ISSN 0963-9292
Ecotoxicology
DOI 10.1007/s10646-014-1214-x
1 23

1 23

Author's personal copy
Ecotoxicology
DOI 10.1007/s10646-014-1214-x
Mei Li • Xiangyu Gao • Bing Wu • Xin Qian •
John P. Giesy • Yibin Cui
Accepted: 16 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 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 gracilis
Introduction
Comet assay
DNA damage
Drinking water
Bioassay
Euglena
Microalgae
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
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 concen-
trations 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
123

Author's personal copy
M. Li et al.
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 multi-
cellular 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 Ha¨der
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 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
;
Sviezˇena´ 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 ° 28
0
20
00
N,
120 ° 13
0
29
00
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 XAD-
2 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

Author's personal copy
Microalga Euglena as a bioindicator extract included equivalents of organic residues that were
1.0
9 , 5.0
9 , and 10 9 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 10
5 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 l mol photon
(m
2 s)
1 irradiance for a period of 6 d.
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 10
6 cells.
Anti-oxidative enzymes assay
Table 1 Content of semi-volatile organic compounds in Meiliang
Bay of Taihu Lake in all seasons ( l g l
1
)
Compounds March June September December
PAHs
PAEs
BETXs
Others
All
0.558
16.067
0.540
0.294
17.459
0.300
7.840
1.200
1.800
11.140
0.735
9.608
0.686
0.045
11.147
0.399
4.428
0.606
0.162
5.675
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.
Activities of enzymes were determined in E. gracili that had been cultured for 6 d and centrifuged at 3,000 9 g 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 12,000 9 g 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 10
6 cells. POD (EC
1.11.1.7) activity was determined by use of the methods
described by Li ( 2000
).
Comet assay
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.
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
Results
Concentrations of SVOCs
Total concentrations of semi-volatile organic chemicals
(SVOCs) in Meiliang Bay, Taihu Lake were 17.459,
11.140, 11.147 and 5.675
l g 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.
Pigments bioassay
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 10
6 cells, respectively.
The concentration of Chl a exposed to the 10 9 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.
1 a).
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 5 9 or 10 9 concentrations of organic extract relative to that of controls
(Fig.
1
b). In June, Chl b content in cells exposed to 1 9 and
123

a
0.05
0.04
0.03
0.02
0.01
0.00
Mar.
Jun.
Sep.
Dec.
**
** b 0.015
CK
Mar.
Jun.
Sep.
Dec.
DMSO 1
Treatment
*
5
***
0.010
*
*
*
10
***
0.005
*
*
*
*
Author's personal copy
M. Li et al.
*** a
40
30
20
10
Mar.
Jun.
Sep.
Dec.
**
**
* b
0
CK DMSO 1
Treatment
5
20
Mar.
Jun.
Sep.
Dec.
10
* ***
10
***
*
***
***
***
0.000
c
0.020
CK DMSO 1
Treatment
5
Mar.
Jun.
Sep.
Dec.
*
10
0
CK DMSO 1
Treatment
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
0.015
*
0.010
0.005
*
0.000
CK DMSO 1
Treatment
5 10
Fig. 1 Concentrations of chlorophylla ( 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
10 9 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 5 9 extract was significantly less
(8 %) than that of the control. In December, Chl b content in cells exposed to 10 9 organic extract was greater (33 %) than that of the control.
123
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 1 9 organic extract (Fig.
1 c).
Antioxidative enzymes assay
There were no statistically significant effects of the organic extracts on SOD activity (Fig.
2 a) except in June, when
SOD activity was 17, 21 % ( P \ 0.01) and 130 %
( P \ 0.001) greater than controls in cells treated with 1 9 ,
5 9 , or 10 9 concentrations of organic extracts, respectively.
In March, exposure of E. gracilis to 1 9 or 5 9 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 10 9 extract. In September, all three concentrations of organic extract caused

