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Toxicity of thifluzamide in earthworm (Eisenia fetida)
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Toxicity of thifluzamide in earthworm (Eisenia fetida) Xiangfeng Yaoa,b,1, Fengwen Zhanga,b,1, Zhihua Qiaoa,b, Haoyong Yua,b, Shiang Suna,b, Xiangdong Lia, Jiwang Zhangc, Xingyin Jianga,b,∗ a b c
College of Plant Protection Shandong Agricultural University, Tai'an, Shandong, 271018, PR China Key Laboratory of Pesticide Toxicology & Application Technique, Tai'an, Shandong, 271018, PR China State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong, 271018, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Thifluzamide Earthworm Mechanism Genotoxicity Growth and reproduction
An increase in the area treated with the fungicide thifluzamide has triggered concerns for soil ecosystem service providers such as earthworms. Here, we assessed effects of thifluzamide on earthworm (Eisenia fetida) biomarker indicators of stress responses and reproduction following exposure to 0, 0.1, 1.0, and 10.0 mg of thifluzamide kg−1 soil for 7, 14, 21, and 28 d (biomarker indicators) and 30 d (reproduction). Growth and reproduction were inhibited by exposure to thifluzamide at 10.0 mg/kg, and the activities of succinate dehydrogenase (SDH) and respiratory chain complex II were inhibited by exposure to 1.0 and 10.0 mg/kg thifluzamide for the majority of the 28-d experiment. Reactive oxygen species (ROS) increased across all thifluzamide treatments, and the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) tended to be inhibited by thifluzamide. Upon exposure to thifluzamide, the activities of catalase (CAT) and guaiacol peroxidase (POD) initially increased and then decreased. Increased levels of malondialdehyde (MDA) were detected only at seven days after exposure, and genotoxicity increased as the thifluzamide concentration increased. The results suggest that thifluzamide presents a potential risk to earthworms at the concentration of 10.0 mg/kg, and its use should be moderated to reduce damage to soil ecosystem function.
1. Introduction Fungicides are a commonly used type of pesticide; however, many cause severe environmental pollution in agricultural systems, particularly in soils (Song et al., 2009). Although originally designed for specific targets, fungicides in the environment can affect non-target organisms (Karl et al., 2006). Thifluzamide, an amide fungicide, inhibits the synthesis of succinate dehydrogenase (SDH) and is widely used in rice production to protect against sheath blight (Rejeb et al., 2001; Chen et al., 2012; Vercesi et al., 2014). Thifluzamide is also applied to other crops to control a range of fungal diseases (Sun et al., 2012; Qin et al., 2013; Shang et al., 2018; Wu et al., 2018). The persistence of thifluzamide in soils is generally low but varies by crop. For example, Ma et al. (2018) reported a thifluzamide half-life of 4.56–15.85 d in cornfield soils following application at the recommended dosage, and the maximum residue in soil samples at corn physiological maturity did not exceed 0.278 mg/kg. Li et al., 2017a) reported a thifluzamide halflife of 11.0–14.0 d in peanut field soils of Shandong, Anhui, and Hunan, China after application at the recommended dosage, with an original
deposition of thifluzamide in these soils of 0.402–0.659 mg/kg. Earlier, Qin et al., 2013 showed that the half-life of thifluzamide in potato field soils in Tianjin and Nanjing, China was 4.92–7.07 d when applied at the recommended and at ×1.5 dose, where the greatest initial residue was 0.9608 mg/kg. Given the high efficacy of thifluzamide, it is expected to be used widely in the future (Liu, 2019); however, there are concerns about its environmental effects. Thifluzamide has been shown to affect zebrafish (Danio rerio) (Yang et al., 2016), and the LC50 (the concentration that is lethal to 50% of the test organisms) of thifluzamide was reported as 1.2 mg/L (96 h) for giant sunfish (Mola mola), 2.9 mg/L (96 h) for cyprinoid (Cyprinus carpio L.), and 2.9 mg/L (96 h) for water flea (Daphnia magna). These LC50 values indicate a moderate level of toxicity (IUPAC, 1997). While thifluzamide clearly poses a moderate risk to aquatic organisms, the effects on soil organisms (e.g., earthworms) likely to result from thifluzamide accumulation through spraying and seed coating remain unclear. Earthworms are widely used as a model bioindicator organism in pesticide ecotoxicological studies because of their environmental sensitivity and essential role in ecosystem function (Basefsky, 1984;
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Corresponding author. College of Plant Protection Shandong Agricultural University, Tai'an, Shandong, 271018, PR China. E-mail address: [email protected] (X. Jiang). 1 These two authors contributed equally to the present study. https://doi.org/10.1016/j.ecoenv.2019.109880 Received 7 August 2019; Received in revised form 17 October 2019; Accepted 25 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Xiangfeng Yao, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109880
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washed with distilled water, transferred to saline-soaked filter papers in Petri dishes, and placed in the dark at 20 °C ± 2 °C to facilitate the discharge of excreta. Earthworm mortality was zero throughout the experiment.
Stürzenbaum et al., 1998). Thus, understanding the effects of fungicides on earthworms is essential to predict the potential consequences of fungicide use in the food chain (Song et al., 2009). Physical and chemical factors that affect the structure and function of soils can have direct or indirect effects on earthworm physiological activity (Rao and Kavitha, 2004; Yasmin and D'Souza, 2007; Song et al., 2009). Furthermore, earthworms may be used to detect the effects of low doses of pollutants and provide a safety threshold for the protection of soil ecosystems (Zheng et al., 2008). Phenotypic observation can directly reflect the effects of pollutants on organisms. In this study, the growth inhibition and reproduction of earthworms were studied. Although the purpose of a fungicide is to inhibit or kill fungal pathogens, the toxic effect may not apply only to fungi (Maltby et al., 2009). Hence, the activities of succinate dehydrogenase (SDH) and respiratory chain complex II in earthworms were examined. Reactive oxygen species (ROS) are produced in organisms exposed to environmental pollutants. Increased ROS levels lead to oxidative damage to proteins, nucleic acids, lipids, and other macromolecules, impairing cellular mechanisms (Sabatini et al., 2009). In this study, we assessed the levels of (1) ROS; (2) antioxidants such as superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD); and (3) indicators of stress such as malondialdehyde (MDA), which reflects the extent of tissue cell trauma (Wang et al., 2016a,b), and glutathione-S-transferase (GST), which reflects the activity of the detoxification system. The comet assay is sensitive enough to reveal DNA damage at very low concentrations, and single-cell gel electrophoresis (SCGE) was used to detect DNA damage in earthworm cell. The biomarkers assessed in this study can be used to evaluate the responses of earthworms exposed to toxic xenobiotics (Lin et al., 2012b). The hazards posed by and genotoxicity of thifluzamide in earthworms have never been studied. Therefore, this study evaluated the toxicity of thifluzamide to earthworms.
2.2. Earthworm growth and reproduction The initial average weight of one replicate of 20 earthworms was recorded prior to being added to the beakers (W0). On days 7, 14, 21 and 28, 20 earthworms were taken from each beaker, and their average weight was recorded as Wt. The change in weight was calculated as:
weight change (%) = (Wt − W0/ W0) × 100%. At day 30, the soils were passed through a 1-mm sieve, and the earthworm cocoons and juveniles were collected and counted. 2.3. Measurement of stress indicators At each sampling occasion (at 7, 14, 21, and 28 d), we randomly selected and removed five earthworms from each of the three replicates for analysis of activity of oxidative stress indicators, three for enzyme and respiratory chain complexes II, one for ROS, one for SCGE. The earthworms were ground in a prechilled mortar prior to preparation for analysis. 2.3.1. Protein content We used the Bradford assay (Bradford, 1976) to quantify protein content, using bovine serum albumin as the standard protein. Light absorption at 595 nm was compared with a plotted standard curve to calculate the protein content. 2.3.2. ROS determination ROS levels were determined based on dichlorohydrofluorescein diacetate (DCFH-DA) fluorescence (Lawler et al., 2003) Earthworms were homogenized in 100 mM of ice-cold potassium phosphate buffer (4 °C, pH 7.4). The mixture was then centrifuged at 3000 rpm for 5 min at 4 °C to recover the liquid phase. The supernatant was recentrifuged at 20,000 rpm for 20 min at 4 °C. After resuspension, DCFH-DA solution was added to the mitochondrial sedimentation and incubated in a water bath, in darkness, for 30 min at 37 °C. The reaction was terminated using 200 μL of 1 mol/L of HCl, and then fluorescence was monitored using a fluorescence spectrophotometer (F-4600, Hitachi, Japan) at excitation and emission wavelengths of 488 and 522 nm, respectively.
