Aquatic Toxicology 146 (2014) 12–19

Contents lists available at ScienceDirect

Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Enantioselective changes in oxidative stress and toxin release in Microcystis aeruginosa exposed to chiral herbicide diclofop acid Jing Ye a,b , Ying Zhang c , Shengwen Chen d , Chaonan Liu a , Yongqiang Zhu a , Weiping Liu b,∗ a

School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Natural Research and Environmental Sciences, Zhejiang University, Hangzhou 310058, China c Department of Environmental Science, East China Normal University, Shanghai 200241, China d School of Urban Development and Environment Engineering, Shanghai Second Polytechnic University, Shanghai 201209, China b

a r t i c l e

i n f o

Article history: Received 4 September 2013 Received in revised form 17 October 2013 Accepted 23 October 2013 Keywords: Diclofop acid Environmental toxicology Enantioselectivity Toxin release

a b s t r a c t Enantioselective oxidative stress and toxin release from Microcystis aeruginosa after exposure to the chiral herbicide diclofop acid were investigated. Racemic diclofop acid, R-diclofop acid and S-diclofop acid induced reactive oxygen species (ROS) generation, increased the concentration of malondialdehyde (MDA), enhanced the activity of superoxide dismutase (SOD) and triggered toxin release in M. aeruginosa to varying degrees. The increase in MDA concentration and SOD activity in M. aeruginosa occurred sooner after exposure to diclofop acid than when the cyanobacteria was exposed to either the R- and the Senantiomer. In addition, enantioselective toxicity of the enantiomers was observed. The R-enantiomer trigged more ROS generation, more SOD activity and more toxin synthesis and release in M. aeruginosa cells than the S-enantiomer. Diclofop acid and its R-enantiomer may collapse the transmembrane proton gradient and destroy the cell membrane through lipid peroxidation and free radical oxidation, whereas the S-enantiomer did not demonstrate such action. R-diclofop acid inhibits the growth of M. aeruginosa in the early stage, but ultimately induced greater toxin release, which has a deleterious effect on the water column. These results indicate that more comprehensive study is needed to determine the environmental safety of the enantiomers, and application of chiral pesticides requires more direct supervision and training. Additionally, lifecycle analysis of chiral pollutants in aquatic system needs more attention to aide in the environmental assessment of chiral pesticides. © 2013 Published by Elsevier B.V.

1. Introduction Cyanobacterial blooms can form in aquatic systems eutrophied by abiotic natural sources such as surface or ground water emerging from sediments naturally rich in nutrients (Phlips et al., 2002). However, recently increasing anthropogenic input of nutrients such as phosphorus and nitrogen from urbanization and agricultural expansion has led to increases in nutrient effluxes into aquatic systems. Cyanobacteria have been quite opportunistic in exploiting these available nutrients which has resulted in more frequent and extensive blooms (Ross et al., 2006). Such blooms of cyanobacteria often cause serious environmental, esthetic, and economic problems such as decreased recreational value of waterways, fish kills, and increased cost of water treatment (Dokulil and Teubner, 2000). Furthermore, causative cyanobacteria can produce microcystins (MCs) or other cyclic hepatotoxins which are toxic to domestic livestock and wildlife around the world. They also pose a serious

∗ Corresponding author. Tel.: +86 571 88982341; fax: +86 571 88982341. E-mail address: [email protected] (W. Liu). 0166-445X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.10.023

health hazard to humans exposed to them when they use contaminated water for drinking, cooking, or recreation (Dittmann and Wiegand, 2006). Among the toxic cyanobacteria Microcystis aeruginosa is the most frequently observed in highly eutrophic lakes (Dai et al., 2009). Certain environmental factors such as pH, temperature, and the amount of phosphorus and nitrogen in the water have been found to affect the production of MCs and have been extensively studied in both batch and continuous cultures (Downing et al., 2005; Ame and Wunderlin, 2005). But other factors, such as the concentration of environmental pollutants including pesticide residues which may be prevalent in aquatic ecosystems, have not been studied in sufficient detail. During the past few decades, chiral pesticides have become an important class of environmental pollutants. It has been estimated that over 40% of pesticides used currently in China have chiral components (Ye et al., 2009a). Large quantities of chiral pesticides can be released into aquatic ecosystem during application or after heavy rainfall and exhibit severe toxicity toward nontarget organisms, including invertebrates (Zhou et al., 2007, 2009; Zhao et al., 2009), fishes (Xu et al., 2008), plants (Zhang et al., 2012; Ye et al.,

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

Cl

O Cl S-diclofop acid

CH 3 O *C H

CH3 C* O

H O C OH

C O HO mirror

13

O

Cl Cl

R-diclof op acid

Fig. 1. Chemical structures of the diclofop acid enantiomers (* indicates chiral position).

