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Evaluation of the toxicity of herbicide topramezone to Chlorella vulgaris: Oxidative stress, cell morphology and photosynthetic activity
Ecotoxicology and Environmental Safety 143 (2017) 129–135
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Evaluation of the toxicity of herbicide topramezone to Chlorella vulgaris: Oxidative stress, cell morphology and photosynthetic activity ⁎
MARK
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Fangfang Zhaoa,b, , Qingqing Xianga,b, Ying Zhoua,b, , Xiao Xua,b, Xinyi Qiuc, Yi Yua,b, Farooq Ahmadd a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou, China c Albert College, 160 Dundas Street West Belleville, Ontario, China d State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Topramezone Flow cytometry Oxidative stress Cell viability Photosynthesis
Topramezone is a new, highly selective herbicide of pyrazole structure for the post-emergence control of broadleaf and grass weeds in corn. In this study, the effects of topramezone on C. vulgaris, especially in relation to the cell growth, oxidative stress, cell morphology and photosynthetic activity were assessed. Results showed that topramezone treatment was detrimental to C. vulgaris growth during the 24–96 h of exposure. The changes in cells pigments content and relative transcript of photosynthesis-related genes, which implies that topramezone disrupted the photosynthetic system. Moreover, topramezone induced membrane permeability in a significant proportion of cells with a maximum damage rate of 40.40%, and morphology of cells was more complicated than the control group. TEM images also revealed that topramezone compromised the integrity of the cells. The data corroborated topramezone induced ROS triggered oxidative stress, leading to an increase of MDA. These results suggested that topramezone could have significant effects on growth and physiological functions in algae species, and we supposed that this herbicide affected all of these parameters and the observed effects can be explained by the generation of oxidative stress. This research helps to understand how topramezone affects C. vulgaris and provides a scientific basis for applications of topramezone in aquatic environment.
1. Introduction Topramezone, [3-(4,5-dihydro-1,2-oxazol-3-yl)-4-mesyl-o-tolyl] (5hydroxy-1-methylpyrazol-4-yl) methanone, is a new, highly selective herbicide of pyrazole structure for the post-emergence control of a wide spectrum of broadleaf weeds and annual grass in corn and wheat, and it has been commercially introduced in 2006 (Grossmann and Ehrhardt, 2007; Schonhammer et al., 2006; Porter et al., 2005). Its efficacy is the result of inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) in target plants, leading to chlorophyll loss and necrosis in the growing shoot tissues (Grossmann and Ehrhardt, 2007). Particularly in their function as protectors of the photosystems against photooxidation, the loss of pigments leads to oxidative degradation of chlorophyll and photosynthetic membranes in growing shoot tissues. Consequently, chloroplast synthesis and function are disturbed (Bo ̈ger and Sandmann, 1998). Surface runoff, direct overspray or drift during herbicide application can result in significant quantities of herbicide entering the aquatic
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environments (Solomon and Thompson, 2003). Occurrence of herbicide in the aquatic environment has become a matter of ecological concern. These substances are not routinely monitored because they are often not included in the environmental legislation and the environmental fate is poorly understood or not studied at all (Zenker et al., 2014). Topramezone is an inhibitor of 4-HPPD which results in elevated serum tyrosine levels. As consequence of these elevated tyrosine levels, histopathological evaluations showed that the thyroid (follicular cell hyperplasia) in rats and dogs, pancreas (diffuse degeneration) in rats, liver (hepatocellular hypertrophy and focal necrosis) in rats and mice, and eyes (chronic keratitis) in rats had dose-dependent increases of the adverse effects (http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs +hsdb:@term+@DOCNO+7500). Hence, topramezone poses a potential risk to human health and the environment. Although topramezone has been in use for several years, we are unaware of any studies that have been reported in the literature as a result of overwater use of topramezone exposure to aquatic organisms. Moreover, topramezone is currently registered for use on corn in China.
Corresponding authors at: College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China. E-mail addresses: zjhz_zff@126.com (F. Zhao), [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.ecoenv.2017.05.022 Received 8 February 2017; Received in revised form 8 May 2017; Accepted 11 May 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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following determinations of the present study. Inhibition of algal growth was monitored as an index to evaluate the toxicity of the material, and EC50 value for growth was calculated, based on growth rate data, using the computer program CompuSyn (Chou and Martin, 2005). Three replicates were performed for each test. When the effects of the herbicide on growth, ultrastructure and gene expression were evaluated, triplicate cultures were prepared for each concentration of topramezone and samples were taken after 24, 48, 72 and 96 h for enzyme and RNA extraction.
Drift and runoff has been identified as the primary routes leading to topramezone residues entering aquatic ecosystems, topramezone residues may also be present in irrigation water and may be phytotoxic to irrigated non-target plants (Li et al., 2011). Thus, with the unprecedented scale of the current use of topramezone-based herbicides, combined with the development of new methods and tools in environmental toxicology, it has become necessary to a supplement to the safety and environmental impacts of this class of pesticides. Algae species are the primary producers in the aquatic ecosystems and form the base of the aquatic food chain, and widely used in research on the response of aquatic ecosystems to chemicals, mainly due to their characteristics of small size, rapid propagation, and sensitivity to toxicants (Oukarroum et al., 2012). Chlorella vulgaris (C. vulgaris) is a unicellular eukaryotic autotrophic organism (primary producer). It is one of the major producers of biomass, oxygen, biofuel feedstock, natural vitamin B12 source globally (Sadiq et al., 2011; Santos et al., 2015). C. vulgaris also act as a useful test subject in the toxicity studies of water-soluble substances, metals, and other pollutants (Qian et al., 2009a, 2009b; Rashkov et al., 2012). To date, however, the effects of topramezone, as a common herbicide, on C. vulgaris, are yet to be understood. For this study, a traditional spectrophotometric method was used to assess the effects of the herbicide topramezone on pigment content of C. vulgaris. To better understand the mechanisms of topramezone as the herbicide, we also assessed the effects on physiological level by measuring ROS production and lipid peroxidation (MDA), subcellular level by observing cell ultra structure and molecular level by analyzing photosynthesis-related gene expression in the algae C. vulgaris, which was regarded as a kind of model freshwater algae, and its genetic information was relative abundant (Qian et al., 2009a, 2009b). Additionally, flow cytometry was used to characterize the response of C. vulgaris cells to topramezone about cell complexity and cell viability, in order to improve understanding of the effects and toxicity mechanisms of this herbicide. This research helps to understand how topramezone affects C. vulgaris, which is to present the current exposure data of herbicide topramezone in the aquatic environment and to critically evaluate our current understanding of its effects in aquatic organisms by employing unicellular micro algae C. vulgaris.