2. Materials and methods We obtained 96% purity thifluzamide from Shandong Kangqiao Biotechnology Co. Ltd (China), and other chemicals, which were of reagent grade, were purchased from the Sigma Chemical Co. (St. Louis, USA) and the Comin Biotechnology Co. Ltd (Suzhou, China). 2.1. Exposure to thifluzamide Earthworms (Eisenia fetida) were purchased from an earthworm farm in Jiangsu, China, and maintained at 20 ± 2 °C for 14 d in a breeding box that contained a mix of soil and cattle manure. Toxicological tests were carried out using artificial soil (OECD, 2004), comprising 10% sphagnum peat moss (Premier), 20% kaolin clay (Fisher Scientific), and 70% sand (grade 70, particle size: 0.1–0.3 mm). The pH of the artificial soil was adjusted to 6.0 ± 0.5 by adding CaCO3 (OECD, 2004). Prior to exposure to thifluzamide, the earthworms (350 ± 10 mg) acclimated for 24 h in the artificial soil. We used final treatment concentrations of 0 (solvent-only control), 0.1, 1.0, and 10.0 (assess the concentration of soil contaminated with uncontrolled thifluzamide) mg of thifluzamide kg−1 of soil, based on previous thifluzamide soil residue data (Qin et al., 2013; Li et al., 2017a; Ma et al., 2018). Thifluzamide was dissolved in acetone and mixed into the artificial soil. The sample was then allowed to stand for 12 h to allow the acetone to evaporate. Next, eight replicates (five for the assessment of earthworm growth and three for the assessment of stress indicators) were created by transferring 500 g of contaminated soil into eight 1-L beakers. Twenty earthworms were then added to each beaker. The moisture content was adjusted to 35% using distilled water, and cow manure was added to the soil surface every week at a rate of 0.5 g per earthworm (Liu et al., 2009). The beakers were maintained at 20 °C ± 2 °C with a daily 12-h:12-h light:dark cycle for 30 d. At 12 h prior to analysis, the earthworms were removed from the beakers,
2.3.3. Activity of target enzyme and oxidative stress indicators Ground earthworms were mixed in 50 mM phosphate buffer (1:10, w/v; pH 7.8) under ice-cold conditions. After the mixture was centrifuged at 10,000 rpm at 4 °C for 15 min, the supernatant and precipitate were prepared for the determination of protein, MDA content, and enzyme activity. The activities of SDH in the supernatant and respiratory chain complex II in the precipitate were determined using an assay kit (Keming, Suzhou, China) following the manufacturer's instructions. The determination of other indicators was carried out using the supernatant with the following methods. SOD activity, based on its ability to inhibit nitroblue tetrazolium chloride (NBT), was determined following Song et al. (2009). The reaction mixture (3 mL), which comprised ethylenediaminetetraacetic acid (EDTA), phosphate buffer (pH 7.8), NBT, methionine, riboflavin, and enzyme supernatant (100 mM, 50 μM, 750 μM, 130 mM, 20 μM, 50 μL), was shaken and absorbance was assayed at 560 nm after 30 min. One unit (U) of SOD activity was defined as the amount of enzyme that inhibited reduction of NBT by 50%. CAT activity was assayed using a modified version of the method described by Xu et al. (1997). Enzyme extract (10 μL) and 3 mL phosphate buffer I (pH 7.0) were added to the reference frame; 10 μL 2
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Fig. 1. Weight change rates in earthworms (Eisenia fetida) upon exposure to thifluzamide at different time intervals. Bars are means ± standard errors (n = 5). Different letters indicate significant differences (p < 0.05) among treatments as determined by the least significant difference (LSD) test.
double-strand DNA breaks following a modified version of the approach used by Singh et al. (1988) and Song et al. (2009). We mixed 40 μL of 60 μL of the supernatant (cell suspension) with 500 μL of 0.7% low melting agar (LMA) in phosphate-buffered saline at 37 °C. This mixture was then pipetted onto a fully frosted slide precoated with 100 mL of 0.8% normal melting agar and allowed to solidify on ice for 15 min. Subsequently, another layer of 50 μL of LMA was added to the slide. The slide was then immersed in a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA at pH 10.0, 1% sodium sarcosinate, 10% dimethyl sulfoxide, and 1% Triton X-100) for 1 h. The slide was then incubated in an electrophoresis tank containing 300 mM NaOH and 1 mM Na2EDTA for 30 min followed by electrophoresis for 15 min at 25 V (300 mA). The slides were neutralized using 0.4 M Tris (pH 7.5) for 15 min and stained with 40 μL ethidium bromide (13 mg/mL) for fluorescence microscopy analysis (Olympus BX51 microscope). Finally, SCGE images (100 cell cores per slide) were analyzed using Comet Assay Software Project (CASP) software (Końca et al., 2003). The extent of DNA damage was quantified based on the olive tail moment (OTM), defined as the product of the distance between the head and tail centers of gravity and the proportion (%) of total DNA in the tail (Song et al., 2009).
enzyme extract and 3 mL 30% (w/v) hydrogen peroxide (H2O2)/phosphate buffer II were added to the sample frame. Ultraviolet absorption at 250 nm was measured every 10 s for 2 min. One unit of CAT activity was defined as the amount of enzyme consumed in the H2O2 buffer in 100 s at 25 °C. POD activity was measured using a modified version of the method described by Kochba et al. (1977). Enzyme liquid was added to 3 mL of a reaction mixture containing 100 mL potassium phosphate buffer (100 mM, pH 6.0), 38 μL 30% H2O2, and 56 μL guaiacol. Absorbance was recorded at 470 nm, and POD activity was expressed as U/mg protein. GST activity was determined using the method of Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The absorbances of glutathione (GSH) and CDNB at 340 nm were measured every 30 s for 3 min. GST activity was expressed as nmol/min/mg protein. MDA content, which is the final product of lipid peroxidation, was determined using the thibabituric acid (TBA) colorimetric method described by Xiang and Wang (1990) with some modifications. Enzyme liquid (200 μL) was mixed with a reaction mixture containing SDS (0.2 mL, 8.1%), acetic acid (1.5 mL, 20%), TBA (1%), and deionized water (1 mL) prior to immersion in a water bath at 95 °C for 1 h. The mixture was then centrifuged at 10,000 rpm for 5 min. The MDA levels in the supernatant were determined at 532 nm and expressed as nmol/ mg protein.
2.4. Statistical analysis There were eight replicates in this experiment, among which five have been used for growth and reproduction tests and three other ones for the biochemical determination of stress indicators and DNA damage. Comet assay images were analyzed with CASP. Differences among treatments were evaluated by analysis of variance (ANOVA) and least significance difference tests at P < 0.05; thifluzamide concentration, exposure time, and their interaction were analyzed using bifactorial ANOVA. The data are presented as mean ± standard error. Statistical analyses were performed with SPSS v18.0 software.
2.3.4. DNA damage SCGE comet assay was used to analyze DNA damage in cells following the method of Eyambe et al. (1991). One replicate of a single earthworm for each treatment ( total three replicates) was rinsed in physiological saline solution and then rinsing in the extrusion medium (5% ethanol, 95% saline, 2.5 mg/mLEDTA, and 10 mg/mL guaiacol glyceryl ether, pH 7.3) to induce spontaneous excretion of the coelomocytes; the coelomocytes were collected by centrifugation of the extracting solution at 3000 g for 10 min. The supernatant was placed on ice prior to the assay. The alkaline protocol for comet assay was used to detect single- and 3
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Fig. 2. Effects of thifluzamide on cocoon production and number of juveniles of earthworms (Eisenia fetida) after 30d exposure. Bars are means ± standard errors (n = 5). Different letters indicate significant differences (p < 0.05) among treatments as determined by the least significant difference (LSD) test.
thifluzamide. Overall, the ROS fluorescence intensity was higher in the groups treated with thifluzamide compared to the control group (Fig. 4).
