Ecotoxicology (2014) 23:633–640
DOI 10.1007/s10646-014-1214-x
Microalga Euglena as a bioindicator for testing genotoxic
potentials of organic pollutants in Taihu Lake, China
Mei Li • Xiangyu Gao • Bing Wu • Xin Qian
John P. Giesy • Yibin Cui
•
Accepted: 16 February 2014 / Published online: 26 February 2014
Ó Springer Science+Business Media New York 2014
Abstract The microalga Euglena was selected as a bioindicator for determining genotoxicity potencies of organic
pollutants in Meiliang Bay of Taihu Lake, Jiangsu, China
among seasons in 2008. Several methods, including the
comet assay to determine breaks in DNA and quantification
of antioxidant enzymes were applied to characterize
genotoxic effects of organic extracts of water from Taihu
Lake on the flagellated, microalga Euglena gracilis. Contents of photosynthetic pigments, including Chl a, Chl
b and carotenoid pigments were inversely proportion to
concentrations of organic extracts to which E. gracilis was
exposed. Organic extracts of Taihu Lake water also
affected activities of superoxide dismutase (SOD) and
peroxidase (POD) of E. gracilis. There were no statistically
significant differences in SOD activities among seasons
except in June but significant differences in POD activities
were observed among all seasons. The metrics of DNA
fragmentation in the alkaline unwinding assay (Comet
M. Li (&) X. Gao B. Wu X. Qian J. P. Giesy Y. Cui
State Key Laboratory of Pollution Control and Resource Reuse,
School of the Environment, Nanjing University, Xianlin
Campus, 163 Xianlin Avenue, Nanjing 210023, China
e-mail: meili@nju.edu.cn
J. P. Giesy
Department of Biomedical Veterinary Biosciences and
Toxicology Centre, University of Saskatchewan,
Saskatoon S7N 5B3, Canada
J. P. Giesy
Zoology Department, Center for Integrative Toxicology,
Michigan State University, East Lansing, MI 48824, USA
J. P. Giesy
Department of Biology and Chemistry, and State Key
Laboratory in Marine Pollution, City University of Hong Kong,
Kowloon, Hong Kong SAR, China
assay), olive tail moment (OTM) and tail moment (TM),
used as measurement endpoints during the genotoxicity
assay were both greater when E. gracilis was exposed to
organic of water collected from Taihu Lake among four
seasons. It is indicated that the comet assay was useful for
determining effects of constituents of organic extracts of
water on E. gracilis and this assay was effective as an early
warning to organic pollutants.
Keywords Comet assay Drinking water Euglena
gracilis DNA damage Bioassay Microalgae
Introduction
With the rapid increase in population, as well as development of industry and agriculture in the region surrounding
Taihu Lake, quality of water in the lake has been decreasing
(Turgeon et al. 2004; Lu et al. 2011, 2013). Several studies
of pollution of Taihu Lake and its catchments have been
conducted (Haruhiko et al. 2005; Zhu et al. 2006; Fu et al.
2009; Qin et al. 2010). Although relatively small concentrations of several organic pollutants have been reported to
occur in water from Taihu Lake (Katsoyiannis and Samara
2005; Zhu et al. 2005; Gao et al. 2011), the risks of these
chemicals on organisms of the lake or potential effects on
people through drinking water had not been assessed. There
is little information on organic pollutants in Meiliang Bay,
Taihu Lake and no information on genotoxicity or potential
of the mixture of organic pollutants to cause damage to
DNA. Quantification of individual chemicals is a first step in
monitoring quality of water, but comparing these concentrations to individual water quality criteria does not take into
consideration chronic exposure to small concentrations of
mixtures (Pellacani et al. 2006). Furthermore, the absence of
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634
methods or standards can preclude identification and quantification of all the residues present. Even if all the constituents could be quantified, there are insufficient water quality
standards and criteria or threshold toxicity reference values
to which to compare concentrations of contaminants such
that the potential adverse effects can be assessed. Finally,
the effects of interactions of the constituents cannot be easily
assessed based on knowledge of concentrations of individual
constituents.
