are still unclear. Phenanthrene, a three ring

PAH, is found in relatively high concentrations in coal tar

contaminated sites and in polluted aquatic environments

(Mai et al. 2002).

The lipophilic PAHs and their derivatives can indi-

rectly disturb the structure and function of biomembranes

through the formation of free radicals (Tukaj and Aks-

mann 2007). Juchau et al. (1992) reported that xenobiotics

sometimes termed ‘‘protertogens’’, which are relatively

non-toxic, can be enzymatically bioactivated in the

embryo to highly toxic, electrophilic or free radical

reactive intermediates. If not detoxified, electrophilic

reactive intermediates can bind covalently to embryonic

cellular macromolecules, while free radical reactive

intermediates can react directly or indirectly with

molecular oxygen to form reactive oxygen species (ROS),

such as superoxide anion (O2•-), hydrogen peroxide

(H2O2), and hydroxyl radical (•OH) (Wells et al. 2005).

Free radicals and ROS are produced under normal phys-

iological conditions and can be detected in all plant

Y. Yin � X. Wang (&) � L. Yang � H. Guo

State Key Laboratory of Pollution Control and Resources Reuse,

School of Environment, Nanjing University,

Nanjing 210093, China

e-mail: yinying@nju.edu.cn

Y. Sun

Department of Hydrosciences, Nanjing University, Nanjing

210093, China

123

Ecotoxicology (2010) 19:1102–1110

DOI 10.1007/s10646-010-0492-1

tissues. Under environmental stress, changes in free rad-

ical processes are expected to occur and these in turn

affect the radical scavenging ability of a plant (Batish

et al. 2006). When ROS generation exceeds the capacity

of the cellular antioxidants it may initiate oxidative

reactions resulting in significant oxidative damage to the

plant. These cytotoxic ROS can strongly disrupt normal

metabolism through the oxidative damage of chlorophyll,

lipids, protein, and nucleic acids (Herbinger et al. 2002;

Selote et al. 2004).

Because internal O2 concentrations are high during

photosynthesis, chloroplasts are especially prone to gen-

erate ROS. Electrons leaked from electron transport chains

can react with O2 during normal aerobic metabolism to

produce ROS. Once produced, O2•- will rapidly dismutate,

either enzymatically or non-enzymatically, to yield H2O2

and O2. In addition, H2O2 and O2•- may interact in the

presence of Fe2? to yield highly reactive •OH (Imlay and

Linn 1988). ROS production and subsequent oxidative

damage may be an important mechanism of toxicity in

organisms exposed to xenobiotics (Livingstone 2001). To

protect them against oxidative stress, plant cells produce

both antioxidant enzymes such as superoxide dismutase

(SOD), catalase (CAT), peroxidase (POD), and glutathi-

one-s-transferase (GST), and non-enzymatic antioxidants

such as glutathione and ascorbate. Some of these constitute

good molecular bioindicators for contaminant-mediated

oxidative stress (Shao et al. 2002). Also, in vivo free rad-

ical formation has been cited as a contributing factor to the

deleterious effects of many chemical pollutants (Li et al.

2008).

Submerged macrophytes can cover large areas and

dominate primary productivity in aquatic environments and

are considered as a compartment for energy fixation and

nutrient flux. The plants growing in polluted water bodies

can absorb toxic xenobiotics, which thus enter into the food

chain, posing a serious threat to human health (Gupta and

Chandra 1998). Their ability to accumulate toxic metals

from water has been well documented (Mishra et al. 2006),

but in general, less is known about the effects of organic

contaminants (such as PAHs).

It is hypothesized that submerged macrophytes may be

adversely affected upon exposure to PAHs, a class of

chemicals known to be environmental pollutants. To test

this assumption, we exposed the macrophyte Ceratophyl-

lum demersum to the PAH phenanthrene under laboratory

conditions and (1) determined the uptake of phenanthrene;

(2) measured free radical production; and (3) examined

changes in biological parameters known to be indicative of

toxicity such as antioxidant enzymes, lipid peroxidation,

and chlorophyll content.

Materials and methods

Plant material and growth conditions

Plants of C. demersum were collected from unpolluted

water bodies and acclimated in 18 glass containers (four

plants per container) containing 4 l 10%-strength Hoag-

lands nutrient solution for 1 month under natural day and

night cycle in shade conditions (Hoagland and Arnon 1950;

Owen and Jones 2001). During the experiment, airflow was

kept constant, the pH was 7.3 ± 0.3, the temperature was

20 ± 2�C, and the hardness of water was about 100 mg/l

as CaCO3. After acclimatization, plants were divided into

six groups. Phenanthrene was spiked with an acetone car-

rier that was kept below 0.01% (v:v). Five groups received

exposure concentrations of 0.01, 0.02, 0.05, 0.07, and

0.1 mg/l (three containers per treatment), while the con-

trols were the three containers without phenanthrene.

