Article pubs.acs.org/est
Enantioselective Physiological Effects of the Herbicide Diclofop on Cyanobacterium Microcystis aeruginosa Jing Ye,†,‡,§ Lumei Wang,§ Zhijian Zhang,‡ and Weiping Liu*,‡ ‡
MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Natural Research and Environmental Sciences, Zhejiang University, Hangzhou 310058, China † School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China § Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, Shanghai Jiao Tong University, Shanghai 200240, China S Supporting Information *
ABSTRACT: Water blooms caused by cyanobacteria are currently major global environmental issues. The outbreaks induced by nutrient elements have attracted much attention; however, the effects of environmental pollutants on the cyanobacteria are themselves poorly understood, especially those due to chiral chemicals. To explore the enantioselective eco-effects of the chiral herbicide diclofop-methyl (DM) and its major metabolite diclofop acid (DA), the physiological characteristics of Microcystis aeruginosa were investigated. The results showed that using both biomass and protein content as growth parameters is necessary to access the impact of the herbicides, that stimulation biomass production by R-DA and S-DA was apparent (nonessential), and that the concentration of 5 mg/L is worth noting. Ultrastructure changes in gas vacuoles, thylakoids, glycogen, cyanophycin granules, poly betahydroxybutyrate, polyhedral body, and lipids indicated different toxicity modes among the four chemicals. The different effects between R-DA and S-DA demonstrated that R-DA probably acts as a proton ionophore shuttling protons across the plasmalemma, whereas S-DA did not demonstrate such action. The toxicity order in the present study is S-DA < R-DA < DM < DA. Stimulation of the growth of M. aeruginosa during the first 3 days by herbicidally inactive S-DA was greater than that due to R-DA, which is adverse to water quality in water bodies. Therefore, using the herbicidally active R-enantiomer is recommended. These results are helpful in understanding the enantioselective effects of chiral pesticides on cyanobacteria, which is important for environmental assessment and protection. It is also helpful for guiding the application of chiral pesticides in agricultural settings.
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INTRODUCTION Outbreaks of water blooms have occurred frequently in inland waters in China recently.1 Generally, the blooms in eutrophic lakes and reservoirs are composed of cyanobacteria. The overproliferation of cyanobacteria may significantly change or degrade ecosystem function. Known negative impacts include attenuating light in the water column, discoloring the water, causing water anoxia, producing toxins, and altering the food web structure.2 Therefore, public health risks brought about by water blooms have gained increasing attention, and water blooms are now an important environmental issue.3,4 The factors causing the blooms have been investigated extensively.5,6 Various approaches to deal with the environmental problems induced by cyanobacteria blooms have also been reported, including nutrient diversion, artificial destratification, sediment oxidation/removal, biomanipulation, etc.7,8 However, studies investigating the physiological effects of environmental pollutants on cyanobacteria, which are important for understanding the relationship between water blooms and xenobiotics, are insufficient. Chiral pesticides, including insecticides and herbicides, have become an important class of environmental pollutants in the © 2013 American Chemical Society
past few decades. It has been estimated that chiral pesticides account for over 40% of pesticides currently used in China.9 Due to the wide application of chiral pesticides, parent compounds and their transformation products may be distributed throughout the environment and may exert enantioselective toxic actions on biota.10 It has been broadly reported that chiral pesticides posed enantioselective ecological effects on nontarget organisms in the environment,11 and they will inevitably impact cyanobacteria in waters. The damaged cyanobacteria cells together with the residues of chiral pesticides in the aquatic system may result in the decline of water quality and cause additional environmental problems mentioned above. Therefore, it is important to understand the enantioselective effects of chiral pesticides on cyanobacteria in order to provide better environmental assessment and protection. Additionally, such understanding will be helpful for guiding the application of chiral pesticides in agriculture. Received: Revised: Accepted: Published: 3893
November 10, 2012 March 8, 2013 March 14, 2013 March 14, 2013 dx.doi.org/10.1021/es304593c | Environ. Sci. Technol. 2013, 47, 3893−3901
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chemicals (diclofop methyl, diclofop acid, R-diclofop acid, Sdiclofop acid) at a range of concentrations. Based on the results, we determined that the toxicity mechanisms among the four chemicals are different. To identify the toxicity mechanism of chiral DA, the ultrastructures of M. aeruginosa were observed. The results of this study provide enhanced insights for evaluating environmental risks posed by DA and reveal the enantioselectivity between the two enantiomers of DA. Further, we speculate that understanding the different physiological effects caused by diclofop on cyanobacteria can predict outbreaks of water blooms and ultimately protect our environment and human health.