2.2. Photosynthetic pigment content The pigments were extracted from the concentrated algal samples in an aqueous solution of acetone (90%, v/v) after exposure to topramezone for 24, 48, 72 and 96 h (Hu et al., 2015), and chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids content were analyzed by using a UNICO 2802 S spectrophotometer at appropriate wavelengths (664, 647 and 480 nm). The resulting absorbance measurements were translated to chlorophylls and carotenoids according to Jeffrey and Humphrey (1975) and Strickland and Parsons (1972), respectively. The equations used to calculate the pigment concentrations in the extract are:
Chlorophyll a = 11.93 A664 − 1.93 A647 Chlorophyll b = 20.36 A647 − 5.50 A664 Carotenoids = 4.0 A480 where total chlorophylls a and b and carotenoids represent the pigment concentrations of extract in μg mL−1, and A664, A647 and A480 represent the absorbances measured at 664, 647 and 480 nm, respectively. 2.3. Real-time PCR analysis RNA was isolated from control and topramezone exposed (20 mg L−1) cells after 96 h，reverse transcript and real-time PCR analysis for three target photosynthesis-related genes (psaB, psbC and rbcL) were carried out according to Qian et al. (2008). 2.4. Ultrastructure of chlorella vulgaris
2. Materials and methods The ultrastructure of chlorella vulgaris, treated and untreated with topramezone, was observed by TEM (H-7650; Hitachi, Japan). After exposure for 96 h, C.vulgaris, treated or untreated with 20 mg L−1, was collected by centrifugation, followed by pre and post fixation, dehydration, infiltration, embedding, and ultrathin sectioning with LEICA EM UC7 ultratome, then stained with uranyl acetate and alkaline lead acetate for TEM analysis (Zhou et al., 2014).
2.1. Culture conditions and algal inhibition C. vulgaris was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in BG-11 medium, under controlled conditions: 24 ± 1 °C in an incubator under illumination at 4500 lx with daily cycles of 12:12 h light: dark cycle. To ensure optimum growth, the cultures were shaken five times per day during incubation. The cell density of cultures was monitored spectrophotometrically at 685 nm (OD685, optical density at 685 nm) every 24 h by use of double beam UV/Vis spectrophotometer (UNICO 2802S) as well as with hemocytometer for avoiding the possible interference coming from UV/vis spectrophotometer. The regression equation for the relationship between cell density (y×1.0×106 cells mL−1) and absorption at 685 nm (x) was calculated as y=24.93x-0.2033 (p < 0.01, R2=0.98). Topramezone (BASF, product number: LS20100068, MW: 363.39, purity: 98%) fresh stock solutions were prepared by dissolving the pure compound in DMSO (purity: 99.0%). Also control cultures were included, to which only DMSO was added. C. vulgaris was cultured in the BG 11 medium at Exponential growth phase algal cells were cultured in 150 mL culture medium in the presence or absence of the topramezone in 250 mL Erlenmeyer flasks with initial cell density of 1.0×106 cells mL−1 for 96 h. A growth inhibition test for C. vulgaris using topramezone concentrations ranged from 0 to 45 mg L−1, was carried out to determine the herbicide concentration used for the
2.5. Perturbations in algal biochemistry 2.5.1. Assays for ROS Excess ROS can damage the cells (Grant and Loake, 2000) such as excessive ROS can cause membrane lipid peroxidation, thereby damaging the cell membrane. ROS production was measured by cellular conversion of non fluorescent 2’7’-dichlorofluorescin diacetate (DCFDA) to the higher conversion of fluorescent compound dichlorofluorescein (DCF) as described by Wang and Joseph (1999). The DCF-DA was used at a final concentration of 10 μM and was incubated with suspended cells for 30 min at room temperature. Then the cells were immediately washed three times with 0.1 M Phosphate Buffer Solution (PBS), and suspended in 500 μL 0.1 M PBS. Fluorescence was observed by using a microplate reader (Molecular Devices SpectraMax, M2) with excitation wavelength at 488 nm and emission wavelength at 525 nm. 2.5.2. Assays for MDA Commercial kits for measurement of Malondialdehyde (MDA) index 130
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was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to manufacturer instructions. The change in biochemistry in response of oxidative stress induced by topramezone was measured.
tion up to 30.1% (25 mg L−1) and 69.5% (45 mg L−1). The EC50 for growth after 96 h value, based on growth rate data was 37.8 mg L−1. The topramezone concentration used in the following analyses after 24, 48, 72 and 96 h of the present study was 20 mg L−1.
2.6. Flow cytometry analyses
3.2. Photosynthetic pigment content and transcriptomic analyses
Flow cytometry (FCM) analyses of C. vulgaris cells were performed on a BD FACSCalibur flow cytometer fitted with 488 nm and 635 nm excitation lasers, detectors of forward (FS) and side (SS) light scatter and four fluorescence detectors corresponding to different wavelength intervals: 515–550 nm (FL1), 564–606 nm (FL2), N 645 nm (FL3) and 645–677 nm (FL4). The 488-nm argon-ion laser was used as excitation source for the probes assayed. Forward scatter (FS, an estimation of cell size) and red fluorescence dot-plots were used to characterize the microalgal population, setting gating levels in order to exclude nonmicroalgal particles (Prado et al., 2011). At least 10,000 gated cells per sample were collected and analyzed using BD CellQuest Pro and FlowJo software. All FCM determinations were performed at least twice and duplicate samples were run on the flow cytometer (Esperanza et al., 2016).
The inhibitory effects of a topramezone (20 mg L−1) on chlorophyll a, chlorophyll b and total carotenoid content in C. vulgaris cells increased after 96 h exposure (Fig. 2A–C). This effect was timedependent, in such a way that C. vulgaris cells exposed to the longer exposure time assayed showed chlorophyll a content partly recovered to 94.2%, 86.1%, 68.8%, and 53.6% respectively with respect to the control. Chlorophyll b content was also affected by the addition of the herbicide, showing a significant reduction with respect to control (p < 0.05) in cultures after exposed to 72 h and 96 h, which was 31.7% and 42.7%. What's more, biomass of cultures assayed showed chlorophyll b content which had almost no decrease after exposure to 48 h with respect to the control (Fig. 2B). The change of carotenoid content in algal cells was indicated in Fig. 2C. The response in carotenoid content when exposed to topramezone was similar to in chlorophyll a content, except that the content of carotenoid at 48 h of treatment group was slightly higher than control group. However, carotenoid content was less affected (decreased respectively 19.5% and 15.2% with respect to control) than chlorophyll a and chlorophyll b by exposing to topramezone after 72 h and 96 h. Fig. 2D showed the effects of topramezone at exposure 96 h on the relative transcript of psaB, psbC and rbcL genes. The abundance of the psaB transcript and psbC transcript were huge affected by the addition of herbicide, showing a significant reduction with respect to control (p < 0.05) in cultures exposed to topramezone concentrations of 20 mg L−1. Following exposure at 96 h, the abundance of the psaB transcript and psbC transcript were decreased to 36.3%, 50.0% of the control, respectively. However, the abundance of the rbcL transcript showed a different response to topramezone which was not significantly different with the control (p > 0.05). Compared with psaB and psbC, the transcript abundance of rbcL assayed, showed a little increase (13.2%) of treatment group without the exception.
