Toxicological effects of pyraclostrobin on the antioxidant defense system and DNA damage in earthworms (Eisenia fetida)

Ecological Indicators 101 (2019) 111–116

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Original Articles

Toxicological effects of pyraclostrobin on the antioxidant defense system and DNA damage in earthworms (Eisenia fetida)

T

Junchao Ma1, Chao Cheng1, Zhongkun Du, Bing Li, Jinhua Wang, Jun Wang, Zuobin Wang, ⁎ Lusheng Zhu College of Resources and Environment, Shandong Agricultural University, Key Laboratory of Agricultural Environment in Universities of Shandong, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Taian 271018, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pyraclostrobin Strobilurin fungicide Oxidative stress Genotoxicity Risk assessment

In recent years, pyraclostrobin has been widely used as a fungicide. However, pesticides remain in soil and water, potentially causing irreversible damage to non-target organisms. Thus, the present study investigated the toxicity of different pyraclostrobin concentrations (0, 0.1, 1.0, and 2.5 mg/kg) on earthworms (Eisenia fetida). On days 7, 14, 21, and 28, reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione-S-transferase (GST), and malondialdehyde (MDA) levels as well as DNA damage were evaluated. The ROS content under 0.1 mg/kg pyraclostrobin treatment first increased and later returned to the control level. High concentrations of pyraclostrobin (1.0 and 2.5 mg/kg) led to excessive ROS during the experiment. Enzyme activities and MDA contents in the experimental groups first increased and then decreased. Except for POD activity with 0.1 mg/kg pyraclostrobin treatment, SOD, POD, and GST activities under pyraclostrobin treatment were inhibited on day 28. However, CAT activities and MDA contents in the experimental groups were similar to control levels on day 28. DNA damage was promoted with increasing concentrations of pyraclostrobin. On days 21 and 28, DNA damage in earthworms treated with 0.1 mg/kg pyraclostrobin decreased. However, DNA damage in earthworms treated with 1.0 and 2.5 mg/kg pyraclostrobin rose slowly after the 14th day. In summary, pyraclostrobin can break the dynamic balance of ROS in the organism, which can affect the antioxidant defense system and ultimately cause DNA damage.

1. Introduction

et al., 2016; Cui et al., 2016; Cusaac et al., 2017; Zhang et al., 2017; Li et al., 2018a). However, more research should be taken to study the toxicity of pyraclostrobin to soil organisms. In soil, earthworms represent the largest component of the animal biomass (Blouin et al., 2013; Kwak et al., 2014; Kim et al., 2016) with an important role in terrestrial ecosystems (Chevillot et al., 2017; Jing et al., 2017). Earthworms are commonly termed ‘ecosystem engineers’ (Blouin et al., 2013). By burrowing activity, earthworms can modify soil structure, which can have significant effects on hydrology, infiltration and aeration (Chan, 2001). Earthworms also participate in decomposition and nutrient cycling (Han et al., 2014). As they ingest soil and are ubiquitously found in soil (Navarro et al., 2017), they are easily affected by contamination (Song et al., 2009). However, soil formation and nutrient cycling are prerequisites for other services (Blouin et al., 2013). For the above reasons, it is meaningful and necessary to study the toxicity of pyraclostrobin to earthworms. In toxicology, Eisenia fetida is considered as a model organism for

Agricultural chemicals are widely used and may affect humans in daily life. However, efficient use of agricultural chemicals is very low, and most of the pesticides applied remain in water or soil ecosystems (Pimentel, 1995; Werf, 1996; Hussain et al., 2009; Zhang et al., 2017). Pesticide residues in soil and water may cause irreversible damage to non-target organisms and ecosystems. Pyraclostrobin is a strobilurin fungicide that has a broader antifungal activity spectrum and higher efficiency and security profiles than do previously used fungicides (Mercader et al., 2008). Pyraclostrobin inhibits mitochondrial respiration by blocking the transmission of electrons between cytochrome b and c1, which leads to death of the target fungus (Fulcher et al., 2014). Numerous studies have researched the toxicity of pyraclostrobin to aquatic and amphibious organisms (Belden et al., 2010; Ding et al., 2011; Morrison et al., 2013; Anitha and Rathnamma, 2016; Cusaac

⁎

Corresponding author at: College of Resources and Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, China. E-mail addresses: [email protected] (J. Wang), [email protected] (J. Wang), [email protected] (L. Zhu). 1 Junchao Ma and Chao Cheng contributed equally to this work. https://doi.org/10.1016/j.ecolind.2019.01.015 Received 15 September 2018; Received in revised form 13 December 2018; Accepted 5 January 2019 1470-160X/ © 2019 Elsevier Ltd. All rights reserved.