Approximately 50% of the water was replaced daily by

fresh phenanthrene solution to minimize contamination

from metabolic waste. After 10 days of treatment, all

plants were collected and washed thoroughly with tap

water and then distilled water. Plant tissues were divided

into several parts and used for the determination of various

parameters.

Determination of phenanthrene

Plant samples were weighed, cut into pieces and homog-

enized with pure anhydrous sodium sulfate in a mortar at

room temperature. The homogenate was extracted by

acetone/hexane (3/2; v/v) under sonication (Shumei Digital

Ultrasonic Cleaner KQ-50E, China, 50W, 40 kHz) for

20 min. The samples were sonicated and centrifuged three

times at 4,000 rpm for 15 min and the supernatants were

collected and combined after centrifugation. The combined

supernatants were concentrated to about 1 ml by a rotary

evaporator, and then cleaned using an anhydrous Na2SO4/

Florisil column and eluted with dichloromethane. The

eluate was evaporated gently to dryness and the residue

was dissolved in methanol for high performance liquid

chromatography (HPLC) analysis.

The concentration of phenanthrene was analyzed by a

Hewlett–Packard (HP) 1100 HPLC equipped with a HP

photodiode array detection, with an absorption wavelength

of 252 nm, and a mobile phase of methanol/water (85/15;

v/v). The column was a Zorbax Eclipse XDB-C8 (4.6 mm

i.d. 9 150 mm length). The recovery of analysis in the

plant was 90.0 ± 1.43% (n = 3). Phenanthrene was not

detected in the control samples.

Bioaccumulation and ROS generation in Coontail Ceratophyllum demersum L. 1103

123

Electron paramagnetic resonance (EPR) spectra

Three parallel samples of leaves frozen in liquid nitrogen

were lyophilized in a Labconco freeze dryer and introduced

into EPR-type quartz tubes (3 mm diameter). The con-

centration of free radicals was measured by an EPR spec-

trometer recording signals with Bruker EMX 10/12 X-band

at room temperature. The measurement settings were as

follows: center field, 3,470 G; scan range, 200 G; modu-

lation frequency, 100 kHz; modulation amplitude, 1.0 G;

receiver gain, 5 9 104; scans, 2 times; microwave power,

20 mW. The EPR signal obtained with these parameters

was used to calculate the height of the central peak,

indicative of the intensity of the free radicals (Garnczarska

and Bednarski 2004).

Determination of superoxide anion

The measurement of superoxide anion was performed by

determining the oxidation of hydroxylammonium chloride

(Wang and Luo 1990). The reaction product from super-

oxide anion and hydroxylammonium was NO2-, which

reacted with p-aminobenzene sulfonic acid anhydrous and

a-naphthylamine, producing an azo dyestuff with absorp-

tion at 530 nm.

Determination of glutathione

The plant material was homogenized in 0.1 M sodium

phosphate–5 mM EDTA buffer (pH 8.0) and 25% H3PO3.

The homogenate was centrifuged (12,000 rpm for 15 min)

at 4�C to obtain the supernatant. Reduced glutathione

(GSH) and oxidized glutathione (GSSG) levels were

determined fluorometrically according to the method of

Hissin and Hilf (1976). The fluorescence intensity was

recorded at 420 nm after excitation at 350 nm on a Hitachi

fluorescence spectrophotometer.

Determination of enzyme activity

Plant material was homogenized in 50 mM phosphate

buffer (pH 7.8) containing 1% polyvinyl pyrrolidine and

0.1 mM EDTA. The homogenate was centrifuged

(10,0009g for 20 min) at 4�C and the supernatant was used

for the antioxidant enzyme assays.

SOD activity was measured by the inhibition of the

photochemical reduction of nitroblue tetrazolium (NBT) at

560 nm (Beauchamp and Fridovich 1971). One unit of

enzyme activity was defined as the quantity of SOD

required to produce 50% inhibition of the reduction of NBT.