2.6.1. Cell complexity Since the forward scattered light (FSC) is correlated with the size or volume of a cell or particle and the sideward scattered light (SSC) is correlated with the intracellular complexity (Shapiro, 1995), aliquots of microalgal cultures were resuspended in phosphate buffered saline solution (PBS, pH 7.4) and analyzed daily by flow cytometry to study the potential alterations in the cell size and intracellular complexity of C. vulgaris cells for each culture up to 24, 48, 72 and 96 h. For each analyzed parameter, data were recorded in a logarithmic scale and results were expressed as mean values obtained from histograms in arbitrary units (a.u). 2.6.2. Cell viability Effects of topramezone on cell viability for C. vulgaris cultures after exposure were detected by means of the method widely used to assess cell viability by FCM, with the fluorochrome propidium iodide (PI). PI is a vital dye that intercalates with double-stranded nucleic acids to produce red fluorescence when excited with blue light. Due to its polarity, this fluorochrome is unable to pass through intact cell membranes. However, when a cell dies and integrity of cell membrane fails, PI is able to enter and stain nucleic acids (Ormerod, 1990). PI penetrates cells with damaged membranes and stains intracellular nucleic acids, producing a bright red fluorescence (620 nm). PI (No. F4170, Sigma-Aldrich, Shanghai, China) was added at a final concentration of 10 μM for 10 min to the cell suspensions (1.0×105 cells mL−1)(Huang et al., 2015).
3.3. Ultrastructure of Algae TEM images revealed the effects of topramezone on the internal structure of C. vulgaris cells. The control group of cell structural was integrity, plasma membrane was close to the cell wall, mitochondria, chloroplast and nucleus were intact (Fig. 3A). The internal structure of algal cells changed after topramezone processing 96 h (Fig. 3B), such as plasmolysis, wrinkling of cell wall, disruption of chloroplast, and looseness of organelles. In some cases (Figs. 3C, 3D), the organelles were blurred, cytoplasm was leaked and plasma membrane was degraded completely. This damaged chloroplasts and membranes in algal cells, thereby severely affected photosynthesis, triggered apoptosis.
2.7. Statistical analysis Experimental data were analyzed by use of the Origin 7.5 and SPSS 17.0 software packages in accordance with the methods provided by the manufacturers of the test kits. Each of the toxicity data sets was compared with its corresponding control followed by ANOVA. The differences were considered statistically significant when p was less than 0.05.
3.4. Cell morphology and membrane integrity Flow cytometric analysis of PI-stained cells were affected by the addition of topramezone to culture medium and the morphological characteristics of C. vulgaris cells were assessed by SSC signals, which provided information regarding cell granularity. Table 1 showed that 20 mg L−1 topramezone affected cell morphology and membrane integrity of C. vulgaris, as indicated by a significant intracellular PI influx and higher SSC signals with respect to control (p < 0.05), respectively. At 24 h, topramezone induced membrane permeability in a significant proportion of C. vulgaris cells with a maximum cell damage rate of 40.40%. Although cells membrane integrity seemed to partially
3. Results 3.1. Algal inhibition Growth inhibition test of C. vulgaris clearly manifested that topramezone treatment induced a significant (p < 0.05) inhibitory effect on the proliferation of C. vulgaris after 96 h (Fig. 1). Growth data showed control cultures indicated significance (p < 0.05) algal growth inhibi131
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Fig. 1. Percentage of growth rate of cultures of C. vulgaris exposed for 96 h to increasing concentrations of topramezone with respect to the control culture (A). Effects of 20 mg L−1 topramezone on the inhibition of C. vulgaris following different exposure times (B). Initial cell density = 1.0×106 cells mL−1 (mean ± SD; n=3).
Fig. 2. Photosynthetic pigment content and transcriptomic, expressed as percentage with respect to control, of C. vulgaris cultures after exposure to 20 mg L−1 topramezone. (A) chlorophyll a, (B) chlorophyll b, (C) carotenoid, (D) psaB, psbC and rbcL genes. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3).
recover following exposure 96 h, it still showed increased membrane permeability of treated cells (14.82%) with respect to control (5.53%). In the meanwhile, complexity of C. vulgaris cells in cultures exposed to topramezone was maintained higher than that observed for control cells.
times higher than the control after 24 h exposure to 20 mg L−1 topramezone, and was significantly increased following longer exposure times (i.e. 48, 72 and 96 h).
3.5. Oxidative stress
Standardised algae growth bioassays remain the preferred technique for assessment of phytotoxic effects in most ecotoxicological studies (Debelius et al., 2008; Nie et al., 2009; Pereira et al., 2009; van Wezel and van Vlaardingen, 2004). In the present study, the results indicated that topramezone had affected growth, physiological and biochemical characteristics in C. vulgaris, including oxidative stress, cell viability
4. Discussion
Fig. 4 showed ROS level increased to 1.83, 2.37, 2.11 and 1.64 times at 24 h, 48 h, 72 h and 96 h, with respect to control (p < 0.05) in 20 mg L−1 topramezone, respectively. The MDA burst at a short exposure time to topramezone，with the maximum level being 1.88 132
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Fig. 3. TEM Photomicrographs of C. vulgaris exposed to topramezone. Untreated (A) and treated (B–D) of 20 mg L−1 topramezone. chloroplast; cw, cell wall; m, mitochondria; n, nucleus; pm, plasma membrane; thy, thylakoids.
45 mg L−1. To determine the acute toxicity of medicines, 96-h EC50 was calculated using growth inhibition as an endpoint for algae exposed. C. vulgaris was more sensitive to herbicide topramezone (EC50 =37.8 mg L−1) than green algae (EC50 =17.2 mg L−1) as reported previously (http://toxnet.nlm.nih.gov/cgibin/sis/search/a?dbs +hsdb:@term+@DOCNO+7500). And the inhibitory effects on C. vulgaris cells growth increased with the increasing topramezone concentration from 5 to 45 mg L−1 (Fig. 1). Spectrophotometric determination of photosynthetic pigment content indicated a significant reduction of chlorophyll content (p < 0.05) in C. vulgaris cells exposed to 20 mg L−1 topramezone assayed, and it was more evident at long exposure time (Figs. 2A, 2B). These results indicated that the ability of algae cells to synthesize chlorophyll was decreased (Bornman and Vogelmann, 1991). It is reported in previous studies that microalgae cells changed their photoautotrophic metabolism under the stress induced by herbicide, and inefficient under these conditions, to a heterotrophic metabolism. (Esperanza et al., 2015;
Table 1 Cell Morphology and Membrane Integrity of C. vulgaris after exposed to topramezone. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3). Exposure time (h)
24 48 72 96
SSC-mean
Permeabilized (%)
Control
Topramezone (20 mg L−1)
Control
Topramezone (20 mg L−1)
145.3(4.73) 127.7(4.51) 140.3(3.21) 137.3(2.08)
178.0(1.73)** 167.3(2.52)** 164.7(2.52)** 166.3(0.57)**
24.4(4.63) 20.2(3.08) 12.4(1.60) 5.5(1.19)
40.4(5.51) 29.4(4.25) 31.6(3.81)* 14.8(1.25)**
and photosynthetic activity. Growth inhibition test of C. vulgaris clearly manifested topramezone was severely toxic and effectively reduced the population by 69.5% at
Fig. 4. Relative expressions of biochemical parameters A and B, Reactive Oxygen Species (A) and Malondialdehyde (B), in C. vulgaris exposure to 20 mg L−1 topramezone. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3).
