Enantioselective toxicity and bioaccumulation of epoxiconazole enantiomers to the green alga Scenedesmus obliquus

Abstract

Enantioselectivity in environmental behaviour of chiral pesticides has become a subject of growing interest. In this study, ecotoxicity and bioaccumulation of epoxiconazole enantiomers to Scenedesmus obliquus was studied. The acute toxicities of epoxiconazole enantiomers were enantioselective. In addition, enantioselectivity was observed from the determined chlorophyll contents, malondialdehyde contents and antioxidant enzyme (superoxide dismutase and catalase) activities of algae cells after incubation with epoxiconazole racemate or single enantiomers. Moreover, the algae cells treated by the enantiomers of epoxiconazole also showed morphological changes, including plasmolysis phenomena, the disappearance of chloroplasts and nuclei, and the accumulation of lipid droplets and starch granules. In a bioaccumulation experiment, an enrichment in (−)-epoxiconazole was observed during the first three days of algal cell exposure to the racemate; however, (+)-epoxiconazole became enriched after four days of exposure. For the individual-enantiomer treatment, no enantiomerization or enantioselectivity occurred, and the accumulation factor values were higher than those of algae exposed to the racemate. Furthermore, Scenedesmus obliquus had positive effects on the dissipation of epoxiconazole in water.

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1. Introduction

Epoxiconazole is a widely used chiral triazole fungicide. Chirality is a common property of pesticides and is present in more than 25% of all known pesticides.¹ For chiral pesticides that consist of two or more enantiomers, usually only one of them exhibits activity against the target, whereas the other compound or compounds are inactive and unwanted burdens to the environment. Generally, enantiomers in an achiral environment display the same physicochemical properties, however, they may behave differently in terms of biological activities and in biologically mediated environmental processes.² However, most chiral pesticides are produced as racemates. Therefore, as the environmental behaviour of chiral agriculture chemicals is receiving increasing attention, it is essential to study the enantioselective behaviours of such pesticides to better understand the risks of chiral pesticides to the environment and public health.

Epoxiconazole, which was first produced by BASF,³ contains two chiral centres and thus presents two diastereoisomers with four stereoisomers. However, the present process of epoxiconazole production gives only one pair of enantiomers with 2R, 3S- and 2S, 3R-configurations. Structures of the two enantiomers are provided in the ESI (Fig. S1†).⁴ This fungicide is used extensively all over the world to control diseases caused by fungi in cereals, sugar beet, peanuts, oilseed rape, apple, and ornamentals.⁵ On the other hand, it can enter into the fresh water cycle and cause water pollution after prolonged and repeated application. Recent studies have shown that the release of agricultural compounds from agrichemicals including epoxiconazole into the aquatic environment via underground water and irrigation has adverse effects on non-target aquatic microorganisms.^6,7

Microalgae, the major aquatic microorganisms that provide oxygen and organic substances to other organisms such as fish and invertebrates,^8–10 are primary producers and occupy the first trophic level in the aquatic food chain. They play an important role in maintaining the balance of aquatic ecosystems.^11,12 The biological adaptation of microalgae to pesticides has been investigated by researchers with respect to growth, physiological parameters and bioaccumulation. At the physiological level, excessive reactive oxygen species (ROS) are produced due to the negative effects of pesticides. This could generate oxidative stress to nucleic acids or lipid proteins, consequently leading to the damage of various cellular organelles. Moreover, various contaminants induce lipid peroxidation, causing membranes to change into malondialdehyde (MDA) and become rigid. For example, algae chloroplasts are rich in polyunsaturated fatty acids, which are potential targets for peroxidation.^13,14 As an important secondary end product of oxidation, MDA is used extensively to determine the level of oxidative stress.^15,16 Of course, ROS is also an important signal to initiate the biological defence mechanism. There are multiple intracellular non-enzymatic antioxidants such as carotenoids, ascorbic acid and vitamin A as well as antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT).^13,17 They can also serve as indicators for determining the extent of a biological defence mechanism. Furthermore, algae have been reported to bioaccumulate and metabolise different types of pollutants; this has caused concern about the transfer of pollutants to upper trophic level organisms through the food chain and spurred interest in developing green algae-based cleaning systems for aquatic environments.¹⁸