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measuring the SOD, CAT, POD, and GST activities and MDA content, and three for SCGE. During the test, mortality was 0%. Twelve hours before tests, the selected earthworms were rinsed with normal saline and kept on clean humid filter paper at 20 ± 1 °C in the dark for gut content excretion.

toxicological experiments by the Organization for Economic Cooperation and Development (OECD, 1984). Reactive oxygen species (ROS) are continuously produced by mitochondria (Essick and Sam, 2010) and consist of hydroxyl radical (OH−), superoxide anion (O2−), and hydrogen peroxide (H2O2), among others. Excess ROS can oxidize proteins, DNA and lipids, negatively influencing cells and contributing to disease progression (Essick and Sam, 2010). Under normal conditions, the ROS content in an organism is kept in balance. When an organism is exposed to exogenous contamination, this balance is broken (Fazio et al., 2014). Excess ROS will be largely eliminated by the antioxidant enzyme system, including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD). SOD converts superoxide radicals (O2−) to O2 and H2O2 (Mittler, 2002). CAT is the main enzyme scavenging H2O2 by converting it into H2O and O2 (Zhang et al., 2015), and changes in its activity is used to reflect the oxidation-reduction equilibrium in cells. POD not only eliminates H2O2 but can also prevent ROS production (Wen et al., 2011). Glutathione-S-transferase (GST), an important detoxification enzyme, also protects organisms from oxidative damage. As a product of lipid peroxidation, malondialdehyde (MDA) is considered to be a sensitive index to determine cellular oxidative damage (Ma et al., 2017). DNA damage can also be measured using single-cell gel electrophoresis (SCGE) (Li et al., 2018b). We have assessed the toxicity of pyraclostrobin on the antioxidant systems and DNA of Eisenia fetida using the above-mentioned biomarkers.

2.3. Enzyme extraction and protein content measurement Enzyme extraction referred the methods of Mishra and Dash (1980), and Han et al. (2014). During the experiment, earthworms were placed in a homogenizer and ground in 50 mM phosphate-buffered saline (1:8, w/v, pH 7.0). The homogenization buffers were centrifuged at 10,000 rpm for 10 min, and the supernatant was used for analysis of protein, SOD, CAT, POD, GST, and MDA. The entire extraction was performed at 4 °C. The Bradford method (Bradford, 1976) was used to detect the protein content with bovine serum albumin as the standard. 2.4. ROS content measurement The assay used for ROS content was the method of LeBel et al. (1992) and Lawler et al., (2003). Three earthworms from each concentration were homogenized using ice-cold phosphate buffer (100 mM, pH 7.4) at 4 °C, and the mixture was centrifuged at 3000 × g at 4 °C for 10 min; the supernatant was centrifuged again at 20,000 × g at 4 °C for 20 min. A mitochondria suspension was obtained after the sediment was resuspended. Next, dichlorofluorescein diacetate solution and the mitochondria suspension were incubated in a water bath (37 °C) for 30 min, and the fluorescence intensity of the specimen was assessed at 488 nm excitation and 522 nm emission using a fluorescence spectrophotometer.

2. Materials and methods 2.1. Chemicals, test organisms and soil Dr. Ehrenstorfer GmbH. (Augsburg, Germany) provided the pyraclostrobin (purity 99%, CAS No. 175013-18-0). All other necessary chemicals were analytically pure and purchased from either Sigma Chemical (St. Louis, Missouri, USA) or Tianjin kaitong chemical Co. (Tianjin, China). Eisenia fetida specimens were purchased from an earthworm breeding facility located in the south campus of Shandong Agricultural University (Shandong, China). Earthworms were acclimated in a culture pot containing cattle manure and artificial soil for 2 weeks under the condition of 20 ± 1 °C. Artificial soil was used as a test substrate, and the pH of the artificial soil was kept within 6.0 ± 0.5 using CaCO3 (OECD, 2004). The moisture of the soil was kept at 35%. Healthy earthworms weighing 300–600 mg were pre-cultured in uncontaminated artificial soil for 24 h before being used for experiments.