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5. Conclusion
González-Barreiro et al., 2004). A decrease in carotenoids content of algae cells was also observed, but not at 48 h exposure (Fig. 2C), revealing that carotenoids have a higher tolerance to topramezone action than chlorophylls. In these cultures, we even observed a slight increase in carotenoid content of biomass at 48 h exposure, which could be an indicator of the protective role of these pigments against oxidative stress, since carotenoids are known to be potent quenchers of reactive oxygen species, particularly singlet oxygen (Prado et al., 2011; Ünyayar et al., 2005). The changes of photosynthetic genes transcription by topqwramezone confirmed the damage of photosynthetic system. Compared with the control group, the transcript abundance of psaB and psbC decreased significantly at 96 h exposure, while the transcript abundance of rbcL increased slight. These results manifested that topramezone might interfere with the function of the photosynthetic electron transport chain from PSI to PSII by forming electron donor–acceptor complexes with the naphthoquinone phylloquinone, which functions as a secondary electron acceptor A in PSI of plants and algae (Leonova et al., 2004). As a result, it could influence the growth of algae indirectly (Qian et al., 2009a, 2009b). Thus, photosynthesis inhibition induced by topramezone toxicity would affect the entire physiological state and cell growth process. Previous research suggested that membrane disruption leads to inhibition of photosynthesis and eventually results in the increasing proportion of death (Huang et al., 2015). Flow cytometry, a technique which allows us to study different morphological and physiological properties of single cells, has been introduced as a useful tool in toxicity tests with microalgae (Franqueira et al., 2000). Results showed that topramezone affected C. vulgaris Cell morphology and membrane integrity (Table 1). Compared with the controls, the cells exposed to topramezone responded with either shrinking or swelling of the cell volume (Huang et al., 2015), the community of cells became more complexly (Table 1), it meant these cells grew unevenly and the interior was irregular. Besides, topramezone treatment group exhibited highly increased levels of PI staining, which was consistent with the conclusion that some interaction of chemicals with cyanobacteria alter cell morphology and compromise membrane integrity, causing leakage of cell components (Hong et al., 2008). Actually, chloroplast and mitochondria are the main source of ROS, which can cause cell damage in various ways. For instance, these radicals can affect membrane permeability, cause damage to DNA and proteins, and generate lipid peroxide signaling molecules. The TEM images of this study also revealed the organelles were blurred, cytoplasm was leaked and plasma membrane was degraded (Fig. 3). Topramezone causes damage to chloroplasts and membranes in algal cells. In the present work, these results indicated that topramezone induced algae cells to produce higher ROS levels compared to the control (Fig. 4A). These excessive ROS were not totally cleared by the algal cells and eventually caused cell damages. Qian et al. have pointed out that MDA is a major peroxidation product and is an indicator of lipid peroxidation, which reflects cellular oxidative damage under environmental stress conditions. Cell membranes are made of unsaturated phospholipids and are vulnerable to oxygen radical attack resulting in MDA accumulation (Qian et al., 2009a 2009b, 2008; Hong et al., 2008). In our study, the levels of MDA increased in C. vulgaris cells with the extension of exposure time (Fig. 4B), which indicated that topramezone induced membrane lipid peroxidation and caused oxidative damage on cell membranes. This phenomenon was also observed by Kong et al. (2013) and Qian et al. (2008). To sun up, the results obtained indicate that this herbicide affects all of these parameters and the observed effects can be explained by the generation of oxidative stress.
On the basis of these experimental results we concluded that topramezone effected oxidative stress, cell morphology and photosynthetic activity in C. vulgaris. Topramezone induced ROS generation, as reactive oxygen species have been implicated in the initiation of membrane damage by lipid peroxidation damaged membrane integrity, which in turn influenced the cell volume, and to a cell component degradation which was reflected by an intracellular complexity increase. This results altered physiological states, thereby severely affecting photosynthesis, triggering apoptosis. These results could be beneficial to understanding the interactions between topramezone and algae species in aquatic environments and evidenced the potential risk of topramezone to the aquatic environment. Further studies should be focused on the toxicity of topramezone at length exposure time. Acknowledgments This work was supported by grants from the Project of Science and Technology in Zhejiang Food and Drug Administration (SP201717) and the Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018). References Bo ̈ger, P., Sandmann, G., 1998. Carotenoid biosynthesis inhibitor herbicides-mode of action and resistance mechanism. Pestic. Outlook 9, 29–35. Bornman, J.F., Vogelmann, T.C., 1991. Effect of UV-B radiation on leaf optical-properties measured with fiber optics. J. Exp. Bot. 42, 547–554. http://dx.doi.org/10.1093/jxb/ 42.4.547. Chou, T.C., Martin, N., 2005. CompuSyn for Drug Combinations: PC Software and User's Guide: a Computer Program for Quantification of Synergism and Antagonism in Drug Combinations and the Determination of IC50 and ED50 and LD50 Values. ComboSyn, Inc., Paramus, NJ. Debelius, B., Forja, J.M., Del Vals, A., Lubián, L.M., 2008. Effect of linear alkylbenzene sulfonate (LAS) and atrazine on marine microalgae. Mar. Pollut. Bull. 57, 559–568. http://dx.doi.org/10.1016/j.marpolbul.2008.01.040. Esperanza, M., Seoane, M., Rioboo, C., Herrero, C., Cid, A., 2015. Chlamydomonas reinhardtii cells adjust the metabolism to maintain viability in response to atrazine stress. Aquat. Toxicol. 165, 64–672. http://dx.doi.org/10.1016/j.aquatox.2015.05. 012. Esperanza, M., Seoane, M., Rioboo, C., et al., 2016. Early alterations on photosynthesisrelated parameters in Chlamydomonas reinhardtii cells exposed to atrazine: a multiple approach study. Sci. Total Environ. 554–555, 237–245. http://dx.doi.org/ 10.1016/j.scitotenv.2016.02.175. 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. http://dx.doi.org/10.1016/s0048-9697(99)00483-0. González-Barreiro, O., Rioboo, C., Cid, A., Herrero, C., 2004. Atrazine-induced chlorosis in Synechococcus elongatus cells. Arch. Environ. Contam. Toxicol. 46, 301–307. http://dx.doi.org/10.1007/s00244-003-2149-z. Grant, J.J., Loake, G.J., 2000. Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol. 124 (1), 21–30. http://dx.doi.org/10. 1104/pp.124.1.21. Grossmann, K., Ehrhardt, T., 2007. On the mechanism of action and selectivity of the corn herbicide topramezone: a new inhibitor of 4-hydroxyphenylpyruvate dioxygenase. Pest Manag. Sci. 63 (5), 429–439. http://dx.doi.org/10.1002/ps.1341. Hong, Y., Hu, H.Y., Li, F.M., 2008. Physiological and biochemical effects of allelochemical ethyl 2-methyl acetoacetate (EMA) on cyanobacterium Microcystis aeruginosa. Ecotoxicol. EnvironSafe 71, 527–534. http://dx.doi.org/10.1016/j.ecoenv.2007.10. 010. Hu, X., Yin, P., Zhao, L., Yu, Q., 2015. Characterization of cell viability in Phaeocystis globosa cultures exposed to marine algicidal bacteria. Biotechnol. Bioprocess Eng. 20 (1), 58–66. http://dx.doi.org/10.1007/s12257-014-0437-2. Huang, H., Xiao, X., Ghadouani, A., et al., 2015. Effects of natural flavonoids on photosynthetic activity and cell integrity in Microcystis aeruginosa. Toxins 7 (1), 66–80. http://dx.doi.org/10.3390/toxins7010066. Jeffrey, S.W., Humphrey, G.F., 1975. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167, 191–194. http://dx.doi.org/10.1016/0022-2860(75) 85046-0. Kong, Y., Xu, X.Y., Zhu, L., 2013. Cyanobactericidal effect of Streptomyces sp. HJC-D1 on Microcystis auruginosa. PLoS One 8, e57654. http://dx.doi.org/10.1371/journal. pone.0057654. Leonova, N.M., Zhukova, E.E., Evstigneeva, R.P., 2004. A model of the quinone-binding site of photosystem I: investigation of charge transfer complexes of phylloquinone and its derivatives with tryptophan. Biofizika 49, 970–978. Li, Y., Dong, F., Liu, X., et al., 2011. Miniaturized liquid-liquid extraction coupled with