CAT and POD activities were measured according to Rao

et al. (1997). One unit of enzyme activity was defined as the

change of absorbance in 60 s at 25�C. GST activity was

measured according to Habig et al. (1974). Enzyme activ-

ity was determined by monitoring changes in absor-

bance at 340 nm (e340 1-chloro-2,4-dinitrobenzene (CDNB)

conjugation = 9.6 mM-1 cm-1) for 2 min at constant

temperature.

Protein was estimated by the method of Bradford

(1976), using bovine serum albumin (BSA) as a standard.

All antioxidant enzyme activities were expressed as units

(U) mg-1 protein. The absorbance was measured with a

Shimadzu UV-220 spectrophotometer.

Determination of MDA and chlorophyll

MDA content was determined according to Peever and

Higgins (1989). The MDA content was calculated by using

its extinction coefficient of 155 mM-1 cm-1.

Chlorophyll content was determined according to Arnon

(1949). Absorbance of extracts was measured at 663 and

645 nm with a Shimadzu UV-220 spectrophotometer.

Statistical analysis

Data were expressed as means ± standard deviation (SD).

Significant differences (p \ 0.05) were determined by the

Student’s t test using Excel 2000 spreadsheet software

(Microsoft). Graphs of free radicals were prepared using

Origin 7.0.

Results

Bioaccumulation of phenanthrene in plants

The concentrations of phenanthrene in C. demersum

increased significantly with increasing exposure concen-

trations (Fig. 1). Regression analysis demonstrated a good

relationship between the phenanthrene contents (y) in

plants of treated groups and the exposure concentration (x)

(R2 = 0.958).

Electron paramagnetic resonance spectra

Typical EPR spectra of the control and phenanthrene-

treated samples consisted of a single peak free radical

signal with a g value of 2.0035 ± 0.0004 and linewidth of

0.9074 ± 0.0441 mT (Fig. 2a). The signal intensities in

the treated samples were significantly higher than the

control (p \ 0.05) (Fig. 2b). The concentrations of free

radicals were about 54% (at 0.01 mg/l) and 74% (at

0.1 mg/l) higher in the exposure groups than those in the

control. A reasonable good correlation was obtained

between the signal intensity of the free radicals and the

exposure concentration (R2 = 0.877).

1104 Y. Yin et al.

123

Superoxide anions

Compared with the control group, phenanthrene exposure

significantly induced generation of O2•- (p \ 0.05) (Fig. 3).

O2•- content increased with an increase in phenanthrene

concentration from 0.01 to 0.05 mg/l, with the maximum

level being 91% higher than the control. Upon exposure

of [0.05 mg/l phenanthrene, the O2•- content began to

decrease but was still higher than the control.

Activities of antioxidant enzymes and glutathione

content

The antioxidant responses of the plants to phenanthrene

exposure are shown in Fig. 4. When the exposure con-

centration of phenanthrene was 0.07 mg/l, the SOD activ-

ity began to descend sharply and reached the lowest value

in the last group (p \ 0.05), with an inhibition rate at 61%

compared with the control. At 0.05 mg/l, CAT activity was

at a maximum, with an activation rate at 51%. Significant

induction of POD activity was observed in all exposure

groups (p \ 0.05), from the 0.02 mg/l group and up, POD

activity began to increase with higher phenanthrene con-

centration. GST activity in the treated groups were also

significantly induced (p \ 0.05) with higher phenanthrene

concentrations, and GST activity was activated at the

0.1 mg/l phenanthrene level, with a value 3.88 times the

control group.

Phenanthrene exposure significantly inhibited the GSH

content (p \ 0.05) (Table 1). Compared with the control

group, 53% of the GSH content was reduced when the

phenanthrene concentration was 0.1 mg/l. GSSG content

was elevated significantly (p \ 0.05) in the treatment

Fig. 1 Phenanthrene accumulation in Ceratophyllum demersum L.

exposed to different concentrations of phenanthrene after 10 days

Fig. 2 a The EPR spectrum of free radicals in Ceratophyllumdemersum L. b Free radical signal intensity in Ceratophyllumdemersum L. during 10 days phenanthrene exposure. * Significantly

different from control, p \ 0.05

Fig. 3 O2•- production rate in Ceratophyllum demersum L. exposed

to phenanthrene for 10 days. * Significantly different from control,

p \ 0.05

Bioaccumulation and ROS generation in Coontail Ceratophyllum demersum L. 1105

123

groups and showed little change after the phenanthrene

concentration reached 0.05 mg/l. Total glutathione content

in the exposure groups was induced compared with the

control (p \ 0.05), reaching the maximum in the 0.05 mg/l

treated group, and then began to decrease. The GSH/GSSG

ratio decreased continuously, and the ratios in the five

exposure groups correlated well with the corresponding

exposure concentration (R2 = 0.988).