2009b; Qian et al., 2009), and algae (Cai et al., 2008). The enantiomers of a chiral compound usually have different ecotoxicities and the environmental fates of these chiral pollutants have also been found to be enantioselective (Ye et al., 2010). Chiral pollutants also impact cyanobacteria in waters. Recently, it has been reported that chiral herbicides pose enantioselective toxicity to M. aeruginosa, but did not quantify the release of the intracellular toxins or identify the mechanism responsible for cell mortality (Ye et al., 2013). Therefore, better understanding about the effects of these chiral pollutants on cyanobacteria is needed. Further, the toxin release caused by this class of compounds also needs to be investigated. Diclofop acid ((R,S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoic acid) is the active form of the herbicide diclofop methyl used on wheat, barley, and golf courses (turf). The total annual domestic usage of diclofop methyl between 1987 and 1996 in the USA was approximately 340,200 kg of active ingredient (a.i.) (RED, 2000a). In China, the usage was between 1 and 5 million kilograms in 2006 (Ye et al., 2009b). Up to 73% of the active ingredient of diclofop methyl falls onto soil surfaces during application rather than on the target weeds (Smith et al., 1986). Under alkaline aquatic conditions, diclofop methyl rapidly hydrolyzes to diclofop acid which has a relatively high solubility in water compared to diclofop methyl making it more prone to enter aquatic systems after precipitation events (RED, 2000b). Therefore, diclofop acid is likely to be present in surface water in significant amounts (Liu et al., 1991). Diclofop acid is a chiral herbicide with one stereogenic center (Fig. 1). A study of the enantioselective herbicidal activity of diclofop acid revealed that the R-enantiomer is approximately twice as active as the racemic mixture against millets and oats (Kurihara et al., 1997). It has been reported that the two enantiomers of diclofop acid posed different ecotoxicities to three freshwater algae and that their degradation in alga cultures was enantioselective (Cai et al., 2008). Diclofop acid was also found to present enantioselective ecotoxicity on rice xiushui 63 seedlings (Ye et al., 2009b), Arabidopsis thaliana (Zhang et al., 2012), and M. aeruginosa (Ye et al., 2013). The results demonstrated that herbicidal activity and environmental safety are not always consistent. Therefore, considering the target (herbicidal activity) and nontarget (environmental safety) bioactivity of chiral pesticides simultaneously is necessary when designing an herbicide application regimen. In the current study oxidative damage and toxin release from M. aeruginosa caused by the chiral pesticide diclofop acid were studied. Diclofop methyl was proposed to induce oxidative stress in susceptible plant tissues and to collapse the transmembrane proton gradient. Cells can develop a set of cellular defense systems via enzymatic and non-enzymatic antioxidants to counteract the toxicity of reactive oxygen species (ROS) like H2 O2 , O2 •− and HO• . In the enzymatic pathways, superoxide dismutase (SOD) is important to scavenge ROS (Shimabukuro et al., 1999). Previously, we studied the physiological effects of diclofop methyl and two enantiomers of diclofop acid on M. aeruginosa and found that the R-diclofop acid and S-diclofop acid pose different toxicity indicated by biomass, protein content and ultrastructural characteristics (Ye et al., 2013). Based on the proposed toxicity mechanism of diclofop methyl on

susceptible plants and the physiological effects on M. aeruginosa studied before, in this study, we speculate that oxidative stress may also be induced by diclofop acid in M. aeruginosa and that the release of toxins will also be increased as a result of cell death. The purpose of this study was to determine whether the chiral pollutant diclofop acid induces enantioselective oxidative stress on M. aeruginosa cells and leads to a significant release of soluble toxin into the surrounding water column. Further, our results will aid in developing a model that can predict outbreaks of water blooms and that ultimately protects our environment and human health. 2. Materials and methods 2.1. Chemicals and cell cultures Diclofop acid {(R,S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate acid} was prepared from diclofop methyl according to Smith (1976) and identified by HPLC. Diclofop methyl with purity ≥97% was generously provided by Iprochem Co., Ltd. (Shenzhen, China). R- and S-diclofop acid (purity ≥99.0%, optical purity ≥94.0%) were synthesized in our laboratory (Cai et al., 2008). Standard MC-LR with purity ≥95% was purchased from Express Technology Co., Ltd. (Beijing, China). The cyanobacteria M. aeruginosa was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, China. The unialgal inoculant was cultured in sterile BG11 medium under an irradiance of 40 ␮mol/m2 s with wavelength range from 400 to 750 nm and a photoperiod of 12 h light/12 h dark at 28 ± 1 ◦ C. The algal cultures were shaken three times per day. Based on our previous results (Ye et al., 2013), a maximum concentration of 5 mg/L was selected for the present study. The oxidative stress tests were carried out using 0, 1, 2, and 5 mg/L of diclofop acid, R-diclofop acid and S-diclofop acid. Three replicates of each concentration were prepared in Erlenmeyer flasks (100 mL) containing 5 mL of algal inoculant and 45 mL of culture medium. The initial algal density in each flask was (5.5–6.7) × 106 cells/mL. 2.2. Analysis of exposure concentrations After the chemicals added into culture medium, the triplicate culture samples were filtered through a 0.45-␮m filter and analyzed by HPLC. The analyses were performed on a Jasco LC-2000 series HPLC system (Jasco, Tokyo, Japan) with a PU-2089 quaternary gradient pump, a CO-2060 column temperature control compartment, and a UV-2075 plus UV/vis detector. The operation conditions were a Kromstar C18 column (4.6 mm × 250 mm, Daicel Chemical Industries, Tokyo, Japan), a flow rate of 0.8 mL/min, a mobile phase of methanol/water (50:50, v/v), a detection wavelength of 254 nm, an injection volume of 10 ␮L, and an oven temperature of 40 ◦ C. 2.3. Detection of intracellular ROS generation Oxidative stress or overproduction of ROS induced by diclofop acid was assessed with 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is converted to DCFH by esterases during up

14

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

Table 1 Relative ROS concentrations in M. aeruginosa cells exposed to varying concentrations of diclofop acid and its enantiomers. Recorded values were calculated using Eq. (1) and are the average of 3 independent trials. Time