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ultra-performance liquid chromatography/tandem mass spectrometry for determination of topramezone in soil, corn, wheat, and water. Anal. Bioanal. Chem. 400 (9), 3097–3107. http://dx.doi.org/10.1007/s00216-011-4967-6. Nie, X., Gu, J., Lu, J., Pan, W., Yang, Y., 2009. Effects of norfloxacin and butylated hydroxyanisole on the freshwater microalga Scenedesmus obliquus. Ecotoxicology 18, 677–684. http://dx.doi.org/10.1007/s10646-009-0334-1. Ormerod, M.G., 1990. Analysis of DNA. General methods. In: Ormerod, M.G. (Ed.), Flow Cytometry. A Practical Approach. Oxford University Press, Oxford, pp. 69–87. Oukarroum, A., Bras, B., Perreault, F., Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicol. Environ. Saf. 78, 80–85. http://dx.doi.org/10.1016/j.ecoenv.2011.11. 012. Pereira, J.L., Antunes, S.C., Castro, B.B., Marques, C.R., Gonc ̧ alves, A.M.M., Gonc ̧ alves, F., Pereira, R., 2009. Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulations versus active ingredient. Ecotoxicology 18, 455–463. http://dx.doi.org/10.1007/s10646-009-0300-y. Porter R.M., Vaculin P.D., Orr J.E., Immaraju J.A., O’Neal W.B., 2005. Topramezone: a new active for postemergence weed control in corn. North Central Weed Science Society Proceedings 60, abstract 93. Prado, R., Rioboo, C., Herrero, C., Cid, A., 2011. Characterization of cell response in Chlamydomonas moewusii cultures exposed to the herbicide paraquat: induction of chlorosis. Aquat. Toxicol. 102, 10–17. http://dx.doi.org/10.1016/j.aquatox.2010.12. 013. Qian, H., Chen, W., Sheng, G.D., Xu, X., Liu, W., Fu, Z., 2008. Effects of glufosinate on antioxidant enzymes, subcellular structure, and gene expression in the unicellular green alga Chlorella vulgaris. Aquat. Toxicol. 88, 301–307. http://dx.doi.org/10. 1016/j.aquatox.2008.05.009. Qian, H.F., Xu, X.Y., Chen, W., Jiang, H., Ji, Y.X., Liu, W.P., Fu, Z.W., 2009a. Allelochemical stress causes oxidative damage and inhibition of photosynthesis in Chlorella vulgaris. Chemosphere 75, 368–375. http://dx.doi.org/10.1016/j. chemosphere.2008.12.040. Qian, H.F., Li, J.J., Sun, L.W., Chen, W., Sheng, G.D., Liu, W.P., Fu, Z.W., 2009b. Combined effect of copper and cadmium on Chlorella vulgaris growth and photosynthesis-related gene transcription. Aquat. Toxicol. 94, 56–61. http://dx.doi. org/10.1016/j.aquatox.2009.05.014. Rashkov, G.D., Dobrikova, A.G., Pouneva, I.D., Misra, A.N., Apostolova, E.L., 2012. Sensitivity of Chlorella vulgaris to herbicides. Possibility of using it as biological receptor in biosensors. Sens. Actuators B: Chem. 161, 151–155. http://dx.doi.org/10. 1016/j.snb.2011.09.088.
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Evaluation of the toxicity of herbicide topramezone to Chlorella vulgaris: Oxidative stress, cell morphology and photosynthetic activity ⁎
MARK
⁎
Fangfang Zhaoa,b, , Qingqing Xianga,b, Ying Zhoua,b, , Xiao Xua,b, Xinyi Qiuc, Yi Yua,b, Farooq Ahmadd a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou, China c Albert College, 160 Dundas Street West Belleville, Ontario, China d State Key Laboratory of Metal Matrix Composites, School of Material Science and Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Topramezone Flow cytometry Oxidative stress Cell viability Photosynthesis
Topramezone is a new, highly selective herbicide of pyrazole structure for the post-emergence control of broadleaf and grass weeds in corn. In this study, the effects of topramezone on C. vulgaris, especially in relation to the cell growth, oxidative stress, cell morphology and photosynthetic activity were assessed. Results showed that topramezone treatment was detrimental to C. vulgaris growth during the 24–96 h of exposure. The changes in cells pigments content and relative transcript of photosynthesis-related genes, which implies that topramezone disrupted the photosynthetic system. Moreover, topramezone induced membrane permeability in a significant proportion of cells with a maximum damage rate of 40.40%, and morphology of cells was more complicated than the control group. TEM images also revealed that topramezone compromised the integrity of the cells. The data corroborated topramezone induced ROS triggered oxidative stress, leading to an increase of MDA. These results suggested that topramezone could have significant effects on growth and physiological functions in algae species, and we supposed that this herbicide affected all of these parameters and the observed effects can be explained by the generation of oxidative stress. This research helps to understand how topramezone affects C. vulgaris and provides a scientific basis for applications of topramezone in aquatic environment.
1. Introduction Topramezone, [3-(4,5-dihydro-1,2-oxazol-3-yl)-4-mesyl-o-tolyl] (5hydroxy-1-methylpyrazol-4-yl) methanone, is a new, highly selective herbicide of pyrazole structure for the post-emergence control of a wide spectrum of broadleaf weeds and annual grass in corn and wheat, and it has been commercially introduced in 2006 (Grossmann and Ehrhardt, 2007; Schonhammer et al., 2006; Porter et al., 2005). Its efficacy is the result of inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) in target plants, leading to chlorophyll loss and necrosis in the growing shoot tissues (Grossmann and Ehrhardt, 2007). Particularly in their function as protectors of the photosystems against photooxidation, the loss of pigments leads to oxidative degradation of chlorophyll and photosynthetic membranes in growing shoot tissues. Consequently, chloroplast synthesis and function are disturbed (Bo ̈ger and Sandmann, 1998). Surface runoff, direct overspray or drift during herbicide application can result in significant quantities of herbicide entering the aquatic
⁎
environments (Solomon and Thompson, 2003). Occurrence of herbicide in the aquatic environment has become a matter of ecological concern. These substances are not routinely monitored because they are often not included in the environmental legislation and the environmental fate is poorly understood or not studied at all (Zenker et al., 2014). Topramezone is an inhibitor of 4-HPPD which results in elevated serum tyrosine levels. As consequence of these elevated tyrosine levels, histopathological evaluations showed that the thyroid (follicular cell hyperplasia) in rats and dogs, pancreas (diffuse degeneration) in rats, liver (hepatocellular hypertrophy and focal necrosis) in rats and mice, and eyes (chronic keratitis) in rats had dose-dependent increases of the adverse effects (http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs +hsdb:@term+@DOCNO+7500). Hence, topramezone poses a potential risk to human health and the environment. Although topramezone has been in use for several years, we are unaware of any studies that have been reported in the literature as a result of overwater use of topramezone exposure to aquatic organisms. Moreover, topramezone is currently registered for use on corn in China.