Alternatively, biological assays can be used to effectively assess potential effects of mixtures on particular
endpoints in wildlife or humans (Ohe et al. 2004) without
the need to quantify all the individual constituents. Also,
bioassay-directed fraction and identification can be used to
determine the potential for effect, to identify sources and
institute remedial actions, can be applied to identify causative agents (De Coen et al. 2000; Snyder et al. 2000;
Hecker and Giesy 2011). Different unicellular and multicellular organisms, including bacteria, protozoa, algae,
invertebrate and fishes have been used for this purpose.
Different physiological responses such as growth rate,
biomass, chlorophyll, fluorescence, and movement and
other behaviors are measurement endpoints that have been
monitored as criteria for the toxicity of given water sample
(Kohler and Arndt 1992).
While some phyto-toxicological research has been
conducted on the effects of organic pollutants on terrestrial
macrophytes, the use of algae as test species has advantages (Djomo et al. 2004). The relatively rapid rates of
growth of algae and their small size make them amenable
to bioassays. Euglena gracilis is a unicellular photosynthetic freshwater flagellate which occurs in many aquatic
ecosystems. Due, in part, to its sensitivity, to a range of
toxicants, it has been shown to be a reliable organism for
use in bioassays to determine to various toxicants,
including metals, herbicides and other organic compounds
(Danilov and Ekelund 2000, 2001; Einicker-Lamas et al.
2002). These findings suggest that E. gracilis can be used
as one of a battery of suitable bioassays. Motility, cell
growth, cell shaper orientation and photosynthetic parameters of E. gracilis can be used as biomarkers in evaluating
the toxicity of toxic substances (Ahmed and Häder 2010).
Induction of reactive oxygen species (ROS)-scavenging
enzymes, such as superoxide dismutase (SOD), peroxidase
(POD), ascorbate peroxidase (APX) and catalase (CAT)
are some of the mechanisms for detoxifying ROS formed
during responses to chemical stressors (Mitter 2002).
Changes in activities of these enzymes can be used as
sensitive, functional biomarkers of exposure and adaptive
responses to pollutants. But algae have not been extensively used as indicator species for studying genotoxicity
caused by environmental pollutants. A few studies of
genotoxicity based on the comet assay have been
123
M. Li et al.
conducted with the flagellated, green algae E. gracilis
(Watanabe and Suzuki 2002; Aoyama et al. 2003; Li et al.
2009), Chlamidomonas reinhardtii (Erbes et al. 1997;
Sviežená et al. 2004) or marine diatoms (Desai et al. 2006).
In the present study, the freshwater green microalga E.
gracilis was used as the test organism to determine the
effect of organic compounds extracted from waters of
Taihu Lake water in each of four seasons. The measurement endpoints considered were growth (cell number),
photosynthetic pigment content, activities of two antioxidant enzymes (SOD and POD), and genotoxicity as measured by fragmentation during alkaline unwinding of DNA.
Materials and methods
Preparation of samples
Twenty liter water samples were collected at 0.5 m beneath
the surface in Meiliang Bay, Taihu Lake (31°280 2000 N,
120°130 2900 E). Water was collected four times during 2008,
at the end of March, June, September, and December,
respectively. Two gram ascorbic acid was added into each
sample and mixed, then 1–2 drops of concentrated hydrochloric acid was added to keep pH \ 2. All samples were
refrigerated at 4 °C and kept in the dark until extraction
treatment. After storing for about 24 h, the water samples
were filtered through gauze and filter paper to remove suspended materials or sediments. Filtered water was then
passed through a column with non-polar neutral resin XAD2 to adsorb organic compounds. The flow rate through the
column was of 30–40 ml min-1. The column was eluted
with methanol, acetone and dichloromethane successively.
The eluate was dried under a gentle stream of nitrogen at
50 °C and re-dissolved in 2.0 ml dimethysulphoxide
(DMSO) (Bian and Kang 1994). The samples were stored in
the dark at -20 °C. Semi-volatile organic compounds
(SVOCs) in the water samples were measured by GC/MS
(GCQ Finnigan MAT, USA). Standard SVOCs and a deuterated internal standard (IS) mixture were both obtained
from Supelco (Bellefont, PA, USA). Equilibrim time was
0.3 min and gasification temperature was 280 °C. Transfer
line was kept at 220 °C and source temperature was 220 °C.