4h 1d 2d 3d 4d 5d 6d 7d 8d 9d

Diclofop acid

R-diclofop acid

S-diclofop acid

1 mg/L

2 mg/L

5 mg/L

1 mg/L

2 mg/L

5 mg/L

1 mg/L

2 mg/L

5 mg/L

112 ± 10 (A, a)a 117 ± 19 (A, a) 110 ± 14 (A, a) 85 ± 24 (A, a) 86 ± 13 (A, ab) 107 ± 6 (A, a) 93 ± 25 (A, a) 102 ± 11 (A, a) 118 ± 19 (A, a) 76 ± 14 (A, a)

103 ± 6 (A, a) 112 ± 5 (A, a) 90 ± 8 (A, a) 85 ± 7 (A, a) 72 ± 9 (A, a) 102 ± 22 (A, a) 98 ± 30 (A, a) 154 ± 30 (A, b) 117 ± 31 (A, a) 67 ± 10 (A, a)

102 ± 10 (A, a) 112 ± 11 (A, a) 125 ± 42 (A, a) 105 ± 27 (AB, a) 124 ± 27 (A, b) 142 ± 39 (A, a) 93 ± 14 (A, a) 117 ± 17 (A, ab) 139 ± 33 (AB, a) 106 ± 11 (A, b)

119 ± 11 (A, a ) 94 ± ± 15 (A, a ) 110 ± 23 (A, a ) 122 ± 15 (A, a ) 82 ± 9 (A, a ) 111 ± 30 (A, a ) 75 ± 22 (A, a ) 108 ± 16 (A, a ) 107 ± 12 (A, a ) 88 ± 7 (A, a )

131 ± 32 (A, a ) 92 ± 7 (A, a ) 97 ± 10 (A, a ) 82 ± 11 (A, b ) 100 ± 10 (B, a ) 108 ± 19 (A, a ) 72 ± 23 (A, a ) 110 ± 18 (AB, a ) 127 ± 30 (A, ab ) 139 ± 35 (B, a )

114 ± 4 (A, a ) 90 ± 3 (B, a ) 80 ± 17 (A, a ) 120 ± 25 (A, ab ) 90 ± 10 (AB, a ) 112 ± 23 (A, a ) 82 ± 17 (A, a ) 107 ± 3 (A, a ) 152 ± 12 (A, b ) 102 ± 28 (AB, a )

124 ± 17 (A, a ) 108 ± 4 (A, a ) 110 ± 39 (A, ab ) 115 ± 31 (A, a ) 92 ± 36 (A, a ) 94 ± 15 (A, a ) 82 ± 15 (A, a ) 95 ± 9 (A, a ) 111 ± 15 (A, a ) 81 ± 5 (A, a )

113 ± 11 (A, a ) 122 ± 5 (B, b ) 133 ± 13 (B, a ) 83 ± 13 (A, a ) 91 ± 25 (AB, a ) 94 ± 7 (A, a ) 60 ± 5 (A, a ) 82 ± 16 (B, a ) 109 ± 19 (A, a ) 86 ± 35 (AB, ab )

110 ± 4 (A, a ) 104 ± 25 (AB, ab ) 89 ± 1 (A, b ) 71 ± 3 (B, a ) 71 ± 13 (B, a ) 83 ± 13 (A, a ) 64 ± 13 (A, a ) 72 ± 24 (A, a ) 92 ± 9 (B, a ) 69 ± 4 (B, b )

a Results are presented as mean ± SD of three independent assays. Italic and bold character indicated statistically significant ROS generation occurs. Different capitalized letters indicate a significant difference (p < 0.05) between individual enantiomers or between an enantiomer and racemate at the same time and the same concentration, while the same letter indicates no significant difference (LSD). Different lowercase letters indicate a significant difference (p < 0.05) between different concentrations of the same analyte (a, b, c for diclofop acid; a , b , c for R-diclofop acid; and a , b , c for S-diclofop acid)

take by cell. DCFH reacts with ROS to produce the highly fluorescent compound, 2 ,7 -dichlorofluorescein (DCF). In living organisms suffering from environmental stress, oxidative stress is caused by both overproduction of ROS and depletion of antioxidants. The ROS level was determined as described by Liu et al. (2004) and He et al. (2002). Samples consisting of 1 mL of either herbicide-treated or untreated (control) algal cells were centrifuged in 1.5 mL tubes and the resulting pellets were resuspended in 1 mL of phosphate buffered saline (PBS) solution (50 mM, pH 7.0). Incubation saline and 1 mL of 10 ␮M DCFH-DA were added to each sample and the solutions were placed in the dark for 60 min at 25 ◦ C. After incubation, the samples were washed twice with fresh PBS. The DCF fluorescence was measured with a fluorescence microplate reader (Tecan Infinite 200, Austria) using an excitation wavelength of 485 nm and emission wavelength of 525 nm. The ROS level was determined after 4 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h and 216 h treatment with the enantiomers of diclofop acid. The relative ROS level (%) was calculated using the formula given below: relative ROS level(%) =

mean DCF fluorescence[herbicide – added] mean DCF fluorescence[control]

× 100

(1)