Lipid peroxidation and chlorophyll content

Changes in MDA content are shown in Fig. 5. The MDA

content was enhanced after phenanthrene exposure. A

slight increase in MDA content in the 0.01 mg/l group was

followed by a significant increase when the exposure

concentration of phenanthrene was 0.02 and 0.05 mg/l

(p \ 0.05). From the 0.07 mg/l exposure group and higher,

the MDA content did not increase further.

Fig. 4 Antioxidant defense

enzyme activity in

Ceratophyllum demersum L.

exposed to phenanthrene for

10 days. * Significantly

different from control, p \ 0.05

Table 1 The changes in GSH and GSSG of Ceratophyllum demersum L. after exposure to phenanthrene (lg g-1 fresh weight)

Group Exposure concentrations of phenanthrene (mg l-1)

0 0.01 0.02 0.05 0.07 0.1

GSH 35.9 ± 5.54 28.0 ± 6.53a 25.0 ± 2.20a 24.5 ± 3.45a 21.3 ± 4.77a 16.8 ± 1.01a

GSSG 5.49 ± 2.09 29.2 ± 2.42a 32.6 ± 5.92a 37.1 ± 2.23a 37.4 ± 4.78a 37.9 ± 4.04a

GSH ? GSSG 41.4 ± 9.09 57.2 ± 8.41a 57.6 ± 7.48a 61.5 ± 3.33a 58.7 ± 9.68a 54.7 ± 4.02a

GSH/GSSG 6.53 ± 0.91 0.96 ± 0.18a 0.85 ± 0.17a 0.71 ± 0.13a 0.60 ± 0.05a 0.46 ± 0.15a

Data are expressed as means ± SD, n = 4a Significantly different from control, p \ 0.05

Fig. 5 MDA content in Ceratophyllum demersum L. exposed to

phenanthrene for 10 days. * Significantly different from control,

p \ 0.05

1106 Y. Yin et al.

123

Changes in chlorophyll content are shown in Fig. 6. The

contents of chlorophyll a and b were significantly increased

in the 0.01 mg/l group (p \ 0.05). As the exposure to

phenanthrene increased, the concentration of chlorophyll

decreased. At 0.1 mg/l phenanthrene, a 31% reduction in

the content of total chlorophyll was noted.

Discussion

Due to low water solubility and strong lipophilic character,

PAHs highly accumulate in the environment and are

preferably concentrated in aquatic organisms (Ghosh et al.

2003). The environmental fate of PAHs is mostly depen-

dent on the molecular size. Generally, an increased

molecular size results in an increase in the octanol/water

partition coefficient (Kow). In this study, phenanthrene was

largely accumulated in submerged plant tissue, probably

because of its higher lipophilicity (logKow = 4.57) and the

lower metabolic ability of the plants. Sun et al. (2006)

reported that phenanthrene rapidly accumulated in fish

tissue and stimulated the production of free radicals in the

liver of Carassius auratus. All of these are examples of the

oxidative stress that may be induced by xenobiotics (Shi

et al. 2005; Qian et al. 2009). However, limited studies

have been undertaken concerning the production of ROS

and the resulting oxidative stresses in aquatic plants in the

presence of phenanthrene exposure.

The EPR method can be used for estimating the type and

content of some stable free radicals, which accumulate in

tissue during oxidative stress. EPR spectroscopy analysis

for the treated plants revealed that the 10 days phenan-

threne exposure promoted the synthesis of free radicals.

The concentration of free radicals increased and clearly

remained far above the control level. The g value of 2.0035

is similar to the signal from the abiotically stressed leaves

of A. thaliana (Muckenschnabel et al. 2002). In contrast,

the g value of other plant tissues exposed to abiotic stres-

ses, e.g., the roots of lupin exposed to lead, or wheat leaves

exposed to elevated ozone concentrations (at 2.0053) were

considerably higher than those in the submerged plants

exposed to phenanthrene in the present work (Reichenauer

and Goodman 2001). The EPR signal implied that the

paramagnetic radicals were derived from a quinone,

although the exact origin of these radicals was not deter-

mined (Garnczarska and Bednarski 2004). To resolve this

problem, further studies are needed using spin-trapping or

other methods to identify other radical species.