Corresponding authors at: College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China. E-mail addresses: zjhz_zff@126.com (F. Zhao), [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.ecoenv.2017.05.022 Received 8 February 2017; Received in revised form 8 May 2017; Accepted 11 May 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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following determinations of the present study. Inhibition of algal growth was monitored as an index to evaluate the toxicity of the material, and EC50 value for growth was calculated, based on growth rate data, using the computer program CompuSyn (Chou and Martin, 2005). Three replicates were performed for each test. When the effects of the herbicide on growth, ultrastructure and gene expression were evaluated, triplicate cultures were prepared for each concentration of topramezone and samples were taken after 24, 48, 72 and 96 h for enzyme and RNA extraction.
Drift and runoff has been identified as the primary routes leading to topramezone residues entering aquatic ecosystems, topramezone residues may also be present in irrigation water and may be phytotoxic to irrigated non-target plants (Li et al., 2011). Thus, with the unprecedented scale of the current use of topramezone-based herbicides, combined with the development of new methods and tools in environmental toxicology, it has become necessary to a supplement to the safety and environmental impacts of this class of pesticides. Algae species are the primary producers in the aquatic ecosystems and form the base of the aquatic food chain, and widely used in research on the response of aquatic ecosystems to chemicals, mainly due to their characteristics of small size, rapid propagation, and sensitivity to toxicants (Oukarroum et al., 2012). Chlorella vulgaris (C. vulgaris) is a unicellular eukaryotic autotrophic organism (primary producer). It is one of the major producers of biomass, oxygen, biofuel feedstock, natural vitamin B12 source globally (Sadiq et al., 2011; Santos et al., 2015). C. vulgaris also act as a useful test subject in the toxicity studies of water-soluble substances, metals, and other pollutants (Qian et al., 2009a, 2009b; Rashkov et al., 2012). To date, however, the effects of topramezone, as a common herbicide, on C. vulgaris, are yet to be understood. For this study, a traditional spectrophotometric method was used to assess the effects of the herbicide topramezone on pigment content of C. vulgaris. To better understand the mechanisms of topramezone as the herbicide, we also assessed the effects on physiological level by measuring ROS production and lipid peroxidation (MDA), subcellular level by observing cell ultra structure and molecular level by analyzing photosynthesis-related gene expression in the algae C. vulgaris, which was regarded as a kind of model freshwater algae, and its genetic information was relative abundant (Qian et al., 2009a, 2009b). Additionally, flow cytometry was used to characterize the response of C. vulgaris cells to topramezone about cell complexity and cell viability, in order to improve understanding of the effects and toxicity mechanisms of this herbicide. This research helps to understand how topramezone affects C. vulgaris, which is to present the current exposure data of herbicide topramezone in the aquatic environment and to critically evaluate our current understanding of its effects in aquatic organisms by employing unicellular micro algae C. vulgaris.
2.2. Photosynthetic pigment content The pigments were extracted from the concentrated algal samples in an aqueous solution of acetone (90%, v/v) after exposure to topramezone for 24, 48, 72 and 96 h (Hu et al., 2015), and chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids content were analyzed by using a UNICO 2802 S spectrophotometer at appropriate wavelengths (664, 647 and 480 nm). The resulting absorbance measurements were translated to chlorophylls and carotenoids according to Jeffrey and Humphrey (1975) and Strickland and Parsons (1972), respectively. The equations used to calculate the pigment concentrations in the extract are:
Chlorophyll a = 11.93 A664 − 1.93 A647 Chlorophyll b = 20.36 A647 − 5.50 A664 Carotenoids = 4.0 A480 where total chlorophylls a and b and carotenoids represent the pigment concentrations of extract in μg mL−1, and A664, A647 and A480 represent the absorbances measured at 664, 647 and 480 nm, respectively. 2.3. Real-time PCR analysis RNA was isolated from control and topramezone exposed (20 mg L−1) cells after 96 h，reverse transcript and real-time PCR analysis for three target photosynthesis-related genes (psaB, psbC and rbcL) were carried out according to Qian et al. (2008). 2.4. Ultrastructure of chlorella vulgaris
2. Materials and methods The ultrastructure of chlorella vulgaris, treated and untreated with topramezone, was observed by TEM (H-7650; Hitachi, Japan). After exposure for 96 h, C.vulgaris, treated or untreated with 20 mg L−1, was collected by centrifugation, followed by pre and post fixation, dehydration, infiltration, embedding, and ultrathin sectioning with LEICA EM UC7 ultratome, then stained with uranyl acetate and alkaline lead acetate for TEM analysis (Zhou et al., 2014).
2.1. Culture conditions and algal inhibition C. vulgaris was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in BG-11 medium, under controlled conditions: 24 ± 1 °C in an incubator under illumination at 4500 lx with daily cycles of 12:12 h light: dark cycle. To ensure optimum growth, the cultures were shaken five times per day during incubation. The cell density of cultures was monitored spectrophotometrically at 685 nm (OD685, optical density at 685 nm) every 24 h by use of double beam UV/Vis spectrophotometer (UNICO 2802S) as well as with hemocytometer for avoiding the possible interference coming from UV/vis spectrophotometer. The regression equation for the relationship between cell density (y×1.0×106 cells mL−1) and absorption at 685 nm (x) was calculated as y=24.93x-0.2033 (p < 0.01, R2=0.98). Topramezone (BASF, product number: LS20100068, MW: 363.39, purity: 98%) fresh stock solutions were prepared by dissolving the pure compound in DMSO (purity: 99.0%). Also control cultures were included, to which only DMSO was added. C. vulgaris was cultured in the BG 11 medium at Exponential growth phase algal cells were cultured in 150 mL culture medium in the presence or absence of the topramezone in 250 mL Erlenmeyer flasks with initial cell density of 1.0×106 cells mL−1 for 96 h. A growth inhibition test for C. vulgaris using topramezone concentrations ranged from 0 to 45 mg L−1, was carried out to determine the herbicide concentration used for the
2.5. Perturbations in algal biochemistry 2.5.1. Assays for ROS Excess ROS can damage the cells (Grant and Loake, 2000) such as excessive ROS can cause membrane lipid peroxidation, thereby damaging the cell membrane. ROS production was measured by cellular conversion of non fluorescent 2’7’-dichlorofluorescin diacetate (DCFDA) to the higher conversion of fluorescent compound dichlorofluorescein (DCF) as described by Wang and Joseph (1999). The DCF-DA was used at a final concentration of 10 μM and was incubated with suspended cells for 30 min at room temperature. Then the cells were immediately washed three times with 0.1 M Phosphate Buffer Solution (PBS), and suspended in 500 μL 0.1 M PBS. Fluorescence was observed by using a microplate reader (Molecular Devices SpectraMax, M2) with excitation wavelength at 488 nm and emission wavelength at 525 nm. 2.5.2. Assays for MDA Commercial kits for measurement of Malondialdehyde (MDA) index 130
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was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to manufacturer instructions. The change in biochemistry in response of oxidative stress induced by topramezone was measured.
tion up to 30.1% (25 mg L−1) and 69.5% (45 mg L−1). The EC50 for growth after 96 h value, based on growth rate data was 37.8 mg L−1. The topramezone concentration used in the following analyses after 24, 48, 72 and 96 h of the present study was 20 mg L−1.