Diclofop methyl {(R,S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate acid methyl ester (DM)} is a post emergence herbicide registered in the United States by Farbwerke Hoechst AG (Frankfurt, Germany) in 1982 for the control of wild oats and annual grasses in wheat and barley. The total annual usage of diclofop methyl in the USA between 1987 and 1996 was approximately 340,200 kg of active ingredient (a.i.), and the total usage in Canada in 1986 was over one million kilograms.12 In China, the usage was one to five million kilograms in 2006.13 It has been reported that up to 73% of the active ingredient may fall onto soil surfaces upon application.14 Therefore, the residues of the parent compound and the acid degradation product may become common in the environment. Under alkaline aquatic conditions, diclofop methyl rapidly hydrolyzes into diclofop acid (DA), which is a polar compound and has a relatively high solubility (23 mg/L at pH 7, 20 °C) in water compared to diclofop methyl (0.8 mg/ L at pH 7, 25 °C).15 Therefore, diclofop acid is likely to be present in surface water in significant amounts.16 Both compounds are chiral herbicides with one stereogenic center (Figure 1). A study on the enantioselective herbicidal
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MATERIALS AND METHODS Chemicals and Cyanobacteria. Diclofop methyl (DM), purity ≥97%, was generously provided by Iprochem Co., Ltd. (Shenzhen, China). Diclofop acid (DA) {(R,S)-2-[4-(2,4dichlorophenoxy)phenoxy]propanoate acid} was prepared from DM according to Smith21 and identified by HPLC. Rand S-diclofop acid (purity ≥99.0%, optical purity ≥94.0%) were synthesized in our laboratory.22 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 μmol/m2·s and a photoperiod of 12 h light/12 h dark at 28 ± 1 °C. Growth Conditions. The growth tests were carried out under different concentrations (0, 0.5, 1, 2, and 5 mg/L) of DA, R-DA, S-DA, and DM. According to our previous study,13 the maximum concentration of 5 mg/L was selected for the present study. 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 Erlenmeyer flasks were maintained at 28 ± 1 °C and a humidity of 60% in a culture chamber with alternation periods of light and dark (12 h/12 h). The irradiance with wavelength range from 400 to 750 nm was kept at 40 μmol/m2·s. The linear equation between cell number and optical density of algal culture at 685 nm was established using UV/vis spectrometer (Jasco V-550, Japan). The initial algal density in every flask was (5.5−6.7) × 106 cells/mL. Algal densities were measured every 24 h to obtain the growth curve under different conditions. Protein Content Determination. The protein content was determined by the Bradford method23 utilizing the principle of protein-dye binding. One mL of M. aeruginosa culture sample was removed from each treatment every three days (day 3, 6, and 9). The cell walls were then disrupted by ultrasound (Microson XL 2000, New York, USA) for 5 min at 4 °C. After disruption, 0.5 mL of the disrupted cells suspension was transferred to a test tube, and the optical density was measured at 595 nm. Ultrastructural Characteristics. The ultrastructure of the cells exposed to different treatments for 72 h were observed by transmission electron microscopy (TEM). The cells were concentrated by centrifugation at 1000 g for 2 min and then embedded into agar. The specimens were fixed with 2.5% glutaraldehyde at 4 °C overnight. Postfixation, the samples were exposed to 1% osmium tetroxide for 1−2 h. Solutions of both fixatives were prepared in 0.1 M phosphate buffer, pH 7.0. The specimens were then dehydrated by a graded series of ethanol solutions (50%, 70%, 80%, 90%, 95%, and 100%) for 15 to 20 min at each step and then transferred to absolute acetone for 20 min. After dehydration, the specimens were placed in a 1:1
Figure 1. Enantiomers of chiral diclofop methyl (DM) and diclofop acid (DA) (* indicates chiral position).