Superoxide anion (O2•-), produced by xanthine oxidase,

tryptophan dioxygenase, diamine oxidase and activated

neutrophils, can be a source of additional harmful ROS

(Di Giulio et al. 1989; Foss et al. 2004). O2•- is a toxic by

product of oxidative metabolism and can interact with

H2O2 to form the highly reactive hydroxyl radical (•OH),

which is thought to be primarily responsible for oxygen

toxicity in the cell (Zini et al. 2007). Fig. 3 showed that

O2•- radical accumulation in C. demersum was significantly

enhanced during exposure to phenanthrene. Previous work

has suggested that phenanthrene could induce ROS such as

O2•- and •OH in the liver of mice and fish, and the for-

mation of ROS and the bioaccumulation of pollutant

showed similar trends with the exposure concentration

(Bagchi et al. 2002; Sun et al. 2006). Our current study

demonstrated that phenanthrene stimulates the production

of ROS in C. demersum (Figs. 2 and 3). With the

increasing exposure concentration, the accumulation of

phenanthrene in plants increased, led to a corresponding

increase in free radicals, biological oxidation–reduction

balance of the body to further break, and ROS increased.

Under conditions of normal healthy growth, plants possess

a number of enzymatic and non-enzymatic detoxification

mechanisms to efficiently scavenge ROS and their reaction

products (Elstner and Osswald 1994). An important feature

of these enzymes and non-enzymatic antioxidants is their

inducibility under oxidative stress. However, severe oxi-

dative stress may suppress the activities of these enzymes,

leading to a loss in compensatory mechanisms with a

consequence of oxidative damage.

SOD, the first enzyme in the detoxifying process, cata-

lyzes the dismutation of O2•- to H2O2 and O2 (Molassiotis

et al. 2006). Oruc and Uner (2000) indicated that induction of

SOD can occur during high production of O2•-, therefore, an

increase in SOD activity indicates an increase in O2•- pro-

duction. According to Mittler (2002), increased SOD activity

may be considered as circumstantial evidence for enhanced

ROS production. In contrast, Dimitrova et al. (1994) reported

that the O2•- by themselves or after their transformation to

Fig. 6 Chlorophyll content in Ceratophyllum demersum L. exposed

to phenanthrene for 10 days. * Significantly different from control,

p \ 0.05

Bioaccumulation and ROS generation in Coontail Ceratophyllum demersum L. 1107

123

H2O2 may induce oxidation of the enzymatic cysteine

resulting in a decrease in SOD activity. Inhibition by SOD

alone does not constitute rigorous proof for involvement of

O2•-. In this study, SOD activity was inhibited gradually in

the exposure groups especially in the two highest concen-

tration groups. This suggests that when O2•- generation

exceeds the elimination ability of SOD, O2•- as well as other

oxyradicals can then attack and inactivate enzymes.

In our study, CAT, POD and GST activities were

increased in the treated groups, which may be an adaptive

trait which helps to overcome the damage to the tissue

metabolism by reducing toxic levels of ROS. The POD and

GST activities were clearly increased in the treated groups

at higher concentrations and were probably a metabolic

adaptation to the phenanthrene exposure and were obvi-

ously a defense against oxidative stress. It was thus con-

sidered that excessive O2•- generation insulted SOD

activity, which decreased the ability of dismutation.

Therefore, only small amounts of H2O2 formed and accu-

mulated which induced CAT but was not enough to inac-

tivate CAT. It could be assumed that POD and GST had

more tolerance than CAT, while SOD was comparatively

susceptible under conditions of oxidative stress.

It has been proven that GSH is one of the most efficient

scavengers of ROS arising as by-products of cellular

metabolism or during oxidative stress (Han et al. 2008).

Total glutathione serves as a prospective biological index

to indicate contaminants exposure (Stein et al. 1992), due

to its function in resisting reactive oxygen toxicity. The

most obvious direct effect of certain pollutants is a

decrease in thiol status, i.e., the ratio of reduced to oxidized

glutathione (GSH/GSSG), due to either direct radical

scavenging or increased peroxidase activity. Phenanthrene

exposure resulted in a significant decrease in GSH levels.

GSSG levels changed alone with GSH levels. The rate of

generation of GSSG was higher than its reduction back to

GSH, thus GSSG accumulated and tGSH was increased in

plants during phenanthrene exposure. The GSH/GSSG

ratios decreased continuously, which correlated well with

the corresponding exposure concentration, partially due to

the high GSSG levels. It may be considered as a sign of

oxidative stress evoked by the ROS. Van der Oost et al.

(2003) demonstrated that GSH and GSSG parameters

should not yet be considered as valid biomarkers but the

GSH/GSSG ratio may be a potential biomarker for oxida-

tive stress in a number of laboratory and field studies.