2.4. Lipid-peroxidation analysis The formation of malondialdehyde (MDA) was used as an indicator of lipid peroxidation. The determination of MDA by the thiobarbituric acid (TBA) reactive substances method was performed as described by Hagege et al. (1990). We purchased lipid peroxidation (MDA) assay kit (Jiancheng bioengineering institute, Nanjing, China) and conducted the measurements according to the instruction. The MDA concentration was measured after 24 h and 48 h of treatments. 2.5. Enzymatic antioxidant assays Algal cells were concentrated by centrifugation at 1500 × g for 10 min at 4 ◦ C and the cell pellets were transferred into 10 mL

centrifuge tubes. The pellets were washed twice with sterilized media and recentrifuged. The recentrifuged cell pellets were resuspended in 2 mL of PBS solution (50 mM, pH 7.0) and homogenized by an ultrasonic cell pulverizer (Microson XL 2000, New York, USA) at 200 W for a total time of 2 min (ultrasonic time: 2 s; rest time: 8 s) while being cooled in an ice-bath. Then the homogenate was centrifuged at 12,000 × g for 10 min at 4 ◦ C. The cell-free enzyme extract supernatant was used for enzymatic and antioxidant measurements. The SOD activity assay was conducted using the method of Beauchamp and Fridovich (1971). We purchased the SOD assay kit (Jiancheng bioengineering institute, Nanjing, China) and conducted the measurements according to the handling instruction. The SOD concentration was determined after 24 h and 48 h of treatments.

2.6. MC-LR extraction and analysis MC-LR is the most common and toxic toxin which contains the l-amino acids leucine (L) and arginine (R). Cells and culture media treated with 5 mg/L of the enantiomers of diclofop acid were separated by filtration through 47 mm GF/C disks (Whatman, Maidstone, Kent, UK). The disks with attached cyanobacterial cells were dried at 50 ◦ C for 12 h. The dried cells were sonicated for 5 min and extracted in methanol for 15 min. The extraction procedure was repeated three times. After filtration, the sample was evaporated to dryness at 45 ◦ C under reduced pressure. The residue consisted of the intracellular extract. The extract was then dissolved in 200 ␮L methanol and stored at −20 ◦ C until analyzed. Aqueous filtrates were preconcentrated on SPE cartridge (Agilent Technologies, NC, USA) packed with 500 mg of octadecylsilane. The solid-phase extraction procedure involved activation of the cartridge with 5 mL of methanol, conditioning with 10 mL of MilliQ water, application of the sample at a flow-rate of 10 mL/min, and rinsing of the cartridge with 5 mL of 20% aqueous methanol. Analytes were subsequently desorbed with 3 mL of methanol. The methanol extract was evaporated to dryness at 45 ◦ C under reduced pressure. The residue was redissolved in 200 ␮L of methanol, and stored at −20 ◦ C until analyzed.

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

15

Fig. 2. MDA concentration in M. aeruginosa cells exposed to 0, 1, 2, and 5 mg/L diclofop acid, R-diclofop acid and S-diclofop acid after 24 h (a) and 48 h (b). Results are presented as mean ± SD of three independent assays (* indicates p < 0.05 and ** indicates p < 0.01 relative to the control by ANOVA). Different capitalized letters above adjacent bars indicate a significant difference (p < 0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference (LSD). Different lowercase letters indicate a significant difference (p < 0.05) between different concentrations of the same analyte (a, b, c for diclofop; a , b , c for R-diclofop acid; and a , b , c for S-diclofop acid). Acetone was included in the assay because it was the solvent for the dichlofop acid.

MC-LR analyses were performed using a Jasco LC-2000 series HPLC system (Jasco, Tokyo, Japan) with a PU-2089 quaternary gradient pump, a CO-2060 column temperature control compartment, and a UV-2075 Plus UV/vis detector. The separation of the toxins was performed using a Hypersil ODS2 C18 column (250 mm × 4.6 mm i.d., 5 ␮m) at a flow rate of 0.8 mL/min, a 60:40 (v:v) methanol/water (acidified to pH 3.2 with trifluoroacetic acid) mobile phase, a detection wavelength of 238 nm, an injection volume of 10 ␮L, and a column-jacket temperature of 25 ◦ C. 2.7. Data analysis Statistical analysis was performed using Origin 8.0 (Microcal Software, Northampton, MA, USA) and SPSS 16.0 (SPSS, USA) to determine the significance among the treatments. p < 0.05 was considered statistically significant. Multiple comparisons between the groups were performed using post hoc test with LSD method.

3. Results 3.1. Exposure concentrations of the chemicals For nominal concentrations of 1, 2, and 5 mg/L, the exposure concentrations of diclofop acid were 0.68 ± 0.015, 1.34 ± 0.014 and 3.82 ± 0.010 mg/L, respectively. The exposure concentrations of R-diclofop acid were 0.66 ± 0.011, 1.30 ± 0.015, and 3.48 ± 0.008 mg/L, respectively. The exposure concentrations of S-diclofop acid were 0.60 ± 0.010, 1.25 ± 0.020, and 3.70 ± 0.012 mg/L, respectively. 3.2. Relative ROS levels in M. aeruginosa cells Table 1 shows the relative ROS concentrations generated in cells exposed to enantiomers of diclofop acid calculated using Eq. (1). Values above 100 indicate that ROS induction occurs. The total

Fig. 3. SOD activities in M. aeruginosa cells exposed to 0, 1, 2, and 5 mg/L diclofop acid, R-diclofop acid and S-diclofop acid after 24 h (a) and 48 h (b). Results are presented as mean ± SD of three independent assays (* indicates p < 0.05 and ** indicates p < 0.01 relative to the control by ANOVA). Different capitalized letters above adjacent bars indicate a significant difference (p < 0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference (LSD). Different lowercase letters indicate a significant difference (p < 0.05) between different concentrations of the same analyte (a, b, c for diclofop acid; a , b , c for R-diclofop acid; and a , b , c for S-diclofop acid). Acetone was included in the assay because it was the solvent for the dichlofop acid.