Scenedesmus obliquus, a widely distributed freshwater microalgae, is one species of the genus Scenedesmus (Chlorophyta). They are colonial and non-motile, generally with four cell arranged in a row. S. obliquus is sensitive to different contaminants and is easy to handle in laboratory cultures. Dewez et al. found that different concentrations of copper can significantly inhibit the growth rate and chlorophyll synthesis of S. obliquus.¹⁹ The toxicities of chiral triazole fungicides including difenoconazole, hexaconazole and myclobutanil to S. obliquus have also been investigated.^20–22 However, data on the enantioselective toxicities and bioaccumulations of epoxiconazole enantiomers in S. obliquus have not been reported.

In this study, 96 h acute toxicity tests were assayed, and the median lethal concentration (EC₅₀) values were calculated to compare the acute toxicities of (−)-, (+)- and rac-epoxiconazole. Additionally, the pigment contents, antioxidant enzyme (CAT and SOD) activities and MDA contents were determined. Changes in the ultrastructural morphologies of algae cells treated with epoxiconazole enantiomers were also studied by transmission electron microscopy (TEM), and possible enantioselective toxic effects of rac-, (−)- and (+)-epoxiconazole on S. obliquus were explored. Furthermore, we evaluated the enantioselectivity of epoxiconazole during its bioaccumulation and degradation in S. obliquus.

2. Materials and methods

2.1. Chemicals and reagents

A reference standard of rac-epoxiconazole (98.4% purity) was provided by the Shanghai Pesticide Research Institute (Shanghai, China). The enantiomers of epoxiconazole were prepared by high-performance liquid chromatography (HPLC; Agilent) with a chiral column (CDMPC-CSP) provided by the Department of Applied Chemistry, China Agricultural University, Beijing. The stock solutions of epoxiconazole and its enantiomers were prepared by dissolving the three compounds in acetone.

Acetone, formic acid, methanol and ethyl acetate were analytical grade and were purchased from Beijing Chemical Reagent Co. Ltd. (Beijing, China). Acetonitrile was HPLC grade and was provided by J. T. Baker (Phillipsburg, NJ, USA). Water was filtered with a 0.22 μm membrane (Milli-Q system, Millipore, Bedford, MA) prior to use.

2.2. Algae culture and growth conditions

S. obliquus was obtained by the Institute of Hydrobiology, the Chinese Academy of Sciences, and cultivated in 100 mL of liquid HB-4 algae growth media in 250 mL flasks at 25 °C in an incubator under 16 : 8 h light : dark cycles and illumination ranging from 3000–4000 lx. The HB-4 medium was prepared according to the Chinese National Environmental Protection Agency Guidelines 201.²³ Details regarding the media are provided in the ESI (Text S1†). The culturing was carried out on a super clean bench, and all of the flasks, culture media and other glassware were sterilized prior to use to ensure an axenic culture. The cultures were shaken three times per day, and the algae cells were periodically inoculated in fresh media to keep the cells in the logarithmic growth phase.

2.3. Algae growth inhibition test

An algae growth inhibition test was carried out to determine the EC₅₀ (the effective pesticide concentration that reduces the population growth rate by 50%), according to the updated Organization for Economic Cooperation and Development (OECD) guideline 201 for freshwater algae and cyanobacteria growth inhibition test.²⁴ The stock solution of epoxiconazole prepared in acetone was dissolved in HB-4 media to achieve the desired series of concentrations. Exponentially growing cells of S. obliquus presented as unicell or four-celled coenobia were inoculated at an initial density of 2 × 10⁵ cells per mL. These concentrations ranged from 0.5 to 20.0 mg L⁻¹ (rac-epoxiconazole and (−)-epoxiconazole: 1, 2, 5, 10, 15 and 20 mg L⁻¹; (+)-epoxiconazole: 0.5, 1, 2, 5, 10 and 15 mg L⁻¹). The controls were grown with no fungicide. Each assay was performed in triplicate. The S. obliquus cell density was monitored for optical density using light with a wavelength of 650 nm at the end of 24, 48, 72 and 96 h, and the inhibition of algae growth was calculated by normalizing the data to the results of the control cultures.