Mas spectra were obtained from m/z = 50–650 base on full
scan with retention time of 4 min.
Treatment of micro-alga
The micro-alga E. gracilis was obtained from Mid-Sweden
University, Sundsvall, Sweden. Experiments were conducted in 250 ml flasks, which had been autoclaved at
121 °C for 20 min. Inoccula were added to medium with or
without organics extracts. Concentrations of organic
Microalga Euglena as a bioindicator
extract included equivalents of organic residues that were
1.09, 5.09, and 109 fold the concentration in the source
water. DMSO was used as a solvent control. The initial cell
density for each experiment was 0.5 9 105 cells ml-1. The
cultures were maintained in 250 ml Erlenmeyer flasks
containing a culture medium (Checcucci et al. 1976) at
23 ± 1 °C under a 12 h light/12 h dark cycle provided by
cool white fluorescence lights at 85–90 lmol photon
(m2 s)-1 irradiance for a period of 6 d.
635
Table 1 Content of semi-volatile organic compounds in Meiliang
Bay of Taihu Lake in all seasons (lg l-1)
Compounds
March
June
0.558
PAEs
16.067
7.840
9.608
4.428
0.540
1.200
0.686
0.606
Others
All
0.735
December
PAHs
BETXs
0.300
September
0.399
0.294
1.800
0.045
0.162
17.459
11.140
11.147
5.675
Quantification of pigments
Pigments were extracted with 80 % acetone and quantified
in accordance with the methods of Jeffrey and Humphrey
(1975) for chlorophyll content and Strickland and Parsons
(1972) for carotenoid pigment content. Concentrations
were expressed as mg pigment per 106 cells.
statistical software. Tukey’s multiple comparisons tests
with a Type I error, P \ 0.05 were applied in assessing
significance in the effect of each treatment. Each experiment was conducted in triplicate.
Results
Anti-oxidative enzymes assay
Concentrations of SVOCs
Activities of enzymes were determined in E. gracili that
had been cultured for 6 d and centrifuged at 3,0009g for
10 min and re-suspended in 1/15 mmol l-1 of pre-cooled
sodium phosphate buffer (pH 7.0). After sonication for
5 min in an ice bath, the cell debris was removed by
centrifugation at 12,0009g for 20 min at 4 °C. SOD (EC
1.15.1.1) activity was determined by use of the ferric
cytochrome c method with xanthine/xanthine oxidase used
as the source of super-oxide radicals, and a unit of activity
was defined as that described in McCord and Fridovich
(1969), which was expressed as U per 106 cells. POD (EC
1.11.1.7) activity was determined by use of the methods
described by Li (2000).
Total concentrations of semi-volatile organic chemicals
(SVOCs) in Meiliang Bay, Taihu Lake were 17.459,
11.140, 11.147 and 5.675 lg l-1 in March, June, September and December, respectively (Table 1). The 25 SVOCs
identified and quantified during this study could be divided
into four classes, including polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), benzenes series (BTEX)
and others. Concentrations of SVOCs were significantly
greater in March than in December. The greatest content
was PAEs, followed by a higher detection content of PAHs
and BTEX. The total concentration of BTEX observed in
June was more than the other two seasons.
Comet assay
Pigments bioassay
The comet assay with E. gracilis was performed following
methods described by Singh et al. (1988), Aoyama et al.
(2003) and Li et al. (2009) with some modifications. The
slides were examined with a fluorescent microscope (BX41,
Olympus, Japan). Three slides for each treatment were
prepared and at least 50 cells were analyzed from each slide.
Images were analyzed according to the method of Collins
et al. (1995) using the comet assay software project. The
effect of dose on Tail Moment and Olive Tail Moment were
analyzed by one-way ANOVA using Origin 8.0 statistical
software with level set at P \ 0.05 and P \ 0.001.
Concentrations of the photosynthetic pigments, Chl a, Chl
b and carotenoid pigements in E. gracilis varied among
seasons and treatments (Fig. 1). Concentrations of Chl
a and Chl b ranged from 1.0 9 10-2 to 5.0 9 10-2 and
4.0 9 10-3 to 1.2 9 10-2 mg per 106 cells, respectively.