Similar to these reports, our results indicated that the GSH/

GSSG ratio was very sensitive to phenanthrene and more

suitable as an indicator of phenanthrene exposure than

tGSH or a single GSH parameter.

MDA content is an important indicator of lipid peroxi-

dation in plant cells. According to Liu (2001), the MDA

content in aquatic plant tissues is positively related to

surfactant concentrations in the solutions, reflecting envi-

ronmental pollution. Liao et al. (2005) also indicated that

MDA content in plant tissues was a useful index to eval-

uate pollution levels and judge the toxic effects of pollu-

tants. In our study, increased MDA revealed that the

organism was in oxidative stress from phenanthrene

exposure. MDA content indicated the prevalence of free

radical reactions in tissues and membrane lipid peroxida-

tion, which could be attributed to the decreases in SOD and

CAT activities. The decreased activities favored accumu-

lation of O2•- and H2O2, which could result in lipid per-

oxidation. This process can be a vicious spiral under

conditions of higher phenanthrene concentrations, which

enhances the toxic effects on plants.

In chloroplasts, ROS can be generated by the direct

transfer of excitation energy from chlorophyll to produce

singlet oxygen (Meloni et al. 2003). ROS are known to

induce pigment bleaching by direct oxidation of chloro-

phyll or by cooxidative processes during lipid peroxidation

where alkoxyl radicals attack pigments resulting in their

oxidation (Elstner and Osswald 1994). Wang et al. (2005)

reported that chloroplasts can accept an electron which is

then donated to O2 creating O2•-, while methyl viologen

treatment in vitro caused chlorophyll damage and leaf

bleaching due to the presence of O2•-. In the present study,

a similar conclusion could be drawn that phenanthrene

treatment caused chlorophyll damage due to ROS. At a low

concentration of phenanthrene exposure, the antioxidant

defense may have been induced by a slight oxidative stress

which activated the metabolic mechanism of C. demersum,

and led to an increase in the chlorophyll content.

In conclusion, we found that phenanthrene could be

bioaccumulated by the submerged plant C. demersum, and

induced production of a large number of free radicals,

resulting in oxidative stress and damage to the tissue of

plants. Additionally, changes in the activities of antioxidant

defense, lipid peroxidation and chlorophyll damage were

found. Among these parameters, the GSH/GSSG ratio was

more suitable as indicator of toxicity in C. demersum under

phenanthrene pollution conditions. Our present result sug-

gested that further studies are needed to illuminate the

mechanisms of accumulation, toxicity and the stress

resistance in aquatic plants exposed to phenanthrene.

Acknowledgments We thank Yunxia Sui for analysis of EPR

spectra. This work was supported by the National Fundamental

Research Foundation of China (2009CB421604) and Natural Science

Foundation of China (40902067).

References

Hoagland PR, Arnon DI (1950) The water culture method for growing

plants without soil. Calif Agric Exp Stn Circ 347:1–32

1108 Y. Yin et al.

123

Aas E, Baussant T, Balk L, Liewenborg B, Andersen OK (2000) PAH

metabolites in bile, cytochrome P4501A and DNA adducts as

environmental risk parameters for chronic oil exposure: a

laboratory experiment with Atlantic cod. Aquat Toxicol 51:

241–258

Arnon DI (1949) Copper enzymes in isolated chloroplasts, polyphe-

noloxidase in Beta vulgaris. Plant Physiol 24:1–15

Bagchi D, Balmoori J, Bagchi M, Ye X, Williams CB, Stohs SJ

(2002) Comparative effects of TCDD, endrin, naphthalene and

chromium (VI) on oxidative stress and tissue damage in the liver

and brain tissues of mice. Toxicol 175:73–82

Batish DR, Singh HP, Setia N, Kaur S, Kohli RK (2006)

2-Benzoxazolinone (BOA) induced oxidative stress, lipid per-

oxidation and changes in some antioxidant enzyme activities in

mung bean (Phaseolus aureus). Plant Physiol Biochem 44:819–

827

Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved

assay and an assay applicable to acrylamide gels. Anal Biochem

44:276–287

Bradford MM (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein–dye binding. Anal Biochem 72:248–254