2.4. Determination of chlorophyll and carotenoids

S. obliquus cells exposed to 1, 5 and 10 mg L⁻¹ of racemate or one of the enantiomers of epoxiconazole for 96 h were collected and centrifuged at 10 000 rpm for 10 min. Chlorophyll a, chlorophyll b and carotenoids were extracted in 80% acetone in the dark for approximately 24 h. The crude extract was centrifuged at 10 000 rpm for 10 min, and OD₄₄₀, OD₆₄₅ and OD₆₆₃ values of the supernatant were measured. The concentrations of chlorophyll and carotenoids were calculated by the equations^25,26 C_A = (12.7OD₆₆₃–2.69OD₆₄₅)/ρ, C_B = (22.9OD₆₄₅–4.68OD₆₆₃)/ρ, C_T = 1000OD₆₅₂/34.5ρ and C_K = (4.7OD₄₄₀–0.27C_T)/ρ, where C_A, C_B, C_T and C_K are the concentrations of chlorophyll a (μg per cell number), chlorophyll b (μg per cell number), total chlorophyll (μg per cell number) and carotenoids (μg per cell number), respectively, and ρ is the density of the algae suspension (cell number per mL⁻¹).

2.5. Preparation of protein extracts

Protein was extracted and measured according to the method of Bradford.²⁷ S. obliquus cells were exposed to 1, 3 and 6 mg L⁻¹ of racemate and individual enantiomers of epoxiconazole for 96 h. Detailed information on the protein sample extraction is provided in the ESI (Text S2†).

2.6. Determination of antioxidant enzyme and malondialdehyde

The supernatant of extract mentioned in 2.5 was used to determine antioxidant enzyme activities and MDA content.

The SOD activity was measured based on its capacity to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) according to Giannopolitis et al.²⁸ Three millilitres of reaction mixture (50 mmol L⁻¹ pH 7.8 phosphate buffer, 0.1 mmol L⁻¹ EDTA–Na₂, 0.13 mol L⁻¹ methionine, 0.75 mol L⁻¹ NBT, 20 mmol L⁻¹ riboflavin and an appropriate aliquot of enzyme extract) were illuminated at a light intensity of 4000 lx, and absorbance was recorded at 560 nm after 20 min.

The CAT activity was determined spectrophotometrically by measuring the disappearance of H₂O₂ at 240 nm over 3 min at 20 °C according to Aebi.²⁹ The reaction mixture contained Tris–HCl (67 mmol L⁻¹, pH 7.4), H₂O₂ (15 mmol L⁻¹) and 50 μL algae extract in a 3 mL volume.

The MDA content was measured based on the thiobarbituric acid (TBA) method using a commercial malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institut, China).

2.7. Observation of algae cell ultrastructure

The algae cells treated by the control (no epoxiconazole added) and epoxiconazole enantiomers (6 mg L⁻¹ rac-, (−)- and (+)-epoxiconazole) were harvested after 96 h for TEM analysis. The detailed methods are provided in the ESI (Text S3†).

2.8. Enantioselective bioaccumulation and degradation of epoxiconazole in algae

To measure the accumulation and degradation of epoxiconazole, S. obliquus was treated with rac-, (−)- and (+)-epoxiconazole at a final concentration of 2 mg L⁻¹ (for the racemate) or 1 mg L⁻¹ (for individual enantiomers) for six days. No 0 d samples for the test groups were taken because no detectable epoxiconazole was found in unspiked algae; thus, the epoxiconazole concentration in the algae or medium could be considered as zero at 0 d. To determine the effect of S. obliquus on the stability of epoxiconazole, pesticide degradation in the presence of algae was compared to that in the control medium containing only chemicals (no algae). All of the flasks were cultured under the same conditions as previously described in 2.2 and conducted in triplicate. Detailed information about the sample extraction and chemical analysis is provided in the ESI (Text S4†).