3. Results 3.1. E. fetida growth and reproduction No significant differences in weight change were observed between different treatments on day 7. Significant decreases in weight were observed at days 14 and 21 in the group treated with 10.0 mg/kg thifluzamide, and the rate of weight gain in the group treated with 1.0 mg/ kg thifluzamide was significantly lower than that of the control group. On day 28, all groups treated with thifluzamide showed growth (weight gain); however, the weight gain rates of the groups treated with 1.0 and 10.0 mg/kg thifluzamide were significantly lower than that of the control group (Fig. 1). Compared to the control group, cocoon production and the number of juveniles were not significantly different in the groups treated with 0.1 and 1.0 mg/kg thifluzamide. In contrast, cocoon production and the number of juveniles were significantly lower in the group treated with 10.0 mg/kg thifluzamide at 30 days after exposure (Fig. 2).
3.4. Antioxidant and detoxification enzyme activity Exposure to most concentrations of thifluzamide inhibited SOD activity, with the exception of on day 28. On day 28, the SOD activities in the treatment groups were similar to that of the control group, and there was a tendency to be activated (1.0 mg/kg; Fig. 5a). On day 7, the earthworms exposed to thifluzamide at all concentrations had greater CAT activities than the earthworms in the control group. In contrast, on day 21, the CAT activities in the treatment groups were lower than in the control group. On day 14, the CAT activity in the group treated with 10.0 mg/kg thifluzamide exceeded that of the control group. On day 28, the CAT activities in the groups treated with 0.1 and 1.0 mg/kg thifluzamide were lower than that of the control, whereas the CAT activities in the other treatment groups were similar to that of the control group (Fig. 5b). Compared to the control, POD activity at day 7 was greater in all treatment groups than in the control group. At day 14, POD activity was greater in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to in the control group. At day 21, POD activity was lower in the groups treated with 0.1 and 1.0 mg/kg thifluzamide compared to the control. At day 28, POD activity was greater in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to the control (Fig. 5c). At all time points, GST activity was lower in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to the control, while GST activity in the group treated with 0.1 mg/kg thifluzamide was lower than in the control group only at day 7 (Fig. 5d).
3.2. Activities of SDH and respiratory chain complexes II Compared to the control, SDH activity was lower at days 7 and 14 in the groups treated with 1.0 and 10.0 mg/kg thifluzamide and at days 21 and 28 in the group treated with 10.0 mg/kg thifluzamide (Fig. 3a). Similarly, the activity of respiratory chain complex II was lower in the groups treated with 1.0 and 10.0 mg/kg thifluzamide at all time points (Fig. 3b); a gradual decrease in the activity of respiratory chain complex II was observed with increasing thifluzamide concentration. Overall, the changes of the activities of SDH and respiratory chain complex II were similar. 3.3. ROS level
3.5. MDA content Compared to the control, the ROS fluorescence intensity was significantly higher on day 7 in the groups treated with 1.0 and 10.0 mg/ kg thifluzamide. Similarly, the ROS fluorescence intensity of all treatment groups was significantly higher than that of the control group at days 14 and 21. On day 28, the ROS fluorescence intensity was only higher than the control in the group treated with 10.0 mg/kg
In all treatment groups, the MDA content only exceeded that of the control group on day 7. However, at days 14, 21, and 28, the MDA content did not differ between the treatment groups and the control group, with the exception of the group treated with 1.0 mg/kg thifluzamide on day 21 (Fig. 6). 4
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Fig. 3. The SDH activity (a) and the activity of respiratory chain complexes II (b) changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3).Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
Fig. 4. ROS level changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
Thifluzamide dose and exposure time had effects on all biomarkers (P < 0.05; Table S2). The weight changes; activities of SDH, respiration chain complex II, SOD, CAT, POD, and GST; ROS level; MDA content; and DNA damage were significantly different among the four time points and among the different thifluzamide concentrations. These differences were also reflected in the one-way ANOVA results, which indicated significant differences between concentrations at the same exposure times.
3.6. E. fetida DNA damage In the control group, the comet head centers were rounded, dense, and shiny, with little or no DNA migration to other regions [Fig. 7A(a)]. Exposure to thifluzamide resulted in large quantities of comet tails in cell nuclei, in which the DNA appeared fluffy, scattered, and mushy [Fig. 7A(b), (c), and (d)]. As the exposure concentration increased, DNA migration and expansion into the nucleus became more pronounced. The OTM of the earthworm's coelomocyte comets gradually increased with increasing thifluzamide concentration (Fig. 7B).
4. Discussion This study investigated growth and reproduction along with a range of biomarkers that indicate environmental stress in earthworms (E. fetida) exposed to different concentrations of thifluzamide over 28 d. The results provide valuable information about subchronic genotoxicity in a key ecosystem service provider.
3.7. Dose-exposure time biochemical responses Interactions between thifluzamide exposure time and dose were observed for all biomarkers (Table S1), indicating that the dose-dependent responses of the biomarkers varied over time (P < 0.05). 5
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Fig. 5. Enzyme activity changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Activity of (a) SOD, (b) CAT, (c) POD, and (d) GST. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
at 5.0 and 10.0 mg/kg for 42 days (Liu et al., 2018) and after exposure to the insecticide cyfluthrin at concentrations exceeding 20 mg/kg (Li et al., 2017b). The high concentration (10.0 mg/kg) of thifluzamide might damage the reproductive systems of earthworms, resulting in the malformation of sperm, loss of fertilization ability, and eventually infertility.
4.1. Toxic effects of thifluzamide on growth and reproduction in E. fetida Earthworm weight is a sensitive indicator of exposure to stress (Xiao et al., 2006; Liu et al., 2018) that may be used to evaluate pollutant toxicity. In this study, the weights of earthworms exposed to 10.0 mg/ kg thifluzamide were lower at days 14 and 21 (Fig. 1). Ye et al. (2016) found that weight loss may be caused by the loss of glycogen, lipids, and proteins upon exposure to toxic chemicals. Givaudan et al. (2014) reported that earthworm detoxification mechanisms involve the removal of exogenous pollutants through energy metabolism. Thus, we posit that exposure to 10 mg/kg thifluzamide may affect the normal metabolism of biomacromolecules in earthworms, with detoxification causing excessive metabolism. Similarly, Liu et al. (2018) found that exposure to 10 mg/kg of the insecticide chlorantraniliprole led to lower earthworm weight after 14 days, while exposure to chlorantraniliprole at 5.0 and 10.0 mg/kg resulted in lower weight after 28 and 42 days. However, in this study, the earthworm weights in all treatment groups indicated growth after 28 days (Fig. 1), possibly because the earthworms had adapted to the stress conditions, or because the thifluzamide had been degraded, allowing normal growth to resume. The number of cocoons and juveniles were lower in the group treated with 10 mg/kg thifluzamide at day 30 (Fig. 2), indicating toxic effects on reproduction. Similar effects on cocoon production and number of juveniles were reported after exposure to chlorantraniliprole
4.2. Effects of thifluzamide on target enzymes While pollutants are known to cause oxidative damage in earthworms, the underlying response mechanisms have been scarcely studied. Wang et al. (2015) found that the neonicotinoid insecticide guadipyr inhibited acetylcholinesterase activity in earthworms. Thifluzamide acts by interfering with succinate ubiquinone reductase (complex II) in the mitochondrial respiration pathway. This inhibition of respiration and the activities of related enzymes reduces electron transfer and energy supply, eventually leading to fungal death (Yang et al., 2016). Similar mechanisms have been reported in other organisms; for example, thifluzamide (Yang et al., 2016) and boscalid (a fungicide) (Qian et al., 2018) inhibited the activities of SDH and mitochondrial respiratory chain complexes in zebrafish. SDH and respiratory chain complex II are embedded in the internal membrane of the mitochondrion; thus, their activities reflect the generation of adenosine triphosphate and mitochondrion function (Leist et al., 1999), 6
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Fig. 6. MDA content changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
which modulate energy generation and may mediate cell apoptosis and necrosis (Zamzami et al., 1995). The activities of SDH and respiratory chain complex II determined in this study revealed a dose-dependent
inhibition effect of thifluzamide on SDH and respiratory chain complex II activity (Fig. 3). Thus, we speculate that thifluzamide acts on the mitochondria of earthworms, inhibits the activities of SDH and Fig. 7. A. Comet assays for coelomocytes of earthworms (Eisenia fetida) (a) control earthworms, (b) nuclei of earthworms exposed to 0.1 mg/kg of thifluzamide, (c) nuclei of earthworm exposed to 1.0 mg/kg of thifluzamide, (d) nuclei of earthworms exposed to 10.0 mg/kg of thifluzamide. B. Effect of thifluzamide on the coelomocytes' comet olive tail moment at different exposure times. Each soil sample was performed in triplicate. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
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responses to pollutants. In this study, GST activity was initially inhibited in earthworms exposed to the low concentration of thifluzamide (0.1 mg/kg) and then gradually recovered. In contrast, the GST activities remained low in the groups exposed to higher levels of thifluzamide (1.0 and 10.0 mg/kg), and a dose–effect relationship was observed (Fig. 5d). This finding supports the results of Lin et al., (2012a), who reported a dose–response relationship for GST activity when earthworms were exposed to the antibiotic chlortetracycline. These findings may be related to the production of intermediate metabolites during detoxification. Such metabolites could alter the composition of the GST subunit, resulting in more consumption of GSH, reduced GST activity, and competition of GST with its substrate (e.g., CDNB) (Egaas et al., 1999). MDA is a product of lipid membrane peroxidation that is formed upon oxidative damage. The MDA content may indirectly indicate the level of free radicals in an organism (Fazeli et al., 2007). In this study, the MDA content in earthworms increased at 7 days after exposure to thifluzamide (Fig. 6). This might result from the earthworms being under oxidative stress, leading to the production of excessive ROS (Fig. 4) and thus the peroxidation of lipid membranes. After 14 days, the MDA levels in the treatment groups did not differ from that of the control group (Fig. 6), perhaps because excess peroxidant radicals were scavenged by activated antioxidant enzymes (CAT and POD), alleviating oxidative stress and minimizing the accumulation of MDA (Schmitt et al., 2007). Likewise, Zhang et al. (2013) found that the peroxidation of lipids in earthworms may be mediated by ROS metabolism as a result of enzymatic toxicity defenses.