Di Giulio RT, Washburn PC, Wenning RJ, Winston GW, Jewell CS

(1989) Biochemical responses in aquatic animals: a review of

determinants of oxidative stress. Environ Toxicol Chem 8:1103–

1123

Dimitrova M St, Tishinova V, Velcheva V (1994) Combined effect of

zinc and lead on the hepatic superoxide dismutase-catalase

system in carp (Cyprinus carpio). Comp Biochem Physiol C

Pharmacol Toxicol Endocrinol 108:43–46

Elstner EF, Osswald W (1994) Mechanisms of oxygen activation

during plant stress. Proc R Soc Edinb Nat Environ 102B:131–

154

Foss BJ, Sliwka HR, Partali V, Cardounel AJ, Zweierb JL,

Lockwoodc SF (2004) Direct superoxide anion scavenging by

a highly water-dispersible carotenoid phospholipid evaluated by

electron paramagnetic resonance (EPR) spectroscopy. Bioorg

Med Chem Lett 14:2807–2812

Garnczarska M, Bednarski W (2004) Effect of a short-term hypoxic

treatment followed by re-aeration on free radicals level and

antioxidative enzymes in lupine roots. Plant Physiol Biochem

42:233–240

Ghosh U, Zimmerman JR, Luthy RG (2003) PCB and PAH speciation

among particle types in contaminated harbor sediments and

effects on PAH bioavailability. Environ Sci Technol 37:2209–

2217

Gupta M, Chandra P (1998) Bioaccumulation and toxicity of mercury

in rooted-submerged macrophyte Vallisneria spiralis. Environ

Pollut 103:327–332

Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases:

the first enzymatic step in mercapturic acid formation. J Biol

Chem 249:7130–7139

Han Y, Zhang J, Chen XY, Gao ZZ, Xuan W, Xu S, Ding X, Shen

WB (2008) Carbon monoxide alleviates cadmium-induced

oxidative damage by modulating glutathione metabolism in the

roots of Medicago sativa. New Phytol 177:155–156

Herbinger K, Tausz M, Wonisch A, Soja G, Sorger A, Grill D (2002)

Complex interactive effects of drought and ozone stress on the

antioxidant defence systems of two wheat cultivars. Plant Physiol

Biochem 40:691–696

Hissin PJ, Hilf R (1976) A fluorometric method for the direct

determination of oxidized and reduced glutathione in tissues.

Anal Biochem 74:214–226

Imlay JA, Linn S (1988) DNA damage and oxygen radical toxicity.

Science 240:1302–1309

Juchau MR, Lee QP, Fantel AG (1992) Xenobiotic biotransformation

bioactivation in organogenesis-stage conceptual tissues: impli-

cations for embryotoxicity and teratogenesis. Drug Metab Rev

24:195–238

Krang A-S (2007) Naphthalene disrupts pheromone induced mate

search in the amphipod Corophium volutator (Pallas). Aquat

Toxicol 85:9–18

Li YH, Jiang B, Zhang T, Mu WM, Liu J (2008) Antioxidant and free

radical-scavenging activities of chickpea protein hydrolysate

(CPH). Food Chem 106:444–450

Liao BH, Liu HY, Zeng QR, Yu PZ, Probst A, Probst JL (2005)

Complex toxic effects of Cd2?, Zn2?, and acid rain on growth of

kidney bean (Phaseolus vulgaris L.). Environ Int 31:891–895

Liu HY (2001) Studies of the harmful effects on aquatic plants and

the biodegradation of surfactants. Doctorate Dissertation, Hunan

Agricultural University, Changsha, China

Livingstone DR (2001) Contaminant-stimulated reactive oxygen

species production and oxidative damage in aquatic organisms.

Mar Pollut Bull 42:656–666

Mai BX, Fu JM, Sheng GY, Kang YH, Lin Z, Zhang G, Min YS,

Zeng EY (2002) Chlorinated and polycyclic aromatic hydrocar-

bons in riverine and estuarine sediments from Pearl River Delta,

China. Environ Pollut 117:457–474

Meloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosyn-

thesis and activity of superoxide dismutase, peroxidase and

glutathione reductase in cotton under salt stress. Environ Exp

Bot 49:69–76

Mishra S, Srivastava S, Tripathi RD, Kumar R, Seth CS, Gupta DK

(2006) Lead detoxification by coontail (Ceratophyllum demersumL.) involves induction of phytochelatins and antioxidant system

in response to its accumulation. Chemosphere 65:1027–1039

Mittler R (2002) Oxidative stress, antioxidants and stress tolerance.