2.9. Data analysis

The accumulation factor (AF), a function of the relative sorptive capacities of the organism versus the surrounding environment, was used to express the bioaccumulation of epoxiconazole enantiomers in S. obliquus. Herein, AF is defined as

AF = C_algae/C_medium,

where C_algae and C_medium are the concentrations of epoxiconazole enantiomers in algae and medium, respectively.

The statistical analysis of the enantioselectivity of epoxiconazole enantiomers was performed using SPSS 20.0. The EC₅₀ values were calculated using the linear regression method.^30,31 The differences between individual treatments were analysed using analysis of variance (ANOVA). The means were analysed using the Dunnett t-test at P < 0.05, and a pairwise multiple-comparison procedure (S–N–K test) was used to compare results at P < 0.05. The data shown are presented as the means ± SD.

3. Results and discussion

3.1. Inhibition of S. obliquus growth

The inhibition of S. obliquus growth by epoxiconazole enantiomers is concentration-dependent. At low concentrations (1 mg L⁻¹ for rac-epxiconazole; 0.5 mg L⁻¹ for (−)- and (+)-epoxiconazole, respectively), the inhibition ratios (percentages of reduced growth relative to the control cultures) after 24 h of exposure for the rac-, (−)- and (+)-epoxiconazole were 5.2%, 2.9% and 7.4%, respectively, and they were 8.4%, 13.7% and 20.9%, respectively, after 96 h of exposure. At high concentrations (20 mg L⁻¹ for rac- and (−)-epoxiconazole; 15 mg L⁻¹ for (+)-epoxiconazole), the inhibition ratios of the rac-, (−)- and (+)-epoxiconazole were 30.3%, 34.2% and 32.7%, respectively, after 24 h of exposure and 90.7%, 90.6% and 89.7%, respectively, after 96 h of exposure. Higher concentrations significantly inhibited algae growth, and inhibition ratio increased with time for all treatments.

The EC₅₀, an index used to evaluate the toxicity of a compound, was calculated from the growth inhibition. The EC₅₀ values decreased with increasing exposure time (Table 1). The EC₅₀ values of rac-epoxiconazole-treated algae after 24 and 48 h (55.416 and 21.711, respectively) were higher than those at 72 and 96 h (9.412 and 5.478, respectively). The same trend was also observed for the (−)- and (+)-epoxiconazole treatments. The differences between the 72 h- and 96 h-EC₅₀ values were small, indicating that the fungicide toxicity was nearly constant after three days.

Table 1 EC₅₀ values of rac-epoxiconazole and its enantiomers

Time (h)	Compound	Regression equation	R^2	EC50^a (mg L^−1)
a EC50, the effective concentration that results in a 50% reduction in population growth compared with the control.
24	rac-Epoxiconazole	y = 0.958x + 3.330	0.947	55.416
	(−)-Epoxiconazole	y = 1.055x + 3.318	0.906	39.271
	(+)-Epoxiconazole	y = 0.936x + 3.557	0.938	34.856
48	rac-Epoxiconazole	y = 1.653x + 2.790	0.956	21.711
	(−)-Epoxiconazole	y = 1.508x + 3.105	0.947	18.045
	(+)-Epoxiconazole	y = 1.168x + 3.639	0.980	14.616
72	rac-Epoxiconazole	y = 1.931x + 3.120	0.953	9.412
	(−)-Epoxiconazole	y = 1.646x + 3.607	0.940	7.023
	(+)-Epoxiconazole	y = 1.428x + 3.982	0.939	5.160
96	rac-Epoxiconazole	y = 2.077x + 3.466	0.975	5.478
	(−)-Epoxiconazole	y = 1.835x + 3.853	0.983	4.218
	(+)-Epoxiconazole	y = 1.405x + 4.466	0.975	2.402

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Significant differences in epoxiconazole enantiomer toxicity were observed at all exposure times. The toxicity at each time point decreased in the following order: (+)-epoxiconazole > (−)-epoxiconazole > racemate. The results of the growth inhibition tests clearly show that the epoxiconazole enantiomers exhibited enantioselective toxicity to S. obliquus. The rac-epoxiconazole is less toxic to S. obliquus than its individual enantiomers.