activity of DA revealed that the R-DA is approximately twice as active as the racemic mixture against millets and oats.17 Based on the enantioselectivity on herbicidal activity, authorities in The Netherlands and Switzerland have revoked the registrations of racemic mixtures of chiral phenoxyalkanoic acids, while approving the registration of single-isomer products. 18 However, this policy did not take enantioselective environmental safety into account. It has been reported that DAenantiomers pose enantioselective ecotoxicity to three freshwater algae and their degradation in alga cultures was also enantioselective.19 Diclofop acid was also found to present enantioselective ecotoxicity on rice xiushui 63 seedlings.13 These two studies showed that herbicidal activity and environmental safety are not always consistent. Therefore, considering target (herbicidal activity) and nontarget (environmental safety) enantioselective bioactivity of chiral pesticides simultaneously is necessary, and such environmental toxicity investigation of chiral pesticides is imminently in need. In this study, the enantioselective physiological effects of diclofop methyl and diclofop acid on the cyanobacteria Microcystis aeruginosa were investigated. M. aeruginosa is the dominant species in summer water blooms in many inland waters in China recently.20 The biomass and the protein content of the cells were determined after exposure to the 4 3894
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Figure 2. Growth curves for M. aeruginosa exposed to DA (a), R-DA (b), S-DA (c), and DM (d). * Indicates p < 0.05 and ** indicates p < 0.01 relative to the control (ANOVA). Different capitalized letters indicate significant differences (p < 0.05) among different concentration exposures on the same day, while the same letter indicates no significant difference (ANOVA).
Figure 3. Synergistic effects of the two enantiomers. R-DA and S-DA alone stimulated biomass production at 0.5 mg/L and 1.0 mg/L, while the racemic mixture of DA exhibited inhibition effect for concentrations equal to the sum of the individual components.
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mixture of absolute acetone and the final resin mixture for 1 h at room temperature and then transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3 h and last to the final resin mixture overnight. Specimens were placed in capsules containing embedding medium and heated at 70 °C for 9 h. The specimen sections were stained by uranyl acetate and alkaline lead citrate for 15 min respectively and observed by TEM (Joel, JEM Model 1230). 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.
RESULTS AND DISCUSSION
The Linear Equation between Cell Number and Optical Density of Algal Culture. The number of cells per milliliter was counted, and the corresponding optical density at 685 nm was measured. Growth Curves of M. Aeruginosa. Growth curves for M. aeruginosa exposed to different forms and concentrations of diclofop are shown in Figure 2a-d. At 0.5 mg/L, DA slightly (not significant) stimulated the production of biomass during the experiment period (Figure 2a). While at the concentration of 1 mg/L and 2 mg/L, the growth of M. aeruginosa was significantly inhibited by DA after day 5. The percent inhibitions on day 9 were 32.6% and 34.8% for 1 mg/L and 3895
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Figure 4. The protein contents of M. aeruginosa cells after treatment for 3 days with different forms of diclofop. * Indicates p < 0.05 and ** indicates p < 0.01 relative to the control (ANOVA). Different capitalized letters indicate significant differences (p < 0.05) between different concentration exposures, while the same letter indicates no significant difference (ANOVA).