respiratory chain complex II, and damages the mitochondrial structure, leading to oxidative damage. 4.3. Effects of thifluzamide on oxidative stress and the antioxidant system The dynamic state of equilibrium of ROS is disrupted by exposure to exogenous pollutants, which drive the accumulation of ROS. Excess ROS levels damage cell structures, nucleic acids, proteins, and lipids (Valko et al., 2006). ROS comprise a series of oxygen free radicals, such as H2O2, superoxide (O2−), and hydroxyl radical (Han et al., 2014). In this study, exposure to thifluzamide caused the ROS levels in earthworms to increase (Fig. 4). Mitochondria are a key source of ROS when cells are subjected to exogenous damage (Piantadosi and Zhang, 1996), and damage to the structure and function of mitochondria may increase levels of free radicals and associated oxidative damage (MartínezFernández et al., 2001). ROS and mitochondrial damage may also be mutually causal because ROS affects the vitality of respiratory chain complexes; this impairs mitochondrial structure and function and may lead to further increases in ROS levels and eventually apoptosis (Liu, 2008). The results of this study indicated that thifluzamide exposure inhibited the activities of SDH and respiratory chain complex II and increased the levels of ROS (Figs. 3 and 4). These results support the findings of the above studies and lead us to speculate that the increases in ROS levels were caused by damage to mitochondrial structure and function. Essential antioxidant enzymes involved in the elimination of ROS include SOD, POD, and CAT, which are frequently used as indicators of ROS production (Zelikoff et al., 1996). SOD is an important component of the primary antioxidant defense mechanism, in which O2− is converted into H2O2 (Han et al., 2014). In this study, except for on day 28, SOD activity in earthworms was inhibited by exposure to thifluzamide, particularly at the high exposure concentrations (1.0 and 10.0 mg/kg; Fig. 5a). Similar effects were reported for the herbicide atrazine (Song et al., 2009). The decreased SOD activity may have been caused by the elimination of highly reactive O2− via its conversion to H2O2 by SOD; however, the elevated SOD activity at day 28 may have arisen from O2 production, which stimulated SOD activity over the longer exposure time. CAT and POD scavenge H2O2 and other free radicals (Han et al., 2014). In this study, CAT activity in earthworms initially increased upon exposure to thifluzamide and then decreased (Fig. 5b). Similarly, Shao et al. (2012) found that exposure to low concentrations of the insecticide endosulfan first increased CAT activity in zebrafish but then inhibited CAT activity over time. This phenomenon may be plausibly explained as follows. A low level of pollutant-related stress produced a little H2O2 soon after exposure, activating CAT; however, CAT activity was later significantly reduced because the pollutant induced more ROS production than CAT could eliminate. Thus, we conclude that thifluzamide induced excess ROS that could not be eliminated by cellular antioxidants, thereby depleting both CAT and SOD. The effects of thifluzamide on POD activity in this study were similar to the effects on CAT activity (Fig. 5c). These results agree with the findings of a study on the acute toxicity and oxidative stress effects of the fungicide pyraclostrobin in earthworms (Wang et al., 2016a,b). Thus, the dual enhancement of POD and CAT activities likely improved the scavenging capacity for H2O2 to restore ROS equilibrium, indicating that the earthworms began to resist potential damage from thifluzamide. The activities of POD and CAT were inhibited later (at days 21 and 28), possibly because the gradual accumulation of H2O2 in the earthworms exceeded the scavenging capacity of CAT and POD, resulting in the inhibition of their activities. GST is a potent detoxifying enzyme that influences antioxidant enzyme activity. GST combines with GSH and electrophilic reagents to eliminate contaminants, scavenge ROS, eliminate metabolites of lipid peroxidation (LPO), and reduce DNA damage (Pickett and Lu, 1989). Thus, GST has been used as a sensitive indicator of oxidative stress
4.4. Genotoxic effects of thifluzamide Exposure to pollutants results in cellular oxidative stress and DNA damage by the action of free radicals (Valavanidis et al., 2006). The extent of this type of damage may be assessed based on OTM, which reflects the distance between the comet head and tail centers (Song et al., 2009). DNA damage tends to be caused by ROS, which leads to the removal of nucleotides, strand breaks, and modifications to nucleotide bases (Cooke et al., 2003). We surmised that DNA damage arose from oxidative stress based on the larger OTM values and ROS accumulation observed in this study (Figs. 4 and 7B), indicating dose–effect relationships. Yang et al. (2016) found that thifluzamide caused mitochondrial DNA damage, leading to toxic effects on zebrafish development and pathological changes through significant alterations in the expressions of some related genes in the mitochondria. We posit that thifluzamide may have a similar mechanism of action on earthworms. Thifluzamide damages nuclear DNA via oxidative stress. It can also directly cause mitochondrial DNA damage due to its effects on mitochondria because mitochondrial DNA is more vulnerable to exogenous damage than nuclear DNA (Liu and Wang, 2013). Disturbances to mitochondrial DNA transcription and translation inhibit the synthesis and expression of mitochondrial DNA coding proteins, which may increase free radicals and lead to greater oxidative damage (the discussion in sections 4.2 and 4.3 provides evidence for this conjecture). Thus, oxidative damage and mitochondrial damage interact to form a vicious cycle (James et al., 1996; Pulkes and Hanna, 2001). 5. Conclusions This study showed toxicity of thifluzamide to earthworms, through effects on mitochondria that induced oxidative stress; the interaction of oxidative damage and mitochondrial damage led to increases in levels of ROS that stimulated the antioxidant and detoxification enzyme systems. Short-term exposure (7 d) to thifluzamide caused LPO in the earthworms, as indicated by high MDA contents. OTM increased with increasing thifluzamide concentration and exposure time. Remarkable DNA damage was induced by oxidative damage and mitochondrial 8
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damage at the evaluated doses. These effects led to lower growth and reproduction rates in earthworms exposed to thifluzamide at 10 mg/kg. Confirmation of the action of thifluzamide on mitochondria requires subcellular-level and molecular-level research to improve our understanding of the risk posed by thifluzamide to soil ecosystems.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Toxicity of thifluzamide in earthworm (Eisenia fetida) Xiangfeng Yaoa,b,1, Fengwen Zhanga,b,1, Zhihua Qiaoa,b, Haoyong Yua,b, Shiang Suna,b, Xiangdong Lia, Jiwang Zhangc, Xingyin Jianga,b,∗ a b c
College of Plant Protection Shandong Agricultural University, Tai'an, Shandong, 271018, PR China Key Laboratory of Pesticide Toxicology & Application Technique, Tai'an, Shandong, 271018, PR China State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong, 271018, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Thifluzamide Earthworm Mechanism Genotoxicity Growth and reproduction
An increase in the area treated with the fungicide thifluzamide has triggered concerns for soil ecosystem service providers such as earthworms. Here, we assessed effects of thifluzamide on earthworm (Eisenia fetida) biomarker indicators of stress responses and reproduction following exposure to 0, 0.1, 1.0, and 10.0 mg of thifluzamide kg−1 soil for 7, 14, 21, and 28 d (biomarker indicators) and 30 d (reproduction). Growth and reproduction were inhibited by exposure to thifluzamide at 10.0 mg/kg, and the activities of succinate dehydrogenase (SDH) and respiratory chain complex II were inhibited by exposure to 1.0 and 10.0 mg/kg thifluzamide for the majority of the 28-d experiment. Reactive oxygen species (ROS) increased across all thifluzamide treatments, and the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) tended to be inhibited by thifluzamide. Upon exposure to thifluzamide, the activities of catalase (CAT) and guaiacol peroxidase (POD) initially increased and then decreased. Increased levels of malondialdehyde (MDA) were detected only at seven days after exposure, and genotoxicity increased as the thifluzamide concentration increased. The results suggest that thifluzamide presents a potential risk to earthworms at the concentration of 10.0 mg/kg, and its use should be moderated to reduce damage to soil ecosystem function.