16

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

Fig. 4. HPLC chromatograms showing of extracellular (a) and intracellular (b) microcystin-LR (detection wavelength 238 nm).

induction percentages for samples exposed to 1 mg/L, 2 mg/L and 5 mg/L of diclofop acid, R-diclofop acid and S-diclofop acid were 66.7%, 60% and 36.7%, respectively. Diclofop acid most strongly induces ROS production, followed by the R-enantiomer and the Senantiomer. After 4 h of exposure, all three compounds induced ROS generation under every concentration. 3.3. MDA production in response to cellular stress MDA, a by-product of lipid peroxidation, was quantified to ascertain the involvement of lipid peroxidation in the toxicity of the diclofop acids (Fig. 2). Most of the exposure levels induced some increase in MDA production relative to the control. At 1, 2, and 5 mg/L, diclofop acid induced 2.16, 0.53 and 9.08-fold increases in MDA compared to the control, respectively; R-diclofop acid did not produce an induction effect; S-diclofop acid induced 0.53, 0.05 and 0.56-fold increases in MDA, respectively. Induction of MDA by the racemic mixture of diclofop acid was greater than that by either the R- or S-enantiomers separately. Of particular interest is that after 24 h of exposure (Fig. 2a), 5 mg/L diclofop acid significantly induced MDA generation. Enantioselectivity was not significantly different between the R- and S-enantiomers. After 48 h of exposure (Fig. 2b), diclofop acid did not significantly induce the production of MDA. In contrast, both the R- and S-enantiomers at 2 mg/L and 5 mg/L induced the MDA production significantly. At 1, 2, and 5 mg/L, the increases were 0.51, 9.51 and 7.26-fold, respectively for R-diclofop acid; and for S-diclofop acid, the increases were 3.63, 8.70 and 11.26-fold, respectively. Statistically significant difference was observed between the two enantiomers at 5 mg/L. 3.4. SOD activity in M. aeruginosa cells To determine whether diclofop acid and its enantiomers affect the antioxidant system, the activity of the antioxidant enzyme

SOD was examined (Fig. 3). After 24 h of exposure, SOD activity in both the 1 mg/L and 5 mg/L diclofop acid treated cells was slightly increased compared to the control, however, the increases were not significant. 1 mg/L R-enantiomer slightly increased SOD activity, whereas the R-enantiomer and the S-enantiomer at 2 mg/L and 5 mg/L decreased the SOD activity relative to the control. After 48 h of exposure, all the three species increased the SOD activity at every concentration. At 2 mg/L, enantioselectivity was observed between the two enantiomers. At 1, 2 and 5 mg/L, the diclofop acid increased the SOD activity 1.39, 3.03 and 1.53-fold, respectively; for R-diclofop acid, the activity increased 1.76, 3.33 and 3.30-fold, respectively; and for S-diclofop acid, the activity increased 1.91, 2.22 and 3.41-fold, respectively. In general, after 48 h SOD induction by diclofop acid was less than that caused by either of the individual enantiomers. 3.5. HPLC detection of MC-LR Cellular extracts and growth medium were analyzed by HPLC. MC-LR contains the Adda residue which includes two conjugated ␲-bonds responsible for a UV absorbance maximum at 238 nm; therefore its UV spectrum is very characteristic (Fig. 4). In control samples, the extracellular MC-LR level remained relatively low and stable (Fig. 5a), while intracellular MC-LR first increased in unison with the cell growth during the first three days and then decreased to a relatively stable level (Fig. 5b). Diclofop acid induced toxin release into the culture medium. On day 5 the MC-LR concentration reached its maximum level which was significantly higher than the control (Fig. 5a). R-diclofop acid significantly increased the release of MC-LR on days 3 and 4 (Fig. 5a). Intracellular MC-LR was also significantly induced by the R-enantiomer (Fig. 5b). The total toxin content peaked on day 4 (Fig. 6). Unlike the R-enantiomer, the S-enantiomer decreased the intracellular toxin synthesis (Fig. 5b). The extracellular MC-LR was also decreased except on day 4 (Fig. 5a).

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

17

shown in Fig. 2. The induction of SOD activity by diclofop acid was also earlier than that by either the R- and S-enantiomer. After 48 h of exposure, SOD activity in R- and S-enantiomer treated cells were higher than in cells treated with the racemic mixture of diclofop acid. The increase of SOD activity in M. aeruginosa cells treated with diclofop acid and its enantiomers implied the activation of the cellular defense system to reduce ROS. Shimabuluro et al. (2001) suggested that the herbicidal mechanism of diclofop methyl lies in the induction of oxidative stress in susceptible plant tissues which, in turn, results in the formation of ROS. Diclofop methyl was observed at a putative membrane binding site to induce senescence, collapse the transmembrane proton gradient, and destroy the cell membrane and other cell components through lipid peroxidation and free radical oxidation. In the present study, the induction of ROS, MDA and SOD in cells treated with diclofop acid, R-diclofop acid or S-diclofop acid suggests that the mechanism for oxidative stress induced by diclofop acid might be connected with excess O2 •− generation that then triggers a free radical chain reaction and aggravates lipid peroxidation of cell membrane components. Zhang et al. (2012) investigated the activity of oxidative stress related enzymes and also found that enantiomers of diclofop acid posed different toxicities to A. thaliana. Diclofop acid is an herbicide considered to be one of the environmental stresses that can lead to oxidative damage in cyanobacteria. When oxidative stress induced ROS levels exceed a threshold, survival of the cells becomes untenable and they eventually die and release their contents to the surrounding water column (Fig. 7). 4.2. Toxin release in response to cell damage

Fig. 5. Extracellular (a) and intracellular (b) MC-LR concentrations in M. aeruginosa cells exposed to no diclofop acid, diclofop acid, R-diclofop acid and S-diclofop acid. Results are presented as mean ± SD of three independent assays (* indicates p < 0.05 and ** indicates p < 0.01 relative to the control by ANOVA). Different capitalized letters above adjacent bars indicate a significant difference (p < 0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference (LSD).