mixture of 1 mg/L of DA (equals 0.5 mg/L R-DA plus 0.5 mg/ L S-DA) exhibited inhibition effect. The results were similar for the 1 mg/L of R-DA plus 1 mg/L S-DA. Lin et al.24 studied the joint acute toxicity of isocarbophos enantiomers to Daphnia magna and found that the joint toxicity of combinations of enantiomers of chiral organophosphorus insecticides may be additive, synergistic, or antagonistic. The joint toxicity of enantiomers of a chiral compound is usually thought to be additive. However, the results in the present study show a synergistic effect. To our best knowledge, before this study, there has not been a study that has attempted to understand such antagonistic or synergistic interactions between enantiomers. It may be attributable to in vivo enantiomerization or other in vivo enantioselective biological reaction. Some enantiomers of a chiral compound are known to undergo chiral inversion or enantiomerization in organisms.25 Alternatively, the presence of one enantiomer may facilitate or hinder the other enantiomer’s binding to the active sites in an organism simply because the enantiomers have similar modes of toxic action.24 The effects between the pair of enantiomers were enantioselective. The biomasses of M. aeruginosa exposed to 0.5 mg/L of R-DA and S-DA were significantly different on days 3 and 4. R-DA was more promotive than S-DA. At 1 mg/ L, the biomasses were also significantly different between the two enantiomers on day 1. At 2 mg/L, the two enantiomers did not exhibit significantly different effects. At 5 mg/L, the biomass treated with DA could not be determined, whereas RDA and S-DA exhibited significantly different effects on day 1 and day 11. Based only on the biomass results above, it can be speculated that S-DA exhibited a greater stimulation effect than the corresponding R-DA. Different biological effects of diclofop enantiomers have been observed in other studies. For example,
2 mg/L, respectively. On day 11, the difference in biomass between the exposure concentration of 1 mg/L and 2 mg/L was significantly different. In contrast, R-DA significantly stimulated the growth of M. aeruginosa from day 4 to day 7 (Figure 2b). The percent stimulations on day 7 were 11.1%, 10.6%, 10.2%, and 14.0% for 0.5, 1, 2, and 5 mg/L, respectively. Interestingly, the stimulations were independent of compound concentration. After day 8, R-DA at 5 mg/L inhibited biomass production relative to the control. The percent inhibitions were 4.0% and 10.3% for days 9 and 11, respectively. After day 7, the stimulations by 2 mg/L R-DA were less than by 0.5 and 1 mg/L R-DA. In general, the growth curves of M. aeruginosa exposed to SDA were similar to those for R-DA (Figure 2c). On day 7 the percent stimulations were 6.4%, 12.7%, 17.3%, and 15.7% for the samples exposed to 0.5, 1, 2, and 5 mg/L S-DA, respectively. Apparently, the stimulation percentages were related to the compound concentration. In contrast to R-DA, S-DA did not significantly inhibit the growth of M. Aeruginosa at any concentration, although at 5 mg/L the growth rate slowed from day 8 onward. DM also significantly stimulated biomass production (Figure 2d). On day 7 the percent stimulations were 12.0%, 12.9%, 15.3%, and 20.5% for the 0.5, 1, 2, and 5 mg/L, respectively. Like S-DA, the stimulation percentages were related to DM concentration. However, the stimulation of the samples treated with 5 mg/L DM decreased to 3.7% on day 9. In general, R-DA, S-DA, and DM stimulated the cell growth of M. aeruginosa, whereas DA exhibited an inhibition effect. The biomass changes demonstrate the synergistic effect between the pair of enantiomers (Figure 3). Both R-DA and S-DA alone exhibited stimulation effects at 0.5 mg/L, while the racemic 3896
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Figure 5. The protein content of M. aeruginosa cells after treatment for 6 days with different forms of diclofop. * Indicates p < 0.05 and ** indicates p < 0.01 relative to the control (ANOVA). Different capitalized letters indicate significant differences (p < 0.05) between different concentration exposures, while the same letter indicates no significant difference (ANOVA).