1. Introduction Fungicides are a commonly used type of pesticide; however, many cause severe environmental pollution in agricultural systems, particularly in soils (Song et al., 2009). Although originally designed for specific targets, fungicides in the environment can affect non-target organisms (Karl et al., 2006). Thifluzamide, an amide fungicide, inhibits the synthesis of succinate dehydrogenase (SDH) and is widely used in rice production to protect against sheath blight (Rejeb et al., 2001; Chen et al., 2012; Vercesi et al., 2014). Thifluzamide is also applied to other crops to control a range of fungal diseases (Sun et al., 2012; Qin et al., 2013; Shang et al., 2018; Wu et al., 2018). The persistence of thifluzamide in soils is generally low but varies by crop. For example, Ma et al. (2018) reported a thifluzamide half-life of 4.56–15.85 d in cornfield soils following application at the recommended dosage, and the maximum residue in soil samples at corn physiological maturity did not exceed 0.278 mg/kg. Li et al., 2017a) reported a thifluzamide halflife of 11.0–14.0 d in peanut field soils of Shandong, Anhui, and Hunan, China after application at the recommended dosage, with an original
deposition of thifluzamide in these soils of 0.402–0.659 mg/kg. Earlier, Qin et al., 2013 showed that the half-life of thifluzamide in potato field soils in Tianjin and Nanjing, China was 4.92–7.07 d when applied at the recommended and at ×1.5 dose, where the greatest initial residue was 0.9608 mg/kg. Given the high efficacy of thifluzamide, it is expected to be used widely in the future (Liu, 2019); however, there are concerns about its environmental effects. Thifluzamide has been shown to affect zebrafish (Danio rerio) (Yang et al., 2016), and the LC50 (the concentration that is lethal to 50% of the test organisms) of thifluzamide was reported as 1.2 mg/L (96 h) for giant sunfish (Mola mola), 2.9 mg/L (96 h) for cyprinoid (Cyprinus carpio L.), and 2.9 mg/L (96 h) for water flea (Daphnia magna). These LC50 values indicate a moderate level of toxicity (IUPAC, 1997). While thifluzamide clearly poses a moderate risk to aquatic organisms, the effects on soil organisms (e.g., earthworms) likely to result from thifluzamide accumulation through spraying and seed coating remain unclear. Earthworms are widely used as a model bioindicator organism in pesticide ecotoxicological studies because of their environmental sensitivity and essential role in ecosystem function (Basefsky, 1984;
∗
Corresponding author. College of Plant Protection Shandong Agricultural University, Tai'an, Shandong, 271018, PR China. E-mail address: [email protected] (X. Jiang). 1 These two authors contributed equally to the present study. https://doi.org/10.1016/j.ecoenv.2019.109880 Received 7 August 2019; Received in revised form 17 October 2019; Accepted 25 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Xiangfeng Yao, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109880
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washed with distilled water, transferred to saline-soaked filter papers in Petri dishes, and placed in the dark at 20 °C ± 2 °C to facilitate the discharge of excreta. Earthworm mortality was zero throughout the experiment.
Stürzenbaum et al., 1998). Thus, understanding the effects of fungicides on earthworms is essential to predict the potential consequences of fungicide use in the food chain (Song et al., 2009). Physical and chemical factors that affect the structure and function of soils can have direct or indirect effects on earthworm physiological activity (Rao and Kavitha, 2004; Yasmin and D'Souza, 2007; Song et al., 2009). Furthermore, earthworms may be used to detect the effects of low doses of pollutants and provide a safety threshold for the protection of soil ecosystems (Zheng et al., 2008). Phenotypic observation can directly reflect the effects of pollutants on organisms. In this study, the growth inhibition and reproduction of earthworms were studied. Although the purpose of a fungicide is to inhibit or kill fungal pathogens, the toxic effect may not apply only to fungi (Maltby et al., 2009). Hence, the activities of succinate dehydrogenase (SDH) and respiratory chain complex II in earthworms were examined. Reactive oxygen species (ROS) are produced in organisms exposed to environmental pollutants. Increased ROS levels lead to oxidative damage to proteins, nucleic acids, lipids, and other macromolecules, impairing cellular mechanisms (Sabatini et al., 2009). In this study, we assessed the levels of (1) ROS; (2) antioxidants such as superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD); and (3) indicators of stress such as malondialdehyde (MDA), which reflects the extent of tissue cell trauma (Wang et al., 2016a,b), and glutathione-S-transferase (GST), which reflects the activity of the detoxification system. The comet assay is sensitive enough to reveal DNA damage at very low concentrations, and single-cell gel electrophoresis (SCGE) was used to detect DNA damage in earthworm cell. The biomarkers assessed in this study can be used to evaluate the responses of earthworms exposed to toxic xenobiotics (Lin et al., 2012b). The hazards posed by and genotoxicity of thifluzamide in earthworms have never been studied. Therefore, this study evaluated the toxicity of thifluzamide to earthworms.
2.2. Earthworm growth and reproduction The initial average weight of one replicate of 20 earthworms was recorded prior to being added to the beakers (W0). On days 7, 14, 21 and 28, 20 earthworms were taken from each beaker, and their average weight was recorded as Wt. The change in weight was calculated as:
weight change (%) = (Wt − W0/ W0) × 100%. At day 30, the soils were passed through a 1-mm sieve, and the earthworm cocoons and juveniles were collected and counted. 2.3. Measurement of stress indicators At each sampling occasion (at 7, 14, 21, and 28 d), we randomly selected and removed five earthworms from each of the three replicates for analysis of activity of oxidative stress indicators, three for enzyme and respiratory chain complexes II, one for ROS, one for SCGE. The earthworms were ground in a prechilled mortar prior to preparation for analysis. 2.3.1. Protein content We used the Bradford assay (Bradford, 1976) to quantify protein content, using bovine serum albumin as the standard protein. Light absorption at 595 nm was compared with a plotted standard curve to calculate the protein content. 2.3.2. ROS determination ROS levels were determined based on dichlorohydrofluorescein diacetate (DCFH-DA) fluorescence (Lawler et al., 2003) Earthworms were homogenized in 100 mM of ice-cold potassium phosphate buffer (4 °C, pH 7.4). The mixture was then centrifuged at 3000 rpm for 5 min at 4 °C to recover the liquid phase. The supernatant was recentrifuged at 20,000 rpm for 20 min at 4 °C. After resuspension, DCFH-DA solution was added to the mitochondrial sedimentation and incubated in a water bath, in darkness, for 30 min at 37 °C. The reaction was terminated using 200 μL of 1 mol/L of HCl, and then fluorescence was monitored using a fluorescence spectrophotometer (F-4600, Hitachi, Japan) at excitation and emission wavelengths of 488 and 522 nm, respectively.