3.2. The pigment content of S. obliquus

Epoxiconazole may influence the synthesis of chlorophyll and carotenoids in algae cells. After S. obliquus was exposed to 1, 5 and 10 mg L⁻¹ of epoxiconazole, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly affected (Fig. 1A–C). At low epoxiconazole concentrations (1 mg L⁻¹), there was a pronounced hormesis for the treatments with rac- and (−)-epoxiconazole. A similar phenomenon was observed in the studies of Cheng et al. and Fisher et al.^20,32 This can be explained by the fact that breakdown of the fungicides may have provided the algae with carbon and nitrogen.³³ At moderate and high concentrations of epoxiconazole (5 and 10 mg L⁻¹), chlorophyll content was significantly decreased compared with the control group (P < 0.05), indicating that the ability of the cells to synthesize chlorophyll was decreased.³⁴ The treatments with (+)-epoxiconazole did not result in pronounced hormesis because the lowest treatment concentration (1 mg L⁻¹) was not a sufficient amount of (+)-epoxiconazole, which has a relatively high growth inhibition (96 h-EC₅₀; 2.402 mg L⁻¹). Chlorophyll reduction is a marker for oxidative stress.^35,36 The impairment of the electron transport chain and replacement of Mg²⁺ ions associated with the tetrapyrrole ring of chlorophyll molecules primarily destruct photosynthetic pigments.³⁷ In general, the above results showed that low concentrations of epoxiconazole promote chlorophyll synthesis, while high concentrations of epoxiconazole inhibit it.

Fig. 1 Chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoid (D) contents in S. obliquus exposed to different concentrations of epoxiconazole racemate and enantiomers (bars are standard error). Different letters (abc) represent statistically significant differences, and * denotes significant difference between control and treatments (S–N–K test, P < 0.05).

The carotenoid content declined with increasing epoxiconazole concentration (Fig. 1D). Carotenoid serves as an antioxidant to scavenge free radicals and reduce damage to cells, cell membranes and their main genetic material.³⁸

The racemate and enantiomers of epoxiconazole had different effects on the chlorophyll and carotenoid concentrations in S. obliquus (Fig. 1). The contents of photosynthetic pigment for all treatments decreased in the following order: racemate > (−)-epoxiconazole > (+)-epoxiconazole. These results are in accord with the growth inhibition results. The change in chlorophyll content took place at the molecular level in cells and occurred much earlier than growth.²⁰ Therefore, chlorophyll may be a more sensitive parameter than growth inhibition when algae are exposed to xenobiotics.³⁹ It could be concluded that epoxiconazole significantly inhibits the photosynthetic pigment content at high concentrations and enhances it at low concentrations. In addition, an enantioselective photosynthetic process may occur when S. obliquus is treated by this chiral fungicide.

3.3. Detection of antioxidant enzyme activities

Various environmental stresses (heavy metals, heat stress and ultraviolet radiation) can induce the production of ROS, which cause oxidative stress to cells. Living organisms have mechanisms to protect against the potentially damaging effects of ROS using a suite of antioxidant enzymes and antioxidant substances. Previous studies have suggested that individual forms of chiral pesticides exhibit different toxicities, which may result from the different reactions of individual enantiomers with enzymes.⁴⁰ SOD and CAT, two important components in preventing the oxidative stress, are typically used as biomarkers to indicate ROS production.^41,42 Therefore, our intention was to use CAT and SOD activities to investigate whether epoxiconazole enantiomers have different effects on S. obliquus at the same dose and exposure time.