2.5. Enzyme assays Based on the method of Giannopolitis and Ries (1977), SOD activity was measured by analyzing the inhibition of nitroblue tetrazolium chloride (NBT) photochemical reduction. CAT activity was assayed as reported by Aebi (1984) and Xu et al. (1997), whereby activity was based on the degradation of H2O2. The measurement of POD activity referred to the methods of Kochba et al. (1977) by noting the change in absorbance at 470 nm when 3 mL of reaction mixture was mixed with the enzyme extract. The assay used for GST activity was the method of Habig et al. (1974). 2.6. MDA content measurement The MDA content was analyzed using the method reported by Ohkawa et al. (1979) and Xiang and Wang (1990). First, the supernatant was added to the reaction mixture containing 1 mL of water, 1.5 mL of 20% acetic acid, 1.5 mL of 1% thiobarbituric acid (TBA), and 0.2 mL of 8.1% sodium dodecyl sulfate (SDS). The mixture was incubated for 1 h at 80–90 °C and then centrifuged for 15 min at 3000 × g. The MDA level was detected at 532 nm.

2.2. Toxicity test According to the China Pesticide Information Network (http:// www.chinapesticide.org.cn), the recommended dose of pyraclostrobin is 56–187.5 g/ha. Previous studies have reported that initial soil deposits of pyraclostrobin residue were 0.06–0.77 mg/kg (Li et al., 2010; Yan et al., 2013; Wang et al., 2014). Ultimately, 0, 0.1, 1.0, and 2.5 mg/ kg were used as treatment concentrations. First, pyraclostrobin was dissolved in acetone, and the appropriate volume of pyraclostrobin solution was mixed well with 10 g artificial soil until the acetone evaporated. The control group was treated with the same volume of acetone. Next, 490 g artificial soil was added and thoroughly mixed. Finally, 500 g of the treated soil was placed into a 1L glass beaker. Six glass beakers were used for each concentration, and each beaker contained 10 earthworms. The beaker was sealed with sealing film with holes in it. During the experiment, earthworms were maintained in an incubator under the following conditions: 12:12 h light/dark and 20 ± 1 °C. On days 7, 14, 21, and 28, nine earthworms from each concentration were randomly chosen for measurement of each experimental index. Three earthworms were used for measuring ROS, three for

2.7. DNA damage measurement Based on the methods of Eyambe et al. (1991), earthworm coelomocytes were collected. First, three earthworms from each treatment were placed in 1 mL of coelomocyte extracting solution for 3 min, which was then centrifuged for 10 min at 3000 × g and 4 °C. The sediment was suspended in 1 mL of phosphate buffer and centrifuged for 10 min at 3000 × g and 4 °C. The coelomocyte fraction was obtained in 1 mL of phosphate buffer (pH 7.4) and used for SCGE. Based on the method reported by Singh et al. (1988), SCGE was employed to analyze DNA damage; microscope slides for which one side was frosted using 100 µL of 0.8% normal melting agarose (NMA) were used for the experiment. Forty microliters of cell suspension was 112

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added to 500 µL of 1% low melting agarose (LMA), and 60 µL of the mixture was titrated onto the frosted side. After 15 min, 50 µL of 0.5% LMA was coated onto the frosted side. When the agarose was cakey, the slides were immersed in lysis solution for 1 h and then soaked in electrophoresis buffer for 30 min. The slides were electrophoresed at 25 V for 15 min. Next, the microscope slides were neutralized using 0.4 M Tris-HCl (pH 7.5) for 15 min and dehydrated in absolute ethyl alcohol. Finally, the slides were stained with 30 µL of ethidium bromide (13 mg/ mL) for 20 min and used for fluorescence microscopy analysis. All treatments were assessed by Comet Assay Software Project (CASP). The olive tail moment (OTM) was used to quantify DNA damage.

activities under 1.0 and 2.5 mg/kg treatments were notably lower than that in the control group on the 28th day. As described in Fig. 2d, GST activities treated with different pyraclostrobin doses were stimulated on the 7th and 14th day. GST activities exposed to low pyraclostrobin concentrations (0.1 and 1.0 mg/ kg) were activated on the 21st day, but GST activity with 2.5 mg/kg pyraclostrobin treatment recovered to a similar level of the control. On day 28, GST activity exposed to 1.0 mg/kg pyraclostrobin concentration was markedly inhibited, whereas those after treated with other concentrations were close to the control.