2. Materials and methods We obtained 96% purity thifluzamide from Shandong Kangqiao Biotechnology Co. Ltd (China), and other chemicals, which were of reagent grade, were purchased from the Sigma Chemical Co. (St. Louis, USA) and the Comin Biotechnology Co. Ltd (Suzhou, China). 2.1. Exposure to thifluzamide Earthworms (Eisenia fetida) were purchased from an earthworm farm in Jiangsu, China, and maintained at 20 ± 2 °C for 14 d in a breeding box that contained a mix of soil and cattle manure. Toxicological tests were carried out using artificial soil (OECD, 2004), comprising 10% sphagnum peat moss (Premier), 20% kaolin clay (Fisher Scientific), and 70% sand (grade 70, particle size: 0.1–0.3 mm). The pH of the artificial soil was adjusted to 6.0 ± 0.5 by adding CaCO3 (OECD, 2004). Prior to exposure to thifluzamide, the earthworms (350 ± 10 mg) acclimated for 24 h in the artificial soil. We used final treatment concentrations of 0 (solvent-only control), 0.1, 1.0, and 10.0 (assess the concentration of soil contaminated with uncontrolled thifluzamide) mg of thifluzamide kg−1 of soil, based on previous thifluzamide soil residue data (Qin et al., 2013; Li et al., 2017a; Ma et al., 2018). Thifluzamide was dissolved in acetone and mixed into the artificial soil. The sample was then allowed to stand for 12 h to allow the acetone to evaporate. Next, eight replicates (five for the assessment of earthworm growth and three for the assessment of stress indicators) were created by transferring 500 g of contaminated soil into eight 1-L beakers. Twenty earthworms were then added to each beaker. The moisture content was adjusted to 35% using distilled water, and cow manure was added to the soil surface every week at a rate of 0.5 g per earthworm (Liu et al., 2009). The beakers were maintained at 20 °C ± 2 °C with a daily 12-h:12-h light:dark cycle for 30 d. At 12 h prior to analysis, the earthworms were removed from the beakers,
2.3.3. Activity of target enzyme and oxidative stress indicators Ground earthworms were mixed in 50 mM phosphate buffer (1:10, w/v; pH 7.8) under ice-cold conditions. After the mixture was centrifuged at 10,000 rpm at 4 °C for 15 min, the supernatant and precipitate were prepared for the determination of protein, MDA content, and enzyme activity. The activities of SDH in the supernatant and respiratory chain complex II in the precipitate were determined using an assay kit (Keming, Suzhou, China) following the manufacturer's instructions. The determination of other indicators was carried out using the supernatant with the following methods. SOD activity, based on its ability to inhibit nitroblue tetrazolium chloride (NBT), was determined following Song et al. (2009). The reaction mixture (3 mL), which comprised ethylenediaminetetraacetic acid (EDTA), phosphate buffer (pH 7.8), NBT, methionine, riboflavin, and enzyme supernatant (100 mM, 50 μM, 750 μM, 130 mM, 20 μM, 50 μL), was shaken and absorbance was assayed at 560 nm after 30 min. One unit (U) of SOD activity was defined as the amount of enzyme that inhibited reduction of NBT by 50%. CAT activity was assayed using a modified version of the method described by Xu et al. (1997). Enzyme extract (10 μL) and 3 mL phosphate buffer I (pH 7.0) were added to the reference frame; 10 μL 2
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Fig. 1. Weight change rates in earthworms (Eisenia fetida) upon exposure to thifluzamide at different time intervals. Bars are means ± standard errors (n = 5). Different letters indicate significant differences (p < 0.05) among treatments as determined by the least significant difference (LSD) test.
double-strand DNA breaks following a modified version of the approach used by Singh et al. (1988) and Song et al. (2009). We mixed 40 μL of 60 μL of the supernatant (cell suspension) with 500 μL of 0.7% low melting agar (LMA) in phosphate-buffered saline at 37 °C. This mixture was then pipetted onto a fully frosted slide precoated with 100 mL of 0.8% normal melting agar and allowed to solidify on ice for 15 min. Subsequently, another layer of 50 μL of LMA was added to the slide. The slide was then immersed in a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA at pH 10.0, 1% sodium sarcosinate, 10% dimethyl sulfoxide, and 1% Triton X-100) for 1 h. The slide was then incubated in an electrophoresis tank containing 300 mM NaOH and 1 mM Na2EDTA for 30 min followed by electrophoresis for 15 min at 25 V (300 mA). The slides were neutralized using 0.4 M Tris (pH 7.5) for 15 min and stained with 40 μL ethidium bromide (13 mg/mL) for fluorescence microscopy analysis (Olympus BX51 microscope). Finally, SCGE images (100 cell cores per slide) were analyzed using Comet Assay Software Project (CASP) software (Końca et al., 2003). The extent of DNA damage was quantified based on the olive tail moment (OTM), defined as the product of the distance between the head and tail centers of gravity and the proportion (%) of total DNA in the tail (Song et al., 2009).
enzyme extract and 3 mL 30% (w/v) hydrogen peroxide (H2O2)/phosphate buffer II were added to the sample frame. Ultraviolet absorption at 250 nm was measured every 10 s for 2 min. One unit of CAT activity was defined as the amount of enzyme consumed in the H2O2 buffer in 100 s at 25 °C. POD activity was measured using a modified version of the method described by Kochba et al. (1977). Enzyme liquid was added to 3 mL of a reaction mixture containing 100 mL potassium phosphate buffer (100 mM, pH 6.0), 38 μL 30% H2O2, and 56 μL guaiacol. Absorbance was recorded at 470 nm, and POD activity was expressed as U/mg protein. GST activity was determined using the method of Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The absorbances of glutathione (GSH) and CDNB at 340 nm were measured every 30 s for 3 min. GST activity was expressed as nmol/min/mg protein. MDA content, which is the final product of lipid peroxidation, was determined using the thibabituric acid (TBA) colorimetric method described by Xiang and Wang (1990) with some modifications. Enzyme liquid (200 μL) was mixed with a reaction mixture containing SDS (0.2 mL, 8.1%), acetic acid (1.5 mL, 20%), TBA (1%), and deionized water (1 mL) prior to immersion in a water bath at 95 °C for 1 h. The mixture was then centrifuged at 10,000 rpm for 5 min. The MDA levels in the supernatant were determined at 532 nm and expressed as nmol/ mg protein.
2.4. Statistical analysis There were eight replicates in this experiment, among which five have been used for growth and reproduction tests and three other ones for the biochemical determination of stress indicators and DNA damage. Comet assay images were analyzed with CASP. Differences among treatments were evaluated by analysis of variance (ANOVA) and least significance difference tests at P < 0.05; thifluzamide concentration, exposure time, and their interaction were analyzed using bifactorial ANOVA. The data are presented as mean ± standard error. Statistical analyses were performed with SPSS v18.0 software.
2.3.4. DNA damage SCGE comet assay was used to analyze DNA damage in cells following the method of Eyambe et al. (1991). One replicate of a single earthworm for each treatment ( total three replicates) was rinsed in physiological saline solution and then rinsing in the extrusion medium (5% ethanol, 95% saline, 2.5 mg/mLEDTA, and 10 mg/mL guaiacol glyceryl ether, pH 7.3) to induce spontaneous excretion of the coelomocytes; the coelomocytes were collected by centrifugation of the extracting solution at 3000 g for 10 min. The supernatant was placed on ice prior to the assay. The alkaline protocol for comet assay was used to detect single- and 3
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Fig. 2. Effects of thifluzamide on cocoon production and number of juveniles of earthworms (Eisenia fetida) after 30d exposure. Bars are means ± standard errors (n = 5). Different letters indicate significant differences (p < 0.05) among treatments as determined by the least significant difference (LSD) test.
thifluzamide. Overall, the ROS fluorescence intensity was higher in the groups treated with thifluzamide compared to the control group (Fig. 4).