The concentration of Chl a exposed to the 109 extracts
were significantly less by 24, 11 and 30 % than that of
control cells in March, June and September, respectively,
except in December which showed no statistically significant differences between the control and any of the treatments with organic extract (Fig. 1a).
Chl b content of E. gracilis was affected differently by
exposure to organic extracts among seasons. In March,
concentrations of Chl b were significantly less (29 and
37 %, respectively) in cells exposed to 59 or 109 concentrations of organic extract relative to that of controls
(Fig. 1b). In June, Chl b content in cells exposed to 19 and
Statistical analysis
Results were expressed as mean ± standard deviation
(SD). Differences between control and treated samples
were analyzed by one-way ANOVA using Origin Pro 8.0
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636
M. Li et al.
**
***
a
Mar.
Jun.
Sep.
Dec.
40
*
0.04
*
30
6
6
Chl a (mg/10 cells)
0.05
SOD ( U/10 cells)
Mar.
Jun.
Sep.
Dec.
a
0.03
*
0.02
**
***
***
10
0.01
0.00
CK
DMSO
1
5
0
10
CK
DMSO
Treatment
Mar.
Jun.
Sep.
Dec.
5
10
b
*
Mar.
Jun.
Sep.
Dec.
20
*
0.010
*
***
6
POD ( U/10 cells)
0.015
1
Treatment
6
Chl b (mg/10 cells)
b
**
**
20
*
*
*
0.005
*
0.000
CK
DMSO
1
5
10
c
0.020
CAR ( mg/10 cells)
Treatment
0.015
Mar.
Jun.
Sep.
Dec.
***
***
***
0
CK
DMSO
1
5
10
Fig. 2 Total SOD activity (a) and POD content (b) of E. gracilis
supplemented with different concentrations of water extracts. Data are
the mean ± SD of three replicates. *P \ 0.05
*
6
Concentrations of carotenoids in E. gracilis were not
affected significantly by exposure to organic extracts in
March or fall, but were affected in June and December. In
June and December, concentrations of carotenoids were
greater than that of the control (18 and 13 %, respectively)
in cells exposed to the 19 organic extract (Fig. 1c).
0.010
*
0.005
0.000
DMSO
*
Treatment
*
CK
***
10
1
5
10
Antioxidative enzymes assay
Treatment
Fig. 1 Concentrations of chlorophyll-a (a), -b (b) and cartotenoids
(c) in E. gracilis treated with different concentrations of water extract.
Data are the mean ± SD of three replicates. *P \ 0.05
109 organic extract was greater (33 and 4 %) than that of
the control. In September, only the concentration of Chl
b in cells exposed to the 59 extract was significantly less
(8 %) than that of the control. In December, Chl b content
in cells exposed to 109 organic extract was greater (33 %)
than that of the control.
123
There were no statistically significant effects of the organic
extracts on SOD activity (Fig. 2a) except in June, when
SOD activity was 17, 21 % (P \ 0.01) and 130 %
(P \ 0.001) greater than controls in cells treated with 19,
59, or 109 concentrations of organic extracts, respectively.
In March, exposure of E. gracilis to 19 or 59 resulted
in 89 and 27 %, greater POD activity, than that of the
control, respectively. In June, POD activity of E. gracilis
was 25 % greater when exposed to 109 extract. In September, all three concentrations of organic extract caused
Microalga Euglena as a bioindicator
a
60
Mar.
Jun.
Sep.
Dec.
50
Olive Tail moment (µm)
637
40
*
*
*
***
30
*
***
20
***
*** *
10
***
***
0
CK
DMSO
1
5
10
Treatment
b
80
*
Mar.
Jun.
Sep.
Dec.
Tail moment (µm)
70
60
50
*
***
*
March exposure to 59 or 109 concentrated organic extract
resulted in 87 and 174 % greater DNA damage, respectively, relative to that of the control. In June, September
and December, based on OTM, there were statistically
significant (P \ 0.05, P \ 0.001) differences among
treatments and the control. Exposure to 19, 59 or 109
resulted in 119, 116 and 206 % in June, 293, 563 and
1,127 % in September, 77.36, 88.22 and 326.20 % in
December more DNA damage than in the controls.