4. Discussion 4.1. Toxicology of diclofop acid The destructive ROS lead to oxidative damage to cellular components such as membrane lipids, nucleic acids and proteins (Halliwell and Gutteridge, 1984; Noctor and Foyer, 1998). MDA is widely used as a biomarker for lipid peroxidation to reflect cellular oxidative damage (Bailly et al., 1996). As shown in Table 1, the ROS levels in M. aeruginosa cells exposed to diclofop acid, R-diclofop acid and S-diclofop acid all increased within 4 h. The acute increase indicated that the cells were in oxidative stress. The increasing MDA concentrations shown in Fig. 2 demonstrated that ROS-mediated oxidative stress caused oxidative damage in M. aeruginosa cells. Diclofop acid produced a larger induction effect than either the R- or S-enantiomer alone after 24 h of exposure. While after 48 h of exposure, the R- and the S-enantiomers exhibited larger induction effects. The results indicate that induction of MDA production by diclofop acid occurred earlier than induction caused by R- and S-enantiomer. SOD provides a first line of defense against ROS (Scandalios, 1993). SOD activities shown in Fig. 3 were similar to the MDA results

M. aeruginosa is a ubiquitous cyanobacteria that has often been linked to toxic blooms worldwide (Jewel et al., 2003; Silva, 2003). However, high concentrations of Microcystis do not necessarily correlate with high levels of microcystins and other toxins in the water column. Many cyanobacteria retain cyanotoxins within their cell structure, and only release these toxins into the surrounding water upon cell lysis (White et al., 2005). The early stages of a toxic bloom are characteristically associated mostly with increased intracellular toxins. As the bloom ages, cell death ensues and the concentration of extracellular toxins increases (Lahti et al., 1997). As shown in Fig. 5 in herbicide free cultivations most of MC-LR was intracellular and the concentration were increased with the growth of the cells during the first three days. Cells treated with diclofop acid (Fig. 5a) or R-diclofop acid (Fig. 5a) showed significant increases in the extracellular MC-LR concentration compared to the control. In contrast, in S-diclofop acid treated cells (Fig. 5b), the intracellular MC-LR concentration was significantly decreased on days 2, 3, and 5 and the extracellular MC-LR concentration remained at a relatively low level similar to the control samples. Under the laboratory conditions studied here, S-diclofop acid induced less MC-LR production than diclofop acid or the R-enantiomer. In photosynthetic organisms ROS are produced in response to a variety of exogenous factors including excessive irradiation and exposure to pesticides, xenobiotics, or pathogens (Xue et al., 2005). The damage caused by ROS includes oxidative lipid peroxidation and destruction of the cell membrane. Our results indicate that toxin release from cultures of M. aeruginosa are stimulated by exposure to diclofop acid and its R-enantiomer. The increase of extracellular MC-LR in cultures of cells treated with diclofop acid or R-diclofop acid supports the hypotheses that diclofop acid acts as a proton ionophore shuttling protons across the plasmalemma and that it collapses the transmembrane proton gradient promoting transfer of the MC-LR from the intracellular to the extracellular fluid (Shimabukuro and Hoffer, 1995; Wright and Shimabukuro, 1987). However, the exact effects of diclofop acid and R-diclofop acid merits further research.

18

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

MC-LR content, mg/L

40

a

extracellular intracellular

control

b

diclofop acid

30 20 10 0

1

2

3

4

5

6

1

2

3

4

5

6

MC-LR content, mg/L

40

c

R-enantiomer

d

S-enantiomer

30 20 10 0 1

2

3 4 time, day

5

6

1

2

3 4 time, day

5

6

Fig. 6. Total MC-LR concentrations (extracellular and intracellular) in M. aeruginosa cells exposed to no diclofop acid (a), diclofop acid (b), R-diclofop acid (c) and S-diclofop acid (d).

4.3. Enantioselective toxicitiy of the R-enantiomer and S-enantiomer In our previous study, we found that the R-enantiomer of diclofop acid was more toxic than the S-enantiomer in their ability to disrupt/modify protein content, chlorophyll a, and ultrastructure characteristic in M. aeruginosa (Ye et al., 2013). However our previous work emphasized the need for additional investigation of enantioselective toxicity of the diclofop acid enantiomers to better understand the mechanism of the toxicity. The present study yielded results consistent with our previous study while providing insight into the mechanism. As mentioned above, the R-enantiomer triggers more ROS generation in M. aeruginosa cells than the Senantiomer. SOD activity in R-enantiomer treated cells was also higher than that in S-enantiomer treated cells after 48 h. The Renantiomer also induces more toxin synthesis and release than the S-enantiomer. All of these results indicate that R-diclofop acid is more toxic than S-diclofop acid to M. aeruginosa, in agreement with

Fig. 7. Schematic diagram showing the induction of ROS production and the subsequent relaes of toxins from M. aeruginosa cells exposed to R-diclofop acid and S-diclofop acid.