2.8. Statistical analysis

As described in Fig. 3, MDA contents first increased and then reached the control level after treatment with different doses of pyraclostrobin. MDA contents increased on days 7, 14, and 21, and on day 28, MDA contents in treatment groups were at the control level.

3.3. Effects of pyraclostrobin on lipid peroxidation

Three replicates were used for each test. All data were processed using SPSS software (Ver 19.0), and all of the results are shown as the means ± standard deviation (SD, n = 3). All data were subjected to the Kruskal-Wallis test. Significances are indicated for p < 0.05.

3.4. Effects of pyraclostrobin on DNA damage As described in Fig. 4, OTM values increased with increasing pyraclostrobin concentration, indicating that pyraclostrobin induced dosedependent DNA damage in earthworms. There were significant differences between treatments. OTM values for earthworms exposed to different doses of pyraclostrobin increased on days 7 and 14. After the 14th day, the OTM value exposed to 0.1 mg/kg pyraclostrobin decreased, but the OTM value exposed to 1.0 mg/kg pyraclostrobin did not change significantly. OTM values in earthworms under the treatment of 2.5 mg/kg pyraclostrobin still increased after the 14th day.

3. Results 3.1. Effects of pyraclostrobin on ROS levels According to the data presented in Fig. 1, the ROS content increased with increasing pyraclostrobin concentration. Under treatment with different doses of pyraclostrobin, ROS contents clearly surpassed the control level during the test, except for the samples under treatment with 0.1 mg/kg pyraclostrobin on the 21st and 28th day.

4. Discussion

3.2. Effects of pyraclostrobin on enzyme (SOD, CAT, POD, GST) activities

4.1. Effects of pyraclostrobin on oxidative stress and the antioxidant system

Fig. 2a shows the changes of SOD. When earthworms were treated with different doses of pyraclostrobin, SOD activities were activated on days 7 and 14. After the 14th day, SOD activities in the experimental groups were inhibited, except for samples under treatment with 0.1 mg/kg pyraclostrobin concentration on the 21st day. Fig. 2b shows that CAT activity in the experimental groups first increased and then decreased over time. Except for CAT activity under treatment with 0.1 mg/kg pyraclostrobin concentration on the 7th day and CAT activity with 2.5 mg/kg pyraclostrobin concentration treatment on the 14th day, CAT activities in experimental groups were stimulated on days 7, 14, and 21. On day 28, CAT activities under different doses of pyraclostrobin treatments were at the control level. According to the data presented in Fig. 2c, POD activity was stimulated by 0.1 mg/kg pyraclostrobin. Except for that after 2.5 mg/kg pyraclostrobin treatment on day 21, POD activities in the 1.0 and 2.5 mg/kg groups were enhanced on days 7, 14, and 21. However, POD

During our study, ROS content increased with increasing concentrations of pyraclostrobin, suggesting disruption of the oxidative balance in earthworms exposed to this fungicide. However, accumulation of ROS decreased after the 14th day, which indicates that antioxidant enzymes in the organisms removed ROS after a period of time. Azoxystrobin and fluoxastrobin are also strobilurin fungicides, the toxicity of which have been examined in earthworms by Han et al. (2014) and Zhang et al. (2018), respectively. In those studies, the changes in ROS described were similar to the results of the present research. During early stages of the experiment, SOD, CAT, and POD activities were stimulated, indicating that these enzymes played a role in resisting oxidative damage. The increased SOD activity enhanced the organisms’ ability to remove ROS. Lee and Lee (2000) reported that increased activity of SOD in an organism may cause accumulation of H2O2, which would enhance CAT activity to promote H2O2 elimination, allowing earthworms to adapt to environmental changes and balance ROS. Increased POD activity also promotes an organism’s ability to eliminate H2O2. Thus, the earthworms started to resist the damage induced by pyraclostrobin. Over time, the accumulation of ROS exceeded the clearing capacity of SOD and affected the synthesis of SOD or its structure. Therefore, SOD activity decreased. Wang et al. (2017) reported that SOD activities under different doses of dimethomorph treatments were first stimulated and then inhibited. On day 28, POD activities exposed to 1.0 and 2.5 mg/kg pyraclostrobin were inhibited. The accumulation of H2O2 may have exceeded the clearing capacity of POD, inhibiting the enzyme. Previous research reported that POD and CAT showed a synergistic role in scavenging H2O2 (Burgeot et al., 1996). Because CAT cannot eliminate excess H2O2, its activity reached the control level at last. Nonetheless, Han et al. (2014) and Zhang et al. (2018) reported different results regarding changes in these enzymes, and these differences may reflect variation in the types of strobilurin