Cai et al.19 studied the toxicity of diclofop on freshwater unicell green alga and found that S-DA was more toxic than R-DA. We previously studied the toxicity of diclofop enantiomers on rice xiushui 63 seedlings13 and revealed that the toxicity on the roots and leaves were reverse. In addition, there are many reports about the enantioselective biotoxicity of chiral insecticides on aquatic invertebrates and human cells.11 These results demonstrate that enantioselective biological effects on organisms in the environment are common characteristics of chiral pesticides. In the present study, the growth curves reveal that both R-DA and S-DA stimulate the growth of M. aeruginosa, which is adverse to water quality in reservoirs and other bodies of water. The stimulation by S-DA was greater than that due to R-DA. In addition, it is worth noting that the maximum optical density varied from 685 to 680 nm. Since cyanobacteria contain only chlorophyll a, the hypochromatic shift suggests damage to the chlorophyll a in the thylakoid. To establish whether the treated cells were composed of damaged cells or healthy cells and to identify the internal toxicology mechanism(s), further studies were needed to investigate the cell characteristics. Therefore, protein determinations and ultrastructure observations were carried out. In particular, the effects of the chemicals at 5 mg/L should be noted, since all the growth curves for M. aeruginosa treated at 5 mg/L all have an inflection point. Protein Content of M. aeruginosa. The protein content of M. aeruginosa cells on day 3 following treatment with different kinds of diclofop are shown in Figure 4. During the period from 0−3 days, the protein content results were consistent with the biomass results. DA stimulated the protein content at 2 mg/L, R-DA exhibited a stimulation effect at 1 and 2 mg/L, and S-DA and DM stimulated protein production at 1, 2, and 5 mg/L. While the four chemicals stimulated protein production on day 3, different results were obtained on day 6 (Figure 5). DA
inhibited the protein content at 1 and 2 mg/L, which was consistent with the biomass results. But for R-DA, S-DA, and DM, the protein content decreased while the biomass increased. The inconsistency between biomass production and protein content indicated that the stimulations by R-DA and S-DA on biomass production may be a “false stimulation”, meaning that a large portion of the biomass may be composed of damaged cells. As noted above, the shift in the wavelength of peak optical density also supports this possibility. Apparently, the number of cells increased, but the protein synthesis was interrupted by diclofop acid enantiomers. These results indicate that using biomass as the only parameter to judge the toxic effect of chemicals may be insufficient and inaccurate. To further explore differences in toxicity effects between the two enantiomers, the ultrastructures of the cells were observed. Ultrastructure Characteristics. The ultrastructure of M. aeruginosa cells exposed to the different chemicals at 5 mg/L were observed with transmission electron microscope (TEM). As mentioned above, the 5 mg/L exposure concentration was particularly worth noting because all the growth curves at that concentration have an inflection point. Therefore, cell samples treated at 5 mg/L with the chemicals were prepared after 72 h of growth. The ultrastructure of the cells are shown in Figure 6. In the control samples (Figure 6A), almost 50% of the cells were in the process of dividing (Figure 6A-d). A layer of mucilage (M) surrounded the cells (Figure 6A-a). Cell wall layers containing LI through LIV were clearly visible (Figure 6Ab, arrow). The photosynthetic apparatus thylakoids (T) were aligned mainly parallel to the cytoplasmic membrane and also surrounded the nucleoplasmic area (N) (Figure 6A-a). A small amount of glycogen (G) was present between the thylakoids. Cyanophycin granules (CG), poly beta-hydroxybutyrate (PBH), polyhedral bodies (PB), and lipid (L) were present in the cells. Gas vacuoles (GV) consisting of packed arrays of cylindrical vesicles with conical ends were easily observed 3897
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Figure 6. Ultrastructure of M. aeruginosa cells in samples after 72-h inoculation with 5 mg/L of the specified herbicide: control (A), DA (B), R-DA (C), S-DA (D), and DM (E). Mucilage-M, thylakoids-T, nucleoplasmic area-N, cyanophycin granules-CG, hydroxybutyrate-PBH, polyhedral bodyPB, and lipid-L were observed.
(Figure 6A-b). The ability of M. aeruginosa to float accounts for a large portion of their biological success, and certainly for much of their contribution to water blooms, so the presence of these organelle are critical to its growth and survival. After 72 h of treatment with 5 mg/L of DA, about 50% of the cell wall layers had disappeared. Other segments were discontinuous and detached from the cytoplasmic membrane (Figure 6B). Most cell structures were seriously disrupted (Figure 6B-a, b). Thylakoids were scarce and arranged both parallel and perpendicular to the cell wall. GV were largely absent, and CG, PBH, PB, and L were not observed except in Figure 6B-a. The most obvious characteristics in cells treated with R-DA after 72 h were the increases in the number of GV and T. Abundant GV were observed in cells (Figure 6C-c). Thylakoids were also abundant and arranged in stripes (Figure 6C-a) both parallel and perpendicular to the cell wall (Figure 6C-a, d). Less than 50% of the cells were in the process of dividing (Figure 6C-b). Cell walls were discontinuous and detached from the cytoplasmic membrane (Figure 6C-c, d, arrow). CG and L were distributed in cells.