Author's personal copy
Microalga Euglena as a bioindicator a
60
50
Mar.
Jun.
Sep.
Dec.
*
*
*
40
***
30
20
***
*
***
*
***
***
***
10
0
CK DMSO 1
Treatment
5 10 b
80
70
30
20
10
0
60
50
40
Mar.
Jun.
Sep.
Dec.
*
***
*
***
*
***
***
***
*
***
***
CK DMSO 1
Treatment
5 10
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
March exposure to 5 9 or 10 9 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 1 9 , 5 9 or 10 9 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.
3
b), the amount of DNA damage in
March was 122 and 238 % ( P \ 0.001) greater than that of the control cells when exposed to 5 9 or 10 9 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 1 9 , 5 9 , or 10 9 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.
Discussion statistically significant differences ( P \ 0.001), between
POD activity of cells exposed to 1 9 , 5 9 , and 10 9 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 10 9 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 1 9 exposure in the March
(Fig.
3 ). When OTM was used as the metric (Fig.
3
a), 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 (Sosak-
Swiderska 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

Author's personal copy
M. Li et al.
consistent with other results where the exposures to lesser concentrations of chemical stressors have caused results in stimulation (Stebbing
1982 ). This effect has been sug-
gested 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
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 (2012ZX07501-
003), 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