2.6. Flow cytometry analyses
3.2. Photosynthetic pigment content and transcriptomic analyses
Flow cytometry (FCM) analyses of C. vulgaris cells were performed on a BD FACSCalibur flow cytometer fitted with 488 nm and 635 nm excitation lasers, detectors of forward (FS) and side (SS) light scatter and four fluorescence detectors corresponding to different wavelength intervals: 515–550 nm (FL1), 564–606 nm (FL2), N 645 nm (FL3) and 645–677 nm (FL4). The 488-nm argon-ion laser was used as excitation source for the probes assayed. Forward scatter (FS, an estimation of cell size) and red fluorescence dot-plots were used to characterize the microalgal population, setting gating levels in order to exclude nonmicroalgal particles (Prado et al., 2011). At least 10,000 gated cells per sample were collected and analyzed using BD CellQuest Pro and FlowJo software. All FCM determinations were performed at least twice and duplicate samples were run on the flow cytometer (Esperanza et al., 2016).
The inhibitory effects of a topramezone (20 mg L−1) on chlorophyll a, chlorophyll b and total carotenoid content in C. vulgaris cells increased after 96 h exposure (Fig. 2A–C). This effect was timedependent, in such a way that C. vulgaris cells exposed to the longer exposure time assayed showed chlorophyll a content partly recovered to 94.2%, 86.1%, 68.8%, and 53.6% respectively with respect to the control. Chlorophyll b content was also affected by the addition of the herbicide, showing a significant reduction with respect to control (p < 0.05) in cultures after exposed to 72 h and 96 h, which was 31.7% and 42.7%. What's more, biomass of cultures assayed showed chlorophyll b content which had almost no decrease after exposure to 48 h with respect to the control (Fig. 2B). The change of carotenoid content in algal cells was indicated in Fig. 2C. The response in carotenoid content when exposed to topramezone was similar to in chlorophyll a content, except that the content of carotenoid at 48 h of treatment group was slightly higher than control group. However, carotenoid content was less affected (decreased respectively 19.5% and 15.2% with respect to control) than chlorophyll a and chlorophyll b by exposing to topramezone after 72 h and 96 h. Fig. 2D showed the effects of topramezone at exposure 96 h on the relative transcript of psaB, psbC and rbcL genes. The abundance of the psaB transcript and psbC transcript were huge affected by the addition of herbicide, showing a significant reduction with respect to control (p < 0.05) in cultures exposed to topramezone concentrations of 20 mg L−1. Following exposure at 96 h, the abundance of the psaB transcript and psbC transcript were decreased to 36.3%, 50.0% of the control, respectively. However, the abundance of the rbcL transcript showed a different response to topramezone which was not significantly different with the control (p > 0.05). Compared with psaB and psbC, the transcript abundance of rbcL assayed, showed a little increase (13.2%) of treatment group without the exception.
2.6.1. Cell complexity Since the forward scattered light (FSC) is correlated with the size or volume of a cell or particle and the sideward scattered light (SSC) is correlated with the intracellular complexity (Shapiro, 1995), aliquots of microalgal cultures were resuspended in phosphate buffered saline solution (PBS, pH 7.4) and analyzed daily by flow cytometry to study the potential alterations in the cell size and intracellular complexity of C. vulgaris cells for each culture up to 24, 48, 72 and 96 h. For each analyzed parameter, data were recorded in a logarithmic scale and results were expressed as mean values obtained from histograms in arbitrary units (a.u). 2.6.2. Cell viability Effects of topramezone on cell viability for C. vulgaris cultures after exposure were detected by means of the method widely used to assess cell viability by FCM, with the fluorochrome propidium iodide (PI). PI is a vital dye that intercalates with double-stranded nucleic acids to produce red fluorescence when excited with blue light. Due to its polarity, this fluorochrome is unable to pass through intact cell membranes. However, when a cell dies and integrity of cell membrane fails, PI is able to enter and stain nucleic acids (Ormerod, 1990). PI penetrates cells with damaged membranes and stains intracellular nucleic acids, producing a bright red fluorescence (620 nm). PI (No. F4170, Sigma-Aldrich, Shanghai, China) was added at a final concentration of 10 μM for 10 min to the cell suspensions (1.0×105 cells mL−1)(Huang et al., 2015).
3.3. Ultrastructure of Algae TEM images revealed the effects of topramezone on the internal structure of C. vulgaris cells. The control group of cell structural was integrity, plasma membrane was close to the cell wall, mitochondria, chloroplast and nucleus were intact (Fig. 3A). The internal structure of algal cells changed after topramezone processing 96 h (Fig. 3B), such as plasmolysis, wrinkling of cell wall, disruption of chloroplast, and looseness of organelles. In some cases (Figs. 3C, 3D), the organelles were blurred, cytoplasm was leaked and plasma membrane was degraded completely. This damaged chloroplasts and membranes in algal cells, thereby severely affected photosynthesis, triggered apoptosis.
2.7. Statistical analysis Experimental data were analyzed by use of the Origin 7.5 and SPSS 17.0 software packages in accordance with the methods provided by the manufacturers of the test kits. Each of the toxicity data sets was compared with its corresponding control followed by ANOVA. The differences were considered statistically significant when p was less than 0.05.
3.4. Cell morphology and membrane integrity Flow cytometric analysis of PI-stained cells were affected by the addition of topramezone to culture medium and the morphological characteristics of C. vulgaris cells were assessed by SSC signals, which provided information regarding cell granularity. Table 1 showed that 20 mg L−1 topramezone affected cell morphology and membrane integrity of C. vulgaris, as indicated by a significant intracellular PI influx and higher SSC signals with respect to control (p < 0.05), respectively. At 24 h, topramezone induced membrane permeability in a significant proportion of C. vulgaris cells with a maximum cell damage rate of 40.40%. Although cells membrane integrity seemed to partially
3. Results 3.1. Algal inhibition Growth inhibition test of C. vulgaris clearly manifested that topramezone treatment induced a significant (p < 0.05) inhibitory effect on the proliferation of C. vulgaris after 96 h (Fig. 1). Growth data showed control cultures indicated significance (p < 0.05) algal growth inhibi131
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Fig. 1. Percentage of growth rate of cultures of C. vulgaris exposed for 96 h to increasing concentrations of topramezone with respect to the control culture (A). Effects of 20 mg L−1 topramezone on the inhibition of C. vulgaris following different exposure times (B). Initial cell density = 1.0×106 cells mL−1 (mean ± SD; n=3).