Based on TM (Fig. 3b), the amount of DNA damage in
March was 122 and 238 % (P \ 0.001) greater than that of
the control cells when exposed to 59 or 109 water
equivalents, respectively. In June, September and December, there were statistically significant differences
(P \ 0.05, P \ 0.001) in TM for all exposure concentrations. TM of cells exposed to 19, 59, or 109 organic
extract were greater than the controls by 154, 192 and
350 % in June, 500, 917 and 2,336 % in September, 101,
46 and 228 % in December greater than that of the controls, respectively.
40
***
30
***
Discussion
***
20
***
*
***
***
10
0
CK
DMSO
1
5
10
Treatment
Fig. 3 Induction of DNA damage (represented as olive tail moment
(a) and tail moment (b) in E. gracilis following exposure to different
concentrations of water extracts in vivo. Data are the mean ± SD of
the three replicates. *P \ 0.05
statistically significant differences (P \ 0.001), between
POD activity of cells exposed to 19, 59, and 109 concentrated organic extract which were 46, 47, and 53 % less
than that of the controls, respectively. In December, the
trend was similar with that of June which was 146 %
greater when exposed to 109 extract.
Comet assay
Organic extracts of water from Meiliang Bay caused concentration-dependent damage to DNA of E. gracilis. The
magnitude of damage was directly proportional to exposure
concentration and statistically significant, regardless of
whether the endpoint monitored was Olive tail moment
(OTM) or tail moment (TM). The response increased
gradually with increasing concentration of organic extract
among all seasons except for the 19 exposure in the March
(Fig. 3). When OTM was used as the metric (Fig. 3a), in
Unicellular algae are ideal for the study of responses to
different environmental factors and free from complications inherent in research with more complex higher plants
(McCormick and Cairns 1994). Furthermore, assays using
microalgae are generally sensitive, rapid and a cost-effective means to evaluate changes in environmental conditions, especially with respect to water pollution (SosakSwiderska et al. 1998) and provide a potentially useful
early warning signal of deteriorating conditions and their
possible causes. Previous studies have shown that most
microalgae display relatively higher sensitivity to changes
in environmental conditions, especially with respect to
water pollution. For these reasons, algae are being widely
used as ecological indicators and phyto-remediation
organisms in polluted aquatic environments (Kelly et al.
1998).
Inhibition of photosynthesis and concomitant changes in
concentrations of photosynthetic pigments has been suggested as the most integrative measure of effects of pollutants on microalgae (Franqueira et al. 2000). In the
present study, organic pollutants caused significant
increases in concentrations of Chl a, Chl b and carotenoid
when E. gracilis was exposed to lesser concentrations of
organic extract, especially in June. However, exposure to
greater concentrations of the organic extracts resulted in
lesser concentrations of these pigments, which suggests a
hormetic effect. Although the reason for the observed
hormesis is unknown, the lesser concentrations of organic
extract stimulated accumulation of chlorophyll, and it is
123
638
consistent with other results where the exposures to lesser
concentrations of chemical stressors have caused results in
stimulation (Stebbing 1982). This effect has been suggested to be due to adaptive responses of organisms to
stressors (Giesy 2001). At greater concentrations the
organisms can no longer respond to the stress and damage
accumulates (Giesy et al. 1988; Giesy and Graney 1989).
The lesser content of chlorophyll observed when E. gracilis
was exposed to greater concentrations could be due to
peroxidation of lipid in membrane of chloroplasts. Destabilization of chloroplast membranes is attributed to
increased peroxidation of the membranes via increased
production of free radicals (Aust et al. 1985). In microalga
cells, formation and accumulation of free radicals can
result in membrane lipid peroxidation leading to structural
alterations in chloroplast and decreased chlorophyll contents, which can hinder growth of cells. Chl a was more
sensitive to the effects of organic pollutants than was Chl b,
and carotenoids, and these results corroborate well with
those reported by Couderchet and Vernet (2003).