3. Results 3.1. E. fetida growth and reproduction No significant differences in weight change were observed between different treatments on day 7. Significant decreases in weight were observed at days 14 and 21 in the group treated with 10.0 mg/kg thifluzamide, and the rate of weight gain in the group treated with 1.0 mg/ kg thifluzamide was significantly lower than that of the control group. On day 28, all groups treated with thifluzamide showed growth (weight gain); however, the weight gain rates of the groups treated with 1.0 and 10.0 mg/kg thifluzamide were significantly lower than that of the control group (Fig. 1). Compared to the control group, cocoon production and the number of juveniles were not significantly different in the groups treated with 0.1 and 1.0 mg/kg thifluzamide. In contrast, cocoon production and the number of juveniles were significantly lower in the group treated with 10.0 mg/kg thifluzamide at 30 days after exposure (Fig. 2).
3.4. Antioxidant and detoxification enzyme activity Exposure to most concentrations of thifluzamide inhibited SOD activity, with the exception of on day 28. On day 28, the SOD activities in the treatment groups were similar to that of the control group, and there was a tendency to be activated (1.0 mg/kg; Fig. 5a). On day 7, the earthworms exposed to thifluzamide at all concentrations had greater CAT activities than the earthworms in the control group. In contrast, on day 21, the CAT activities in the treatment groups were lower than in the control group. On day 14, the CAT activity in the group treated with 10.0 mg/kg thifluzamide exceeded that of the control group. On day 28, the CAT activities in the groups treated with 0.1 and 1.0 mg/kg thifluzamide were lower than that of the control, whereas the CAT activities in the other treatment groups were similar to that of the control group (Fig. 5b). Compared to the control, POD activity at day 7 was greater in all treatment groups than in the control group. At day 14, POD activity was greater in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to in the control group. At day 21, POD activity was lower in the groups treated with 0.1 and 1.0 mg/kg thifluzamide compared to the control. At day 28, POD activity was greater in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to the control (Fig. 5c). At all time points, GST activity was lower in the groups treated with 1.0 and 10.0 mg/kg thifluzamide compared to the control, while GST activity in the group treated with 0.1 mg/kg thifluzamide was lower than in the control group only at day 7 (Fig. 5d).
3.2. Activities of SDH and respiratory chain complexes II Compared to the control, SDH activity was lower at days 7 and 14 in the groups treated with 1.0 and 10.0 mg/kg thifluzamide and at days 21 and 28 in the group treated with 10.0 mg/kg thifluzamide (Fig. 3a). Similarly, the activity of respiratory chain complex II was lower in the groups treated with 1.0 and 10.0 mg/kg thifluzamide at all time points (Fig. 3b); a gradual decrease in the activity of respiratory chain complex II was observed with increasing thifluzamide concentration. Overall, the changes of the activities of SDH and respiratory chain complex II were similar. 3.3. ROS level
3.5. MDA content Compared to the control, the ROS fluorescence intensity was significantly higher on day 7 in the groups treated with 1.0 and 10.0 mg/ kg thifluzamide. Similarly, the ROS fluorescence intensity of all treatment groups was significantly higher than that of the control group at days 14 and 21. On day 28, the ROS fluorescence intensity was only higher than the control in the group treated with 10.0 mg/kg
In all treatment groups, the MDA content only exceeded that of the control group on day 7. However, at days 14, 21, and 28, the MDA content did not differ between the treatment groups and the control group, with the exception of the group treated with 1.0 mg/kg thifluzamide on day 21 (Fig. 6). 4
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Fig. 3. The SDH activity (a) and the activity of respiratory chain complexes II (b) changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3).Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
Fig. 4. ROS level changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
Thifluzamide dose and exposure time had effects on all biomarkers (P < 0.05; Table S2). The weight changes; activities of SDH, respiration chain complex II, SOD, CAT, POD, and GST; ROS level; MDA content; and DNA damage were significantly different among the four time points and among the different thifluzamide concentrations. These differences were also reflected in the one-way ANOVA results, which indicated significant differences between concentrations at the same exposure times.
3.6. E. fetida DNA damage In the control group, the comet head centers were rounded, dense, and shiny, with little or no DNA migration to other regions [Fig. 7A(a)]. Exposure to thifluzamide resulted in large quantities of comet tails in cell nuclei, in which the DNA appeared fluffy, scattered, and mushy [Fig. 7A(b), (c), and (d)]. As the exposure concentration increased, DNA migration and expansion into the nucleus became more pronounced. The OTM of the earthworm's coelomocyte comets gradually increased with increasing thifluzamide concentration (Fig. 7B).
4. Discussion This study investigated growth and reproduction along with a range of biomarkers that indicate environmental stress in earthworms (E. fetida) exposed to different concentrations of thifluzamide over 28 d. The results provide valuable information about subchronic genotoxicity in a key ecosystem service provider.
3.7. Dose-exposure time biochemical responses Interactions between thifluzamide exposure time and dose were observed for all biomarkers (Table S1), indicating that the dose-dependent responses of the biomarkers varied over time (P < 0.05). 5
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Fig. 5. Enzyme activity changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Activity of (a) SOD, (b) CAT, (c) POD, and (d) GST. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
at 5.0 and 10.0 mg/kg for 42 days (Liu et al., 2018) and after exposure to the insecticide cyfluthrin at concentrations exceeding 20 mg/kg (Li et al., 2017b). The high concentration (10.0 mg/kg) of thifluzamide might damage the reproductive systems of earthworms, resulting in the malformation of sperm, loss of fertilization ability, and eventually infertility.
4.1. Toxic effects of thifluzamide on growth and reproduction in E. fetida Earthworm weight is a sensitive indicator of exposure to stress (Xiao et al., 2006; Liu et al., 2018) that may be used to evaluate pollutant toxicity. In this study, the weights of earthworms exposed to 10.0 mg/ kg thifluzamide were lower at days 14 and 21 (Fig. 1). Ye et al. (2016) found that weight loss may be caused by the loss of glycogen, lipids, and proteins upon exposure to toxic chemicals. Givaudan et al. (2014) reported that earthworm detoxification mechanisms involve the removal of exogenous pollutants through energy metabolism. Thus, we posit that exposure to 10 mg/kg thifluzamide may affect the normal metabolism of biomacromolecules in earthworms, with detoxification causing excessive metabolism. Similarly, Liu et al. (2018) found that exposure to 10 mg/kg of the insecticide chlorantraniliprole led to lower earthworm weight after 14 days, while exposure to chlorantraniliprole at 5.0 and 10.0 mg/kg resulted in lower weight after 28 and 42 days. However, in this study, the earthworm weights in all treatment groups indicated growth after 28 days (Fig. 1), possibly because the earthworms had adapted to the stress conditions, or because the thifluzamide had been degraded, allowing normal growth to resume. The number of cocoons and juveniles were lower in the group treated with 10 mg/kg thifluzamide at day 30 (Fig. 2), indicating toxic effects on reproduction. Similar effects on cocoon production and number of juveniles were reported after exposure to chlorantraniliprole
4.2. Effects of thifluzamide on target enzymes While pollutants are known to cause oxidative damage in earthworms, the underlying response mechanisms have been scarcely studied. Wang et al. (2015) found that the neonicotinoid insecticide guadipyr inhibited acetylcholinesterase activity in earthworms. Thifluzamide acts by interfering with succinate ubiquinone reductase (complex II) in the mitochondrial respiration pathway. This inhibition of respiration and the activities of related enzymes reduces electron transfer and energy supply, eventually leading to fungal death (Yang et al., 2016). Similar mechanisms have been reported in other organisms; for example, thifluzamide (Yang et al., 2016) and boscalid (a fungicide) (Qian et al., 2018) inhibited the activities of SDH and mitochondrial respiratory chain complexes in zebrafish. SDH and respiratory chain complex II are embedded in the internal membrane of the mitochondrion; thus, their activities reflect the generation of adenosine triphosphate and mitochondrion function (Leist et al., 1999), 6
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Fig. 6. MDA content changes in earthworms (Eisenia fetida) earthworms exposed to thifluzamide. Pro is the abbreviation of protein. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
which modulate energy generation and may mediate cell apoptosis and necrosis (Zamzami et al., 1995). The activities of SDH and respiratory chain complex II determined in this study revealed a dose-dependent
inhibition effect of thifluzamide on SDH and respiratory chain complex II activity (Fig. 3). Thus, we speculate that thifluzamide acts on the mitochondria of earthworms, inhibits the activities of SDH and Fig. 7. A. Comet assays for coelomocytes of earthworms (Eisenia fetida) (a) control earthworms, (b) nuclei of earthworms exposed to 0.1 mg/kg of thifluzamide, (c) nuclei of earthworm exposed to 1.0 mg/kg of thifluzamide, (d) nuclei of earthworms exposed to 10.0 mg/kg of thifluzamide. B. Effect of thifluzamide on the coelomocytes' comet olive tail moment at different exposure times. Each soil sample was performed in triplicate. Bars are means ± standard errors (n = 3). Different letters indicate significant differences between treatments of the same day (at the p < 0.05 level).