Author's personal copy
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 The authors declare that they have no conflict
References
Aguilera J, Bischof K, Karsten U, Hanelt D, Wiencke C (2002)
Seasonal variation in ecophysiological patterns in macroalgae from an Arctic fjord. II. Pigment accumulation and biochemical defence systems against high light stress. Mar Biol 140:
1087–1095
Ahmed H, Ha¨der DP (2010) A fast algal bioassay for assessment of copper toxicity in water using Euglena gracilis . J Appl Phycol
22:785–792
Akcha F, Arzul G, Rousseau S, Bardouil M (2008) Comet assay in phytoplankton as biomarker of genotoxic effects of environmental pollution. Mar Environ Res 6:59–61
Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol 107:1049–1054
Aoyama K, Iwahori K, Miyata N (2003) Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair. Mutat Res 538:155–162
Aust SD, Marehouse LA, Thomas CE (1985) Role of metals in oxygen radical reactions. J Free Radic Biol Med 1:3–25
Bian YM, Kang ZQ (1994) Systematic analysis for the trace nonvolatile organic compounds in drinking water. J Chin Mass
Spectrom Soc 15:1–8
Butow BJ, Wynne D, Tel-Or E (1997) Antioxidative protection of
Peridinium gatunense in Lake Kinneret: seasonal and daily variation. J Phycol 33:780–786
Checcucci A, Colombetti G, Ferrara R, Lenci F (1976) Action spectra for photo accumulation of green and colorless Euglena : evidence for identification of receptor pigments. Photochem Photobiol
23:51–54
Chen YG, Chen HG, Wu YL, Li ZL, Sun LW, Qu MM (2007)
Toxicity evaluation of Meiliang Bay, Lake Taihu, China drinking water source. Hydrobiologia 581:297–303
Chen CS, Hseu YC, Liang SH, Kuo JY, Chen SC (2008) Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay. Hazard Mater 153:351–356
Collins AR, Dusinska M, Ma AG, Fleming I (1995) The comet assay modified to detect DNA-base oxidation and repair of DNAdamage in cellular and subcellular systems. J Cell Biochem
21A:347
Couderchet M, Vernet G (2003) Pigments as biomarkers of exposure to the vineyard herbicide flazasulfuron in freshwater algae.
Ecotoxicol Environ Saf 55:271–277
Danilov R, Ekelund N (2000) Applicability of growth rate, cell shape, and motility of Euglena gracilis as physiological parameters for bioassessment at lower concentrations of toxic substances: an experimental approach. Environ Toxicol 16:78–83
Danilov RA, Ekelund NGA (2001) Responses of photosynthetic efficiency, cell shape and motility in Euglena gracilis (Euglenophyceae) to short-term exposure to heavymetals and pentachlorophenol. Water Air Soil Pollut 132:61–73
De Coen WM, Janssen CR, Giesy JP (2000) Biomarker applications in ecotoxicology: bridging the gap between toxicology and ecology, new microbiotests for routine toxicity screening and biomonitoring. In: Persoone G, Jenssen C, De Coen W (eds)
New microbiotests for routine toxicity screening and biomonitoring klewer.
Academic/Plenum Publishers, New York, pp 13–25
Desai SR, Verlecar XN, Nagarajappa, Goswami U (2006) Genotxicity of cadmium in marine diatiom Chaetoceros tenuissimus using the alkaline comet assay. Ecotoxicology 15:359–363
Djomo JE, Dauta A, Ferrier V, Narbonne JF, Monkiedje A, Njine T,
Garrigues P (2004) Toxic effects of some major polyaromatic hydrocarbons found in crude oil and aquatic sediments on
Scenedesmus subspicatus . Water Res 38:1817–1821
Einicker-Lamas M, Mezian GA, Fernandes TB, Silva FLS, Guerra F,
Miranda K, Attias M, Oliveira MM (2002) Euglena gracilis as a model for the study of Cu
2 ?
and Zn
2 ?
toxicity and accumulation in eukaryotic cells. Environ Pollut 120:779–786
Erbes M, Wessler A, Obst U (1997) Wild a detection of primary DNA damage in Chlamidomonas reinrardtii by means of modified microgel electrophoresis. Environ Mol Mutagen 30:448–458
Franqueira D, Orosa M, Torres E, Herrero C, Cid A (2000) Potential use of flow cytometry in toxicity studies with microalgae. Sci
Total Environ 247:119–126
Fu GX, Xiao LZ, Jian T (2009) Determination of microcystin-LR in water from Lake Tai, China. Bull Environ Contam Toxicol
82:230–233
Gao XY, Shi XR, Cui YB, Li M, Zhang RF, Qian X, Jiang Y (2011)
Organic pollutants and ambient severity for the drinking water source of western Taihu Lake. Ecotoxicology 20:959–967
Giesy JP (2001) Hormesis—does it have relevance at the population, community or ecosystem levels of organization? Hum Exp
Toxicol 20:517–520
Giesy JP, Graney RL (1989) Recent developments in and intercomparisons of acute and chronic bioassays. Hydrobiologia 188:21–60
Giesy JP, Versteeg DJ, Graney RL (1988) A review of selected clinical indicators of stress-induced changes in aquatic organisms. In: Evans MS (ed) Toxic contaminants and ecosystem health: a great lakes focus. Wiley, New York, pp 169–200
Haruhiko N, Yuko H, Masahiro K (2005) Concentrations and compositions of organochlorine contaminants in sediments, soils, crustaceans, fishes and birds collected from Lake Tai,
Hangzhou Bay and Shanghai city region, China. Environ
Pollution 133:415–429
Hecker M, Giesy JP (2011) Effect-directed analysis of Ah-receptor mediated toxicants, mutagens and endocrine disruptors in sediments and biota. In: Brack W (ed) Effect-directed analysis of complex environmental contamination, 15 vol of the handbook of environmental chemistry. Springer, Berlin, pp 285–313
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equation for determining chlorophyll a , b , c
1
Pflanz 167:194–204 and c
2
. Biochem Physiol
Katsoyiannis A, Samara C (2005) Persistent organic pollutants
(POPs) in the conventional activated sludge treatment process: fate and mass balance. Environ Res 97:245–257
Kelly MG, Cazaubon A, Coring E (1998) Recommendations for the routine sampling of diatoms for water quality assessments in
Europe. J Appl Phycol 10:215–224
Klobuear GLV, Pavlica M, Erben R, Pape D (2003) Application of the micronucleus and comet assays to mussel Dreissena polymorpha haemocytes for genotoxicity monitoring of freshwater environments. Aquat Toxicol 64:15–23
Kohler A, Arndt U (1992) Bioindikatoren fu¨r Umweltbelastungen
Neue Aspekte und Entwicklungen. Hohenheimer Umwelttagung
24:251
Li HS (2000) Principles and techniques of plant physiological biochemical experiment. Beijing Higher Education Press, China, pp 134–137 (in Chinese)
123