SOD could catalyse the disproportionation of O²⁻ to O₂ and H₂O₂ at a very quick rate.⁴³ In the present study, the SOD activities determined from the epoxiconazole racemate and enantiomers were significantly higher than that of the control group (P < 0.05) for the three tested concentrations (Fig. 2B). In addition, SOD activity increased with increasing epoxiconazole concentration. These results may result from the effect of epoxiconazole on the SOD gene or the indirect increase in the level of O²⁻ radicals.⁴⁴ Moreover, the SOD activity of algae exposed to (+)-epoxiconazole was higher than those of the algae exposed to the other two compounds at all of the three concentrations, probably indicating that higher efficiency of SOD was triggered by (+)-epoxiconazole.

Fig. 2 Effects of different doses of rac-epoxiconazole, (−)-epoxiconazole and (+)-epoxiconazole on (A) SOD activity, (B) CAT activity and (C) MDA content in S. obliquus (bars are standard error). Different letters (abc) represent statistically significant differences, and * denotes significant difference between control and treatments (S–N–K test, P < 0.05).

CAT could catalyse the production of H₂O from H₂O₂. As shown in Fig. 2A, the CAT activity of S. obliquus exposed to epoxiconazole was significantly enhanced compared to the control group (P < 0.05). CAT activity increased with increasing concentration of rac- and (−)-epoxiconazole, and the CAT activity of (−)-epoxiconazole-treated algae was higher than that of the racemate-treated algae. This may be due to the defence mechanism of algae cells to prevent subsequent damage, and it is in accordance with other toxicity results in which the (−)-epoxiconazole is more toxic than its racemate. However, for the cells treated with (+)-epoxiconazole, the CAT activities displayed an increase–decrease trend with increasing fungicide concentration; that is, the catalase activity for the high-concentration treatment (6 mg L⁻¹) was lower than that in the other treatments, indicating that (+)-epoxiconazole was the most toxic enantiomer in the present study. Colt et al. reported that once the oxidative stress causes dramatic changes in resistant enzyme activity and the bioaccumulation of H₂O₂, the CAT activity is inhibited.⁴⁵ The SOD/CAT system is one of the main complementary ROS-scavenging systems, and the product of SOD is detoxified by CAT and APX.^14,46

3.4. Effect on MDA content

To determine the lipid peroxidation of algae cells caused by ROS, the MDA contents of S. obliquus cells treated by epoxiconazole for 96 h are illustrated in Fig. 2C. The MDA contents of the treated cells at the three tested concentrations were higher than those of the control group (P < 0.05); that is, the algae cells suffered oxidative lesions in lipids after epoxiconazole exposure, and the antioxidant enzymes induced by epoxiconazole were not able to completely eliminate ROS within 96 h. Moreover, the MDA contents increased with increasing rac- and (−)-epoxiconazole concentrations, and the MDA values of algae treated by (−)-epoxiconazole were all larger than those of algae treated by rac-epoxiconazole (P < 0.05). These results matched those from our toxicity test indicating that the (−)-epoxiconazole is more toxic than the rac- epoxiconazole and therefore induced the cells to generate more ROS. Nevertheless, the MDA contents decreased with increasing (+)-epoxiconazole concentration, but they were still higher than those of the control group at the three test concentrations. At moderate and high epoxiconazole concentrations (3 and 6 mg L⁻¹), the MDA values for the (+)-epoxiconazole treatments were lower than those for the (−)-epoxiconazole treatments (P < 0.05), showing a significant enantioselectivity.

3.5. Ultrastructural observation of S. obliquus

The TEM analysis showed that the racemate and two enantiomers of epoxiconazole affected cell morphology and exhibited enantioselective toxicity to S. obliquus. The TEM results for the test and control treatments are shown in Fig. 3. The typical control S. obliquus cell (Fig. 3A) was shuttle-shaped and enclosed with a rigid cell wall. The peripherally located chloroplasts took up almost half of the cell, in which thylakoids can be seen. The nucleolus and nucleus vacuole existed in an undamaged nucleus with an intact nucleus membrane. Oval-shaped mitochondria could be seen in the cytoplasm of some cells (not shown).