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responses to pollutants. In this study, GST activity was initially inhibited in earthworms exposed to the low concentration of thifluzamide (0.1 mg/kg) and then gradually recovered. In contrast, the GST activities remained low in the groups exposed to higher levels of thifluzamide (1.0 and 10.0 mg/kg), and a dose–effect relationship was observed (Fig. 5d). This finding supports the results of Lin et al., (2012a), who reported a dose–response relationship for GST activity when earthworms were exposed to the antibiotic chlortetracycline. These findings may be related to the production of intermediate metabolites during detoxification. Such metabolites could alter the composition of the GST subunit, resulting in more consumption of GSH, reduced GST activity, and competition of GST with its substrate (e.g., CDNB) (Egaas et al., 1999). MDA is a product of lipid membrane peroxidation that is formed upon oxidative damage. The MDA content may indirectly indicate the level of free radicals in an organism (Fazeli et al., 2007). In this study, the MDA content in earthworms increased at 7 days after exposure to thifluzamide (Fig. 6). This might result from the earthworms being under oxidative stress, leading to the production of excessive ROS (Fig. 4) and thus the peroxidation of lipid membranes. After 14 days, the MDA levels in the treatment groups did not differ from that of the control group (Fig. 6), perhaps because excess peroxidant radicals were scavenged by activated antioxidant enzymes (CAT and POD), alleviating oxidative stress and minimizing the accumulation of MDA (Schmitt et al., 2007). Likewise, Zhang et al. (2013) found that the peroxidation of lipids in earthworms may be mediated by ROS metabolism as a result of enzymatic toxicity defenses.
respiratory chain complex II, and damages the mitochondrial structure, leading to oxidative damage. 4.3. Effects of thifluzamide on oxidative stress and the antioxidant system The dynamic state of equilibrium of ROS is disrupted by exposure to exogenous pollutants, which drive the accumulation of ROS. Excess ROS levels damage cell structures, nucleic acids, proteins, and lipids (Valko et al., 2006). ROS comprise a series of oxygen free radicals, such as H2O2, superoxide (O2−), and hydroxyl radical (Han et al., 2014). In this study, exposure to thifluzamide caused the ROS levels in earthworms to increase (Fig. 4). Mitochondria are a key source of ROS when cells are subjected to exogenous damage (Piantadosi and Zhang, 1996), and damage to the structure and function of mitochondria may increase levels of free radicals and associated oxidative damage (MartínezFernández et al., 2001). ROS and mitochondrial damage may also be mutually causal because ROS affects the vitality of respiratory chain complexes; this impairs mitochondrial structure and function and may lead to further increases in ROS levels and eventually apoptosis (Liu, 2008). The results of this study indicated that thifluzamide exposure inhibited the activities of SDH and respiratory chain complex II and increased the levels of ROS (Figs. 3 and 4). These results support the findings of the above studies and lead us to speculate that the increases in ROS levels were caused by damage to mitochondrial structure and function. Essential antioxidant enzymes involved in the elimination of ROS include SOD, POD, and CAT, which are frequently used as indicators of ROS production (Zelikoff et al., 1996). SOD is an important component of the primary antioxidant defense mechanism, in which O2− is converted into H2O2 (Han et al., 2014). In this study, except for on day 28, SOD activity in earthworms was inhibited by exposure to thifluzamide, particularly at the high exposure concentrations (1.0 and 10.0 mg/kg; Fig. 5a). Similar effects were reported for the herbicide atrazine (Song et al., 2009). The decreased SOD activity may have been caused by the elimination of highly reactive O2− via its conversion to H2O2 by SOD; however, the elevated SOD activity at day 28 may have arisen from O2 production, which stimulated SOD activity over the longer exposure time. CAT and POD scavenge H2O2 and other free radicals (Han et al., 2014). In this study, CAT activity in earthworms initially increased upon exposure to thifluzamide and then decreased (Fig. 5b). Similarly, Shao et al. (2012) found that exposure to low concentrations of the insecticide endosulfan first increased CAT activity in zebrafish but then inhibited CAT activity over time. This phenomenon may be plausibly explained as follows. A low level of pollutant-related stress produced a little H2O2 soon after exposure, activating CAT; however, CAT activity was later significantly reduced because the pollutant induced more ROS production than CAT could eliminate. Thus, we conclude that thifluzamide induced excess ROS that could not be eliminated by cellular antioxidants, thereby depleting both CAT and SOD. The effects of thifluzamide on POD activity in this study were similar to the effects on CAT activity (Fig. 5c). These results agree with the findings of a study on the acute toxicity and oxidative stress effects of the fungicide pyraclostrobin in earthworms (Wang et al., 2016a,b). Thus, the dual enhancement of POD and CAT activities likely improved the scavenging capacity for H2O2 to restore ROS equilibrium, indicating that the earthworms began to resist potential damage from thifluzamide. The activities of POD and CAT were inhibited later (at days 21 and 28), possibly because the gradual accumulation of H2O2 in the earthworms exceeded the scavenging capacity of CAT and POD, resulting in the inhibition of their activities. GST is a potent detoxifying enzyme that influences antioxidant enzyme activity. GST combines with GSH and electrophilic reagents to eliminate contaminants, scavenge ROS, eliminate metabolites of lipid peroxidation (LPO), and reduce DNA damage (Pickett and Lu, 1989). Thus, GST has been used as a sensitive indicator of oxidative stress
4.4. Genotoxic effects of thifluzamide Exposure to pollutants results in cellular oxidative stress and DNA damage by the action of free radicals (Valavanidis et al., 2006). The extent of this type of damage may be assessed based on OTM, which reflects the distance between the comet head and tail centers (Song et al., 2009). DNA damage tends to be caused by ROS, which leads to the removal of nucleotides, strand breaks, and modifications to nucleotide bases (Cooke et al., 2003). We surmised that DNA damage arose from oxidative stress based on the larger OTM values and ROS accumulation observed in this study (Figs. 4 and 7B), indicating dose–effect relationships. Yang et al. (2016) found that thifluzamide caused mitochondrial DNA damage, leading to toxic effects on zebrafish development and pathological changes through significant alterations in the expressions of some related genes in the mitochondria. We posit that thifluzamide may have a similar mechanism of action on earthworms. Thifluzamide damages nuclear DNA via oxidative stress. It can also directly cause mitochondrial DNA damage due to its effects on mitochondria because mitochondrial DNA is more vulnerable to exogenous damage than nuclear DNA (Liu and Wang, 2013). Disturbances to mitochondrial DNA transcription and translation inhibit the synthesis and expression of mitochondrial DNA coding proteins, which may increase free radicals and lead to greater oxidative damage (the discussion in sections 4.2 and 4.3 provides evidence for this conjecture). Thus, oxidative damage and mitochondrial damage interact to form a vicious cycle (James et al., 1996; Pulkes and Hanna, 2001). 5. Conclusions This study showed toxicity of thifluzamide to earthworms, through effects on mitochondria that induced oxidative stress; the interaction of oxidative damage and mitochondrial damage led to increases in levels of ROS that stimulated the antioxidant and detoxification enzyme systems. Short-term exposure (7 d) to thifluzamide caused LPO in the earthworms, as indicated by high MDA contents. OTM increased with increasing thifluzamide concentration and exposure time. Remarkable DNA damage was induced by oxidative damage and mitochondrial 8
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damage at the evaluated doses. These effects led to lower growth and reproduction rates in earthworms exposed to thifluzamide at 10 mg/kg. Confirmation of the action of thifluzamide on mitochondria requires subcellular-level and molecular-level research to improve our understanding of the risk posed by thifluzamide to soil ecosystems.
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