Author's personal copy
M. Li et al.
Li M, Hu C, Gao X, Xu Y, Qian X, Brown MT, Cui Y (2009)
Genotoxicity of organic pollutants in source of drinking water on microalga Euglena gracilis . Ecotoxicology 18:669–676
Lu G, Yan Z, Wang Y, Chen W (2011) Assessment of estrogenic contamination and biological effects in Lake Taihu. Ecotoxicology 20:974–981
Lu G, Yang X, Li Z, Zhao H, Wang C (2013) Contamination by metals and pharmaceuticals in northern Taihu Lake (China) and its relation to integrated biomarker response in fish. Ecotoxicology 22:50–59
Mazhoudi S, Chaoui A, Ghorbal MH, Ferjani EE (1997) Response of antioxidant enzymes to excess copper in tomato ( Lycopersicon esculentum , Mill). Plant Sci 127:129–137
McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biochem 244:
6049–6055
McCormick PV, Cairns JJ (1994) Algae as indicators of environmental change. J Appl Phycol 6:509–526
Mitchelmore CL, Chipman JK (1998) DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutat Res 399:135–147
Mitter R (2002) Oxidatve stress, antioxidants and stress tolerance.
Trends Plant Sci 7:405–410
Ohe T, Watanabe T, Wakabayashi K (2004) Mutagens in surface waters: a review. Mutat Res 567:109–149
Pellacani C, Buschini A, Furlini M, Poli P, Rossi C (2006) A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters. Aquat Toxicol 77:1–10
Qin BQ, Zhu GW, Gao GA (2010) Drinking water crisis in Lake
Taihu, China: linkage to climatic variability and lake management. Environ Manage 45:105–112
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191
Snyder SA, Snyder EM, Villeneuve D, Kannan K, Aillalobos A,
Blankenship AL, Giesy JP (2000) Instrumental and bioanalytical measures of endocrine disruptors in water. In: Keith LH, Jones-
Lepp TL, Needham Larry L (eds) Analysis of environmental endocrine disruptors: ACS symposium series 747. American
Chemical Society, Washington, DC, pp 73–95
Sosak-Swiderska B, Tyrawska D, Maslikowska B (1998) Microalgal ecotoxicity test with 3,4-dichloroaniline.
Chemosphere
37:2975–2982
Stebbing ARD (1982) Hormesis—the stimulation of growth by low levels of inhibitors. Sci Total Environ 22:213–234
Strickland JD, Parsons TR (1972) A practical handbook of seawater analysis. Bull Fish Res Board Can 167:201–203
Sviezˇena´ B, Ga´lova´ E, Kusenda B, Slaninova´ M, Vlcˇek D, Dusˇinska´
M (2004) The comet assay and troubles with its application in the green alga Chlamidomonas reinhardtii . Czech Phycol
Olomouc 4:163–174
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A,
Kobayashi H (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol
Mutagen 35:206–221
Turgeon S, Rodriguez MJ, Thriault LP (2004) Perception of drinking water in the Quebec City region (Canada): the influence of water quality and consumer location in the distribution system.
J Environ Manage 70:363–373
Watanabe M, Suzuki T (2002) Involvement of reactive oxygen stress in cadmium-induced cellular damage in Euglena gracilis . Comp
Biochem Physiol C 131:491–500
Weckx J, Clijesters H (1996) Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper. Physiol
Plantarum 96:506–512
Wu YL, Chen HG, Li ZL, Sun LW (2008) Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay. J Environ Sci 20:487–491
Zhu G, Chi QQ, Qin BQ (2005) Heavy-metal contents in suspended solids of Meiliang Bay, Taihu Lake and its environmental significances. J Environ Sci 17:672–675
Zhu JP, Susan P, Yong LF (2006) Phthalate esters in human milk: concentration variations over a 6-month postpartum time.
Environ Sci Technol 40:5276–5281
123