our previous results. Besides, we also found R-diclofop acid and S-diclofop acid shown a synergistic effect in joint toxicity, which means diclofop acid (the racemic mixture) is more toxic than Renantiomer or S-enantiomer (Ye et al., 2013). The present study yielded results consistent with our previous study. Enantioselective toxicity of diclofop acid has also been observed in freshwater algae (Cai et al., 2008), rice seedlings (Ye et al., 2009b), and A. thaliana (Zhang et al., 2012). Such studies demonstrate that the enantioselective effects of the chiral herbicide diclofop acid are more general and negatively impact a wide range of nontarget organisms. Therefore, it is essential to simultaneously consider both the target (herbicidal activity) and nontarget (environmental safety) enantioselective bioactivity of chiral pesticides to ensure adequate environmental protection. In conclusion, when M. aeruginosa was exposed to the chiral herbicide diclofop acid, the intracellular ROS and MDA levels were significantly increased. In turn, the enzymatic antioxidant SOD quickly increased in an effort to minimize the potentially destructive conditions. These results are consistent with a mechanism in which ROS-induced oxidative damage might be the fatal factor in the phytotoxicity of diclofop acid on M. aeruginosa. MC-LR release was significantly increased in cells treated with diclofop acid and R-diclofop acid, while S-diclofop acid proved to be less toxic to the cells. The toxin release from cell lysis was caused by the toxicity of diclofop acid. We hypothesized that diclofop acid collapses the transmembrane proton gradient and initiates destruction of the cell membrane through lipid peroxidation and free radical oxidation. The difference in toxicities between R-diclofop acid and Sdiclofop acid were enantioselective. We previously found that S-diclofop acid stimulates the growth of M. aeruginosa during the first 3 days after exposure, which is adverse to water quality. As a result, we recommended using the herbicidally active R-enantiomer (Ye et al., 2013). The results in the present study also demonstrate that R-diclofop acid inhibits the growth of M. aeruginosa during the early stages which is helpful to control the

J. Ye et al. / Aquatic Toxicology 146 (2014) 12–19

harmful alga, and the eventual increase in the amount of toxin released has a severely deleterious effect on the water column. Clearly, a thorough and detailed evaluation of the lifecycle of diclofop acid in aquatic system needs more investigation. In addition, the effects of diclofop acid on toxin production and release require more comprehensive survey to help develop the best methods for utilizing this valuable herbicide. Conflict of interest The authors declare no competing financial interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (21307082, 20977062), the project of Science and Technology Commission of Shanghai Municipality, China (11ZR1435600); Innovation Program of Shanghai Municipal Education Commission (13YZ116) and Shanghai Municipal Science and Technology Commission, some local universities capacity building project (12120503700). References Ame, M.V., Wunderlin, D.A., 2005. Effects of iron, ammonium and temperatures on microcystin content by a natural concentrated Microcystis aeruginosa population. Water, Air, Soil Pollut. 168, 235–248. Bailly, C., Benamar, A., Corbineau, F., Come, D., 1996. Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging. Physiol. Plant. 97, 104–110. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. J. 44, 276–287. Cai, X.Y., Liu, W.P., Sheng, G.Y., 2008. Enantioselective degradation and ecotoxicity of the chiral herbicide diclofop in three freshwater alga cultures. J. Agric. Food Chem. 56, 2139–2146. Dai, R., Liu, H., Qu, J., Zhao, X., Hou, Y., 2009. Effects of amino acids on microcystin production of the Microcystis aeruginosa. J. Hazard. Mater. 161, 730–736. Dittmann, E., Wiegand, C., 2006. Cyanobacterial toxins-occurrence, biosynthesis and impact on human affairs. Mol. Nutr. Food Res. 50, 7–17. Dokulil, M.T., Teubner, K., 2000. Cyanobacterial dominance in lakes. Hydrobiologia 438, 1–12. Downing, E.G., Meyer, C., Gehringger, M.M., Van de Venter, M., 2005. Microcystin content on Microcystis aeruginosa is modulated by nitrogen uptake rate relative to specific growth rate or carbon fixation rate. Environ. Toxicol. 20, 257–262. Hagege, D., Nouvelot, A., Boucaud, J., Gasper, T., 1990. Malondialdehyde titration with thiobarbiturate in plant extracts: avoidance of pigment interference. Photochem. Anal. 1, 86–89. Halliwell, B., Gutteridge, J.M.C., 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1–14. He, Y.Y., Klisch, M., Hader, D.P., 2002. Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage. Photochem. Photobiol. 76, 188–196. Jewel, M.A.S., Affan, M.A., Khan, S., 2003. Fish mortality due to cyanobacterial bloom in an aquaculture pond in Bangladesh. Pakistan J. Biol. Sci. 6, 1046–1050. Kurihara, N., Miyamoto, J., Paulson, G.D., Zeeh, B., Skidmore, M.W., Hollingworth, R.M., Kuiper, H.A., 1997. Chirality in synthetic agrochemicals: bioactivity and safety consideration. Pure Appl. Chem. 69, 1335–1348. Lahti, K., Rapala, J., Fardig, M., Niemela, M., Sivonen, K., 1997. Persistance of cyanobacterial hepatotoxin, Microcystin-LR, in particulate material and dissolved in lake water. Water Res. 31, 1005–1012. Liu, W.P., Chen, Z.W., Xu, H.Q., Shi, Y.Y., 1991. Determination of diclofop-methyl and diclofop residues in soil and crops by gas chromatography. J. Chromatogr. A 547 (1/2), 509–515.