Fig. 3 Representative photos from transmission electron micrographs (TEM) of control cells (A) and cells exposed to rac-epoxiconazole (B), (−)-epoxiconazole (C) and (+)-epoxiconazole (D) after 96 h. ¹Cell wall, ²chloroplast, ³nucleus membrane, ⁴nucleus, ⁵cytoplasm, ⁶plasmolysis, ⁷starch, ⁸lipid droplet.

The cells exposed to epoxiconazole enantiomers (Fig. 3B–D) had deformed shapes and damaged cell walls. Serious plasmolysis phenomena were observed, and some cytoplasm leaked into the vacuity, especially for the (+)-epoxiconazole-treated cells. This may be explained by the disruption of cell plasma membranes by the epoxiconazole enantiomers, resulting in the loss of cell rigidity and permeability.⁶ Nucleolus and thylakoids profiles were hardly seen in most of the treated cells, and the chloroplasts not only were more obscure but also exhibited changes in ultrastructural morphology, which may be due to the loss of chlorophyll or carotenoids.⁴⁷ These changes are in accord with the pigment content results obtained when the algae cells were exposed to medium or high concentrations of epoxiconazole. According to the report of Yang et al. carbohydrates and lipids are usually the intracellular places in which chemical energy is stored in algae cells.⁴⁸ This energy reserve can increase under adversity such as nutrient and toxicant stress. In Fig. 3C and 4D, the number and size of starch grains in the chloroplast increased, and some of them even formed a strong ring. Plenty of lipid droplets appeared in the cytoplasm, indicating crucial changes in starch and lipid metabolism when algae cells were exposed to the racemate and enantiomers of epoxiconazole. Interestingly, the cells treated with (−)-epoxiconazole produced more lipid droplets than cells treated by rac- or (+)-epoxiconazole; however, the cells grown in the presence of the (+)-enantiomer of epoxiconazole were more crowded with starch grains than those exposed to the other forms of epoxiconazole, indicating an enantioselective damage to cell morphology. Based on the above results, (+)-epoxiconazole is more toxic to S. obliquus cell than (−)-epoxiconazole, which is in accordance with the other toxicity tests.

Fig. 4 Accumulation curves for epoxiconazole in S. obliquus cells treated with rac-epoxiconazole (bars are standard error). * Denotes significant difference between washed algae and unwashed algae (A) or between the two enantiomers in washed algae (B) at the same time point (S–N–K test, P < 0.05).

3.6. Bioaccumulation and dissipation of epoxiconazole in S. obliquus

The concentrations of epoxiconazole enantiomers in the control media were provided in the ESI (Fig. S2†), which were slightly reduced. After a six-day treatment, the enantiomer concentrations dissipated slowly and steadily; the dissipation of the (−)-epoxiconazole was less than 28%. For the (+)-epoxiconazole, the concentration was stable in the medium without algae and decreased to 89% after the treatment period. This feeble dissipation might be the result of abiotic removal. In addition, no significant difference between the concentrations of epoxiconazole enantiomers was observed during the first three days. From days four to six, the concentration of (−)-epoxiconazole was less than that of (+)-epoxiconazole. In addition, the concentrations of both epoxiconazole enantiomers in media with algae were always less than those in the media without algae, and a relatively high concentration on (+)-epoxiconazole was observed during the entire incubation period, suggesting that enantioselective bioaccumulation occurs in algae.