Trends Plant Sci 7:405–410

Molassiotis A, Sotiropoulos T, Tanou G, Diamantidis G, Therios I

(2006) Boron-induced oxidative damage and antioxidant and

nucleolytic responses in shoot tips culture of the apple rootstock

EM 9 (Malus domestica Borkh). Environ Exp Bot 56:54–62

Muckenschnabel I, Goodman BA, Williamson B, Lyon GD, Deighton

N (2002) Infection of leaves of Arabidopsis thaliana by Botrytiscinerea: changes in ascorbic acid, free radicals and lipid

peroxidation products. J Exp Bot 53:207–214

Oruc EO, Uner N (2000) Combined effects of 2, 4-D and

azinphosmethyl on antioxidant enzymes and lipid peroxidation

in liver of Oreochromis niloticus. Comp Biochem Physiol C

Pharmacol Toxicol Endocrinol 127:291–296

Owen AG, Jones DL (2001) Competition for amino acids between

wheat roots and rhizosphere microorganisms and the role

of amino acids in plant N acquisition. Soil Biol Biochem

33:651–657

Peever TL, Higgins VJ (1989) Electrolyte leakage, lipoxygenase, and

lipid peroxidation induced in tomato leaf tissue by specific and

non specific elicitors from Cladosporium fluvum. Plant Physiol

90:867–875

Qian HF, Chen W, Sun LW, Jin YX, Liu WP, Fu ZW (2009)

Inhibitory effects of paraquat on photosynthesis and the response

to oxidative stress in Chlorella vulgaris. Ecotoxicology 18:537–

543

Rao MV, Paliyath G, Ormrod DP, Murr DO, Watkins CB (1997)

Influence of salicylic acid on H2O2 production, oxidative, stress,

and H2O2-metabolizing enzymes. Plant Physiol 115:137–149

Reichenauer TG, Goodman BA (2001) Stable free radicals in ozone-

damaged wheat leaves. Free Radic Res 35:93–101

Selote DS, Bharti S, Khanna-Chopra R (2004) Drought acclimation

reduces O2-� accumulation and lipid peroxidation in wheat

seedlings. Biochem Biophys Res Commun 314:724–729

Bioaccumulation and ROS generation in Coontail Ceratophyllum demersum L. 1109

123

Shao Y, Rudolf SSW, Richard YCK (2002) Biodegradation and

enzymatic responses in the marine diatom Skeletonema costatumupon exposure to 2,4-dichlorophenol. Aquat Toxicol 59:191–200

Shi HH, Wang XR, Luo Y, Su Y (2005) Electron paramagnetic

resonance evidence of hydroxyl radical generation and oxidative

damage induced by tetrabromobisphenol A in Carassius auratus.

Aquat Toxicol 74:365–371

Stein JE, Collier TK, Reichert WL, Casillas E, Hom T, Varanasi U

(1992) Bioindicators of contaminant exposure and sublethal

effects: studies with benthic fish in Puget sound, Washington.

Environ Toxicol Chem 11:701–714

Sun YY, Yu HX, Zhang JF, Yin Y, Shi HH, Wang XR (2006)

Bioaccumulation, depuration and oxidative stress in fish Caras-sius auratus under phenanthrene exposure. Chemosphere 63:

1319–3027

Tukaj Z, Aksmann A (2007) Toxic effects of anthraquinone and

phenanthrenequinone upon Scenedesmus strains (green algae) at

low and elevated concentration of CO2. Chemosphere 66:480–

487

Van der Oost R, Beyer J, Vermeulen NPE (2003) Fish bioaccumu-

lation and biomarkers in environmental risk assessment: a

review. Environ Toxicol Pharmacol 13:57–149

Wang AG, Luo GH (1990) Quantitative relation of hydroxylamine

and superoxide anion radicals in plants. Chin Plant Physiol

Commun 6:55–57

Wang FZ, Wang QB, Kwon SY, Kwak SS, Su WA (2005) Enhanced

drought tolerance of transgenic rice plants expressing a pea

manganese superoxide dismutase. J Plant Physiol 162:465–472

Wells PG, Bhuller Y, Chen CS, Jeng W, Kasapinovic S, Kennedy JC,

Kim PM, Laposa RR, McCallum GP, Nicol CJ, Parman T, Wiley

MJ, Wong AW (2005) Molecular and biochemical mechanisms

in teratogenesis involving reactive oxygen species. Toxicol Appl

Pharmacol 207:S354–S366

Zini R, Berdeaux A, Morin D (2007) The differential effects of

superoxide anion, hydrogen peroxide and hydroxyl radical on

cardiac mitochondrial oxidative phosphorylation. Free Radic Res

41:1159–1166

1110 Y. Yin et al.

123