19

Liu, M.J., Wang, Z., Li, H.X., Wu, R.C., Liu, Y.Z., Wu, Q.Y., 2004. Mitochondrial dysfunction as an early event in the process of apoptosis induced by woodfordin I in human leukemia K562 cells. Toxicol. Appl. Pharmacol. 194, 141–155. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Phlips, E.J., Bledsoe, E., Badylak, S., Frost, J., 2002. The distribution of potentially toxic cyanobacteria in Florida. In: Johnson, D., Harbison, R.D. (Eds.), Proceedings of the Health Effects of Exposure to Cyanobacteria Toxins. State of the Science, Saratoga, FL, pp. 16–21. Qian, H.F., Hu, H., Mao, Y., Ma, J., Zhang, A.P., Liu, W.P., Fu, Z.W., 2009. Enantioselective phytotoxicity of the herbicide imazethapyr in rice. Chemosphere 76, 885–892. 2000a. R.E.D facts diclofop-methyl. United Stated Environmental Protection Agency, Washington, DC, September, http://www.epa.gov/opp00001/ chem search/reg actions/reregistration/fs PC-110902 1-Sep-00.pdf 2000b. Reregistration Eligibility Decision (RED) diclofop-methyl. United Stated Environmental Protection Agency, Washington, DC, September, http://www. epa.gov/pesticides/chem search/reg actions/reregistration/red PC-110902 1-Sep-00.pdf Ross, C., Santiago-Vazquez, L., Paul, V., 2006. Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aerugiosa. Aquat. Toxicol. 78, 66–73. Scandalios, J.G., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7–12. Shimabukuro, R.H., Hoffer, B.L., 1995. Enantiomers of diclofop-methyl and their role in herbicide mechanism of action. Pestic. Biochem. Physiol. 51, 68–82. Shimabukuro, R.H., Biewer, K.A., Davis, D.G., Hoffer, B.L., 1999. Physiological responses of resistant and susceptible plants to diclofop-methyl and its antagonism by 2,4-D and antioxidants. Physiol. Plant. 107, 68–76. Shimabuluro, R.H., Davis, D.G., Hoffer, B.L., 2001. The effect of diclofop-methyl and its antagonist, Vitamin E, on membrane lipids in oat (Avena sativa L.) and leafy spurge (Euphorbia esula L.). Pestic. Biochem. Physiol. 69, 13–26. Silva, E.I.L., 2003. Emergence of a Microcystis bloom in an urban water body, Kandy Lake. Sri Lanka Curr. Sci. 85, 723–725. Smith, A.E., 1976. Etherification of the hydrolysis product of the herbicide diclofopmethyl in methanol. J. Agric. Food Chem. 24, 1077–1078. Smith, A.E., Grover, R., Cessna, A.J., Shewchuk, S.R., Hunter, J.H., 1986. Fate of diclofop-methyl after application to a wheat field. J. Environ. Qual. 15 (3), 234–238. White, S.H., Duivenvoorden, L.J., Fabbro, L.D., 2005. A decision-making framework for ecological impacts associated with the accumulation of cyanotoxins (cylindrospermopsin and microcystin). Lake Reserv. Manage. 10, 25–37. Wright, J.P., Shimabukuro, R.H., 1987. Effects of diclofop and diclofop-methyl on the membrane potentials of wheat and oat coleoptiles. Plant Physiol. 85, 188–193. Xu, C., Zhao, M.R., Liu, W.P., Chen, S.W., Gan, J.Y., 2008. Enantioselectivity in zebrafish embryo toxicity of the insecticide acetofenate. Chem. Res. Toxicol. 21, 1050–1055. Xue, L., Zhang, Y., Zhang, T., Wang, X., 2005. Effects of enhanced ultraviolet-B radiation on algae and cyanobacteria. Crit. Rev. Microbiol. 31, 79–89. Ye, J., Wu, J., Liu, W., 2009a. Enantioselective separation and analysis of chiral pesticides by high-performance liquid chromatography. Trac-Trend Anal. Chem. 28, 1148–1163. Ye, J., Zhang, Q., Zhang, A.P., Wen, Y.Z., Liu, W.P., 2009b. Enantioselective effects of chiral herbicides diclofop acid on rice xiushui 63 seedling. Bull. Environ. Contam. Toxicol. 83, 85–91. Ye, J., Zhao, M., Liu, J., Liu, W., 2010. Enantioselectivity in environmental risk assessment of modern chiral pesticides. Environ. Pollut. 158, 2371–2383. Ye, J., Wang, L., Zhang, Z., Liu, W., 2013. Enantioselective physiological effects of the herbicide diclofop on cyanobacterium Microcystis aeruginosa. Environ. Sci. Technol. 47, 3893–3901. Zhang, Q., Zhao, M., Qian, H., Lu, T., Zhang, Q., Liu, W., 2012. Enantioselective damage of diclofop acid mediated by oxidative stress and acetyl-CoA acrboxylase in nontarget plant Arabidopsis thaliana. Environ. Sci. Technol. 46, 8405–8412. Zhao, M.R., Wang, C., Liu, K.K., Sun, L.W., Li, L., Liu, W.P., 2009. Enantioselectivity in chronic toxicology and accumulation of the synthetic pyrethroid insecticide bifenthrin in Daphnia magna. Environ. Toxicol. Chem. 28, 1475–1479. Zhou, S.S., Lin, K.D., Yang, H.Y., Li, L., Liu, W.P., Li, J., 2007. Stereoisomeric separation and toxicity of a new organophosphorus insecticide chloramidophos. Chem. Res. Toxicol. 20, 400–405. Zhou, S.S., Lin, K.D., Jin, M.Q., Ye, J., Liu, W.P., 2009. Separation and toxicity of salithion enantiomers. Chirality 27, 922–928.