The bioaccumulation results of epoxiconazole in algae are shown in Fig. 4. More epoxiconazole was found in the unwashed algae (algae extracted directly) than in the washed algae (algae washed in fresh culture solution before extraction; Fig. 4A). This result indicated that part of the epoxiconazole in unwashed algae might be presence in the remaining fluids between the cells or absorbed on cell surfaces rather than located intracellularly. For washed algae, a significant enantioselectivity was observed during the bioaccumulation experiment. During the first four days, the epoxiconazole enantiomers accumulated drastically with increasing time, and the relatively high concentration on (−)-epoxiconazole was observed. Subsequently, the concentrations of epoxiconazole enantiomers decreased with time, and (+)-epoxiconazole became a higher concentration than (−)-epoxiconazole (P < 0.05). Because the epoxiconazole enantiomers did not accumulate steadily in the algae, biodegradation must simultaneously occur during accumulation, possibly due to epoxiconazole metabolism caused by the detoxification mechanism of algae.⁴⁹ The change in epoxiconazole enantioselectivity after the first three days might be explained by enantiomerization, i.e., the transformation of (S)-enantiomer to (R)-enantiomer or vice versa. Another possible explanation is the activation of a stronger detoxification mechanism for (−)-epoxiconazole after three days, decreasing the toxicity of (−)-epoxiconazole with respect to (+)-epoxiconazole.

When the algae cells were treated with the single pure enantiomers of epoxiconazole, no indication of enantiomerization was found. Thus, we can be sure that enantioselectivity indeed occurred in the algae cells. The concentrations of individual enantiomers in the medium without algae were provided in the ESI (Fig. S3†), which were higher than those in the medium with algae, and more enantiomer was detected in the unwashed algae than in the washed-algae (Fig. 5A). Similar results were obtained in the treatments with racemate. Nevertheless, the concentrations of (+)- and (−)-epoxiconazole in algae did not indicate enantioselectivity for the single pure enantiomers of epoxiconazole (Fig. 5B). Moreover, the AF values of epoxiconazole enantiomers in algae treated with racemate were lower than those in algae treated with the individual enantiomers, especially on the third day of exposure (Fig. 6). This might be due to a competitive mechanism between the two epoxiconazole enantiomers when they were added as a mixture. This can also explain why the individual enantiomers of epoxiconazole were more toxic than the racemate. More in-depth research about the competitive mechanism should be studied in the future.

Fig. 5 Accumulation curves for epoxiconazole in S. obliquus cells treated with (−)- or (+)-epoxiconazole (bars are standard error). * Denotes significant difference between washed algae and unwashed algae (A) or between the two enantiomers in washed algae (B) at the same time point (S–N–K test, P < 0.05).

Fig. 6 Calculated AF values for epoxiconazole enantiomers in S. obliquus cells treated with rac-, (−)- or (+)-epoxiconazole (bars are standard error).

4. Conclusions

In this study, the epoxiconazole enantiomers exhibited enantioselective toxicity to S. obliquus. The EC₅₀ values of epoxiconazole enantiomers followed the order: (+)-epoxiconazole > (−)-epoxiconazole > rac-epoxiconazole. Epoxiconazole enantiomers significantly inhibits the photosynthetic pigment content at high concentrations and enhances it at low concentrations. The carotenoid content declined with increasing epoxiconazole concentration. Differences between the effects of epoxiconazole enantiomers to the antioxidant enzymes (SOD and CAT) actibities and MDA content can be also found. Enantioselective toxical effects of epoxiconazole to the S. obliquus were investigated further. In addition, cell wall and organelle changes as well as accumulated starch grains and lipid droplets were observed in algae cells after treatment with epoxiconazole racemate and enantiomers. Moreover, the bioaccumulation and degradation of epoxiconazole in S. obliquus was studied, and no enantiomerization was found. The presence of S. obliquus had a positive effect on the dissipation of epoxiconazole enantiomers in algae suspension. Enantioselectivity occurred when algae cells were exposed to racemate, with a preferential accumulation of (−)-epoxiconazole from days one to three and an enrichment in (+)-epoxiconazole after day four. However, no enantioselectivity was detected when algae cells were exposed to individual epoxiconazole enantiomers. The calculated AF values indicated more bioaccumulation of algae cells after exposure to individual enantiomers of epoxiconazole. Thus, a competitive mechanism between the two epoxiconazole enantiomers may exist when they are added as a mixture.

Acknowledgements

This work was supported by a fund from the National Natural Science Foundation of China (Contract Grant number: 41201499 and 21177154).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12617k

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