Fig. 2. Photosynthetic pigment content and transcriptomic, expressed as percentage with respect to control, of C. vulgaris cultures after exposure to 20 mg L−1 topramezone. (A) chlorophyll a, (B) chlorophyll b, (C) carotenoid, (D) psaB, psbC and rbcL genes. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3).
recover following exposure 96 h, it still showed increased membrane permeability of treated cells (14.82%) with respect to control (5.53%). In the meanwhile, complexity of C. vulgaris cells in cultures exposed to topramezone was maintained higher than that observed for control cells.
times higher than the control after 24 h exposure to 20 mg L−1 topramezone, and was significantly increased following longer exposure times (i.e. 48, 72 and 96 h).
3.5. Oxidative stress
Standardised algae growth bioassays remain the preferred technique for assessment of phytotoxic effects in most ecotoxicological studies (Debelius et al., 2008; Nie et al., 2009; Pereira et al., 2009; van Wezel and van Vlaardingen, 2004). In the present study, the results indicated that topramezone had affected growth, physiological and biochemical characteristics in C. vulgaris, including oxidative stress, cell viability
4. Discussion
Fig. 4 showed ROS level increased to 1.83, 2.37, 2.11 and 1.64 times at 24 h, 48 h, 72 h and 96 h, with respect to control (p < 0.05) in 20 mg L−1 topramezone, respectively. The MDA burst at a short exposure time to topramezone，with the maximum level being 1.88 132
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Fig. 3. TEM Photomicrographs of C. vulgaris exposed to topramezone. Untreated (A) and treated (B–D) of 20 mg L−1 topramezone. chloroplast; cw, cell wall; m, mitochondria; n, nucleus; pm, plasma membrane; thy, thylakoids.
45 mg L−1. To determine the acute toxicity of medicines, 96-h EC50 was calculated using growth inhibition as an endpoint for algae exposed. C. vulgaris was more sensitive to herbicide topramezone (EC50 =37.8 mg L−1) than green algae (EC50 =17.2 mg L−1) as reported previously (http://toxnet.nlm.nih.gov/cgibin/sis/search/a?dbs +hsdb:@term+@DOCNO+7500). And the inhibitory effects on C. vulgaris cells growth increased with the increasing topramezone concentration from 5 to 45 mg L−1 (Fig. 1). Spectrophotometric determination of photosynthetic pigment content indicated a significant reduction of chlorophyll content (p < 0.05) in C. vulgaris cells exposed to 20 mg L−1 topramezone assayed, and it was more evident at long exposure time (Figs. 2A, 2B). These results indicated that the ability of algae cells to synthesize chlorophyll was decreased (Bornman and Vogelmann, 1991). It is reported in previous studies that microalgae cells changed their photoautotrophic metabolism under the stress induced by herbicide, and inefficient under these conditions, to a heterotrophic metabolism. (Esperanza et al., 2015;
Table 1 Cell Morphology and Membrane Integrity of C. vulgaris after exposed to topramezone. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3). Exposure time (h)
24 48 72 96
SSC-mean
Permeabilized (%)
Control
Topramezone (20 mg L−1)
Control
Topramezone (20 mg L−1)
145.3(4.73) 127.7(4.51) 140.3(3.21) 137.3(2.08)
178.0(1.73)** 167.3(2.52)** 164.7(2.52)** 166.3(0.57)**
24.4(4.63) 20.2(3.08) 12.4(1.60) 5.5(1.19)
40.4(5.51) 29.4(4.25) 31.6(3.81)* 14.8(1.25)**
and photosynthetic activity. Growth inhibition test of C. vulgaris clearly manifested topramezone was severely toxic and effectively reduced the population by 69.5% at
Fig. 4. Relative expressions of biochemical parameters A and B, Reactive Oxygen Species (A) and Malondialdehyde (B), in C. vulgaris exposure to 20 mg L−1 topramezone. Asterisks indicate statistically significant differences with respect to the values of control cultures (p < 0.05) (mean ± SD; n=3).
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5. Conclusion
González-Barreiro et al., 2004). A decrease in carotenoids content of algae cells was also observed, but not at 48 h exposure (Fig. 2C), revealing that carotenoids have a higher tolerance to topramezone action than chlorophylls. In these cultures, we even observed a slight increase in carotenoid content of biomass at 48 h exposure, which could be an indicator of the protective role of these pigments against oxidative stress, since carotenoids are known to be potent quenchers of reactive oxygen species, particularly singlet oxygen (Prado et al., 2011; Ünyayar et al., 2005). The changes of photosynthetic genes transcription by topqwramezone confirmed the damage of photosynthetic system. Compared with the control group, the transcript abundance of psaB and psbC decreased significantly at 96 h exposure, while the transcript abundance of rbcL increased slight. These results manifested that topramezone might interfere with the function of the photosynthetic electron transport chain from PSI to PSII by forming electron donor–acceptor complexes with the naphthoquinone phylloquinone, which functions as a secondary electron acceptor A in PSI of plants and algae (Leonova et al., 2004). As a result, it could influence the growth of algae indirectly (Qian et al., 2009a, 2009b). Thus, photosynthesis inhibition induced by topramezone toxicity would affect the entire physiological state and cell growth process. Previous research suggested that membrane disruption leads to inhibition of photosynthesis and eventually results in the increasing proportion of death (Huang et al., 2015). Flow cytometry, a technique which allows us to study different morphological and physiological properties of single cells, has been introduced as a useful tool in toxicity tests with microalgae (Franqueira et al., 2000). Results showed that topramezone affected C. vulgaris Cell morphology and membrane integrity (Table 1). Compared with the controls, the cells exposed to topramezone responded with either shrinking or swelling of the cell volume (Huang et al., 2015), the community of cells became more complexly (Table 1), it meant these cells grew unevenly and the interior was irregular. Besides, topramezone treatment group exhibited highly increased levels of PI staining, which was consistent with the conclusion that some interaction of chemicals with cyanobacteria alter cell morphology and compromise membrane integrity, causing leakage of cell components (Hong et al., 2008). Actually, chloroplast and mitochondria are the main source of ROS, which can cause cell damage in various ways. For instance, these radicals can affect membrane permeability, cause damage to DNA and proteins, and generate lipid peroxide signaling molecules. The TEM images of this study also revealed the organelles were blurred, cytoplasm was leaked and plasma membrane was degraded (Fig. 3). Topramezone causes damage to chloroplasts and membranes in algal cells. In the present work, these results indicated that topramezone induced algae cells to produce higher ROS levels compared to the control (Fig. 4A). These excessive ROS were not totally cleared by the algal cells and eventually caused cell damages. Qian et al. have pointed out that MDA is a major peroxidation product and is an indicator of lipid peroxidation, which reflects cellular oxidative damage under environmental stress conditions. Cell membranes are made of unsaturated phospholipids and are vulnerable to oxygen radical attack resulting in MDA accumulation (Qian et al., 2009a 2009b, 2008; Hong et al., 2008). In our study, the levels of MDA increased in C. vulgaris cells with the extension of exposure time (Fig. 4B), which indicated that topramezone induced membrane lipid peroxidation and caused oxidative damage on cell membranes. This phenomenon was also observed by Kong et al. (2013) and Qian et al. (2008). To sun up, the results obtained indicate that this herbicide affects all of these parameters and the observed effects can be explained by the generation of oxidative stress.
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