Cells have evolved efficient strategies to counteract
oxidative stress. Antioxidant enzymes are one of the
components of the systems that prevent oxidative stress in
plants, with activity of one or more of these enzymes are
generally greater when exposed to stressful conditions and
the elevated activities correlate to increased stress tolerance
(Allen 1995; Mazhoudi et al. 1997). Enzymatic and nonenzymatic reactions of free radical scavengers minimize
the cellular oxidations. The enzymes SOD, CAT and POD
can control cellular levels of reactive oxygen species
(ROS) (Weckx and Clijesters 1996). All aerobic organisms
possess the means to protect themselves from the toxic
effects of reduced oxygen species. The significant changes
in SOD and POD activities observed in this study are
consistent with the micro-alga having been under oxidative
stress as the result of exposure to chemicals in the organic
extract. Different micoralga species exhibit different
responses of antioxidant among seasons (Butow et al.
1997). In the case of the green alga Monostroma aff.
arcticum, there is significantly greater SOD activity
throughout summer (Aguilera et al. 2002). In the current
study, changes in SOD of E. gracilis in response to exposure to organic extracts of water were more sensitive in
June than in other seasons, while POD activity was less in
June and greater in September.
Genotoxicity has also been measured directly in evaluated drinking water for environmental biomonitoring or
clinical applications (Singh et al. 1988; Tice et al. 2000).
The comet assay, is now widely used to measure DNA
damage, during biological surveillance, and genotoxicity
evaluation for different kinds of field studies, and it is
becoming a primary tool for pollutant biomonitoring in the
aquatic environment as well (Mitchelmore and Chipman
123
M. Li et al.
1998; Klobuear et al. 2003; Akcha et al. 2008). Eukaryotic
cells are generally used in the comet assay and thus few
reports describe the applicability of microorganisms. The
comet assay has been used to determine damage to DNA in
the algae, Chlamydomonas reinhadtii (Erbes et al. 1997)
and E. gracilis (Watanabe and Suzuki 2002; Aoyama et al.
2003). Therefore, due to its ease of culturing, standardization of inoculum, availability in micro-alga, and its wide
distribution in aquatic ecosystems E. gracilis was used as
an ecological indicator to detect genotoxicity of organic
extracts in the drinking water source. The results of the
comet assay showed that the organic extracts of water
collected during all four seasons could induce DNA damage to the micro-alga E. gracilis, which might affect longterm survival. Damage to DNA in June and September was
greater than that in March and December, when relatively
little damage was observed regardless of whether the
endpoint monitored was OTM or TM. While the algae cells
with fast growth, rapid reproduction and metabolism
activity in June and September, which is relatively sensitive, were easy to be damaged. The other reason may be
due to the greater concentrations of BTEX in June and
September, which could produce oxidative DNA damage
in human lymphocytes (Chen et al. 2008). However,
compared with other assays, the micro-alga E. gracilis is
more sensitive than human peripheral blood (Wu et al.
2008), mouse spermatid cells (Chen et al. 2007) and zebra
fish embryos (Chen et al. 2007) and it can be used as water
environment monitoring and evaluation.
Conclusions
In this study, organic extracts of water used for drinking
water, collected in all four seasons from Taihu Lake could
affected activities of antioxidant enzymes (SOD and POD)
and induce DNA damage in E. gracilis. The results indicated that the comet assay of E. gracilis could be a biomarker for environmental monitoring and early warning,
especially for evaluating mutagenic/carcinogenic changes
of individuals exposed in organic pollutants, and microalga
E. gracilis could be used as a bioindicator for testing
genotoxic potentials of organic pollutants in Taihu Lake.
Acknowledgments This work was supported by National Major
Project of Science & Technology Ministry of China (2012ZX07501003), National Basic Research Program of China (973 Program, No.
2008CB418003), National Natural Science Foundation of China (Nos.
21077052, 31072217) and Science and Technology Support Program
of Jiangsu Province (BE2012737). The authors wish to acknowledge
the support of an instrumentation grant from the Canada Foundation
for Infrastructure. Prof. Giesy was supported by the program of 2012
‘‘High Level Foreign Experts’’ (#GDW20123200120) funded by the
State Administration of Foreign Experts Affairs, the P.R. China to
Nanjing University and the Einstein Professor Program of the Chinese
Microalga Euglena as a bioindicator
Academy of Sciences. He was also supported by the Canada Research
Chair program, a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine
Pollution, City University of Hong Kong.
Conflict of interest
of interest.
>
The authors declare that they have no conflict
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