In contrast, the ultrastructure of cells exposed to S-DA was similar to the control (Figure 6D). CG, GV, PB, PBH, and L were clearly detected. Thylakoids were arranged parallel and perpendicular to the cytoplasmic membrane (Figure 6D-b). Cell walls were detached from the cytoplasmic membrane, but no discontinuities were observed (Figure 6D-a, c). Half of the cells were in the process of dividing (Figure 6D-d). Cell walls were seriously damaged after treated with DM (Figure 6E). Many were detached from the cytoplasmic membrane. Although CG and L could be observed, the number was decreased. Abundant thylakoids were arranged perpendicular to the cytoplasmic membrane (Figure 6E-b, c) and cells exhibited asymmetric division (Figure 6E-b, d). The four chemicals caused different physiological effects on M. Aeruginosa, and the effects between R-DA and S-DA were shown to be enantioselective. First, gas vacuoles were collapsed by treatment with DA or DM; however, they increased in number when cells were treated with R-DA and changed little when treated with S-DA. Gas vesicles provide buoyancy to aquatic prokaryotes and GV walls are formed entirely of protein.26 The collapse of GV in DA or DM treated cells may due to the disruption of the GV walls. Jones27 reported that gas 3898
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treated samples, suggesting either overproduction of the enzymes responsible for degrading this polysaccharide or a reduction in the amount of enzymes involved in glycogen production and storage. This result is also consistent with the R-DA being more toxic than S-DA. In conclusion, differences in ultrastructural characteristics of M. aeruginosa cells exposed to the 4 closely related chemicals suggest that the toxicity of the four chemicals is different and that the mechanisms of their toxicity are also different. DA was the most toxic, followed by DM, R-DA, and S-DA. Enantiomers usually exhibit different effects and toxicity on target and nontarget organisms. Often, the target herbicidal activity receives much more attention than the environmental risks. In the present study, we investigated the physiological characteristics of M. aeruginosa cells exposed to different forms of diclofop, including R-DA, S-DA, a racemic mixture of DA, and the mother compound DM. Our results demonstrated that (1) using only biomass growth as an indicator of toxicity to cyanobacteria is not sufficient. Protein content is an effective additional indicator providing useful insight into the overall toxicity. The concentration of 5 mg/L is especially worth noting because of the pronounced inhibition to M. aeruginosa biomass production observed after several days of growth. (2) The shift in the wavelength of maximum cell optical density from 685 to 680 nm demonstrates that the damage of chlorophyll a exists in thylakoid. (3) Ultrastructure changes in the cells, including changes to cell walls, gas vacuoles, thylakoids, glycogen, cyanophycin granules, poly beta-hydroxybutyrate, polyhedral body, and lipids, indicate different toxicity mechanisms among the four compounds. The differences between R-DA and S-DA indicate that R-DA probably acts as a proton ionophore, whereas S-DA does not have such action. (4) The toxicity to environmentally harmful M. aeruginosa at environmentally significant concentrations is DA > DM > RDA > S-DA, which indicates that the herbicidally active Renantiomer is more environmentally friendly than the Senantiomer based on our present study. Therefore, using enantiomer-pure diclofop (R-diclofop methyl) in agriculture is recommended.
vesicles collapsed when exposed to the vapors of various organic solvents, e.g., chloroform, ethanol, or ether. Jones and Jost28 noted that the gas vacuole membrane protein seems to be more difficult to solubilize than the proteins of other membranes with only strongly protic solvents causing appreciable solubilization. Both diclofop methyl and diclofop acid are known/thought to be specific proton ionophores that shuttle protons across the plasmalemma. Additionally oxidative membrane catabolism by free radical lipid peroxidation induced by diclofop and its coupling to the effect of diclofop on the transmembrane proton gradient were proved to be the mechanisms of diclofop toxicity.29 Wright and Shimabukuro30 also indicated that AOPP herbicides modulate cytosolic pH by increasing the permeability of the plasmalemma to protons. The damage to the cell walls and GV walls observed in the present ultrastructural characteristics study suggesting that the toxicity mechanism of DM and DA probably lies in the proton ionophore hypothesis. However, the exact mechanism is uncertain and merits further research. The differences between R-DA and S-DA were significant. The number of GV in cells treated with R-DA was much larger than in cells treated with S-DA, indicating different mechanisms for the two enantiomers. The vesicles are often induced by ionic deficiency31 and may occupy as much as 60−70% of the cell volume. Jost suggested that they may be a means for concentrating ions within cells by decreasing the intracellular solution volume.32 R-DA likely acted as a proton ionophore shuttling protons across the plasmalemma, therefore increasing the number of gas vacuoles. Whereas S-DA does not act as a proton ionophore, and the change in number of gas vacuoles was not obvious. Therefore, S-DA is largely nontoxic to the growth of the cells. In contrast, R-DA is toxic to M. aeruginosa. Second, under normal conditions thylakoids are closed discs surrounding and aligned parallel with the nucleoplasmic area (Figure 6A). However, after treatment with any of the four chemicals, the arrangement of the thylakoids changes and become both parallel and perpendicular to the cytoplasmic membrane (Figure 6B-6E). Several studies have reported that thylakoids occurring in bundles perpendicular to the cell wall could represent a mechanism to protect the cell from excessive irradiation and attack by superoxide anions.33,34 Unfortunately, there is no literature available on the morphologic changes of thylakoids in cells exposed to pesticides. However, our observations are consistent with this change in thylakoid orientation serving as a protection mechanism. Our observations indicate significant differences between the enantioselective effects of the enantiomers leading to our conclusion that R-DA is more toxic than S-DA. Third, other intracellular components including CG, G, PB, PBH, and L are also affected by exposure to the different chemicals. CG serves as a nitrogen reserve.35 In DA treated samples, CG almost completely disappeared. However, in RDA treated samples, CG are both more obvious and more numerous (Figure 6C). In S-DA and DM treated samples, CG are also easily observed. The disappearance of CG in DA treated samples is probably because the cells utilized the nitrogen supplies during growth and division under stressed conditions, whereas our observation that R-DA treated cells had distinct CG may indicate specific availability of nitrogen storage.36 Glycogens, a major product of photosynthesis by blue-green algae, are found between the photosynthetic lamellae. The number of G was decreased greatly in DA, DM, and R-DA
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ASSOCIATED CONTENT
S Supporting Information *
Three tables of the biomass of M. aeruginosa exposed to DA, RDA, and S-DA at concentration of 0.5, 1, 2, and 5 mg/L. The significance analysis on the biomass of M. aeruginosa exposed to DA, R-DA, and S-DA. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86 571 8898 2740. Fax: +86 571 8898 2341. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2009CB421603); the project of Science and Technology Commission of Shanghai Municipality, China (Grant No. 11ZR1435600); the Fund of the Key laboratory of Urban Agriculture (South), Ministry of Agriculture, China; Shanghai Young College Teacher Training Subsidy Scheme, 3899
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China (Grant No. yyy11017); the National Natural Science Foundation of China (Grant No. 20977062); and the Shanghai Institute of Technology Scientific Research Foundation for Introduced Talent, China (Grant No. YJ2011-50).
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ABBREVIATIONS DA diclofop acid {(R,S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate acid} DM diclofop methyl {(R,S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate acid methyl ester} TEM transmission electron microscope M mucilage T thylakoid N nucleoplasmic area CG cyanophycin granule PBH hydroxybutyrate PB polyhedral body L lipid G glycogen GV gas vacuole AOPP aryloxyphenoxy propionic acids
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dx.doi.org/10.1021/es304593c | Environ. Sci. Technol. 2013, 47, 3893−3901
Environmental Science & Technology
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dx.doi.org/10.1021/es304593c | Environ. Sci. Technol. 2013, 47, 3893−3901