Fig. 1. Effects of pyraclostrobin on ROS contents in Eisenia fetida. The standard deviation (SD) is shown as error bars. Pr, protein. Different letters above columns indicate significant differences at p < 0.05 between treatments. 113

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Fig. 2. Effects of pyraclostrobin on SOD activity (a), CAT activity (b), POD activity (c) and GST activity (d) in Eisenia fetida. The standard deviation (SD) is shown as error bars. Pr, protein. Different letters above columns indicate significant differences at p < 0.05 between treatments. 0mg/kg

20.0 16.0

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Fig. 3. Effects of pyraclostrobin on MDA contents in Eisenia fetida. The standard deviation (SD) is shown as error bars. Pr, protein. Different letters above columns indicate significant differences at p < 0.05 between treatments.

Fig. 4. Effects of pyraclostrobin on DNA damage in Eisenia fetida. The standard deviation (SD) is shown as error bars. Different letters above columns indicate significant differences at p < 0.05 between treatments.

fungicide used (Zhang et al., 2017). Glutathione-S-transferase (GST) detoxifies ROS to protect cells against DNA damage (Oliveira et al., 2009). Additionally, GST eliminates products of the lipid peroxidation reaction, such as MDA (NegreSalvayre et al., 2008). GST activities at different pyraclostrobin doses were activated on days 7 and 14, increasing with pyraclostrobin concentration. This may constitute the adaptive response of Eisenia fetida to

pyraclostrobin. In the early stages, it is helpful for an organism to reduce the damage induced by exogenous substrates. On day 28, GST activities after treatment with pyraclostrobin were close to those of the control, and earthworms may adapt to a toxic environment over time. Zhang et al. (2018) also reported a similar result when researching fluoxastrobin-induced oxidative damage in earthworms. Additionally, 114

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Acknowledgments

the variation in GST is similar to that of ROS and MDA. After the 14th day, SOD activities in the experimental groups were inhibited. In addition, the MDA contents in the experimental groups increased on day 21 and later decreased. However, GST activities under treatment with different doses of pyraclostrobin were stimulated on day 21 and closed to the control level on day 28. By comprehensively considering the changes of these biomarkers in different periods, we can better understand how this fungicide affects the earthworms and which biomarkers are useful for resisting oxidative damage induced by pyraclostrobin. The observed changes of these biomarkers in our study suggest that GST detoxifies excess ROS and eliminates MDA. GST played a key role in reducing oxidative damage induced by pyraclostrobin. Malondialdehyde (MDA) levels can reflect the degree of lipid peroxidation (Zheng et al., 2016). The increase of MDA content indicated the occurrence of oxidative damage. However, the MDA contents in the experimental groups returned to the control level on day 28. These results demonstrate the joint action of SOD, CAT, POD, and GST. Liu et al. (2014) also reported a similar result when researching metalaxylM-induced oxidative damage in earthworms.

This work was supported by National Key R&D Program of China [grant numbers 2016YFD0800202 and 2017YFD0200307]; National Natural Science Foundation of China [grant numbers 41771282 and 41701279]; Natural Science Foundation of Shandong Province, China [grant numbers ZR2017MD005 and ZR2017BB075] and the Special Funds of Taishan Scholar of Shandong Province, China. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. https://doi.org/10. 1016/S0076-6879(84)05016-3. Anitha, A., Rathnamma, V.V., 2016. Toxicity evaluation and protein levels of fish labeo rohita exposed to pyraclostrobin 20%wg (carbamate). Int. J. Adv. Res. 4, 967–974. Belden, J., McMurry, S., Smith, L., Reilley, P., 2010. Acute toxicity of fungicide formulations to amphibians at environmentally relevant concentrations. Environ. Toxicol. Chem. 29, 2477–2480. https://doi.org/10.1002/etc.297. 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4.2. Genetic toxic effects of pyraclostrobin on earthworms We found that OTM increased with increasing pyraclostrobin concentration, which indicated a greater degree of DNA damage. OTM values also increased with ROS accumulation on days 7 and 14, though accumulation of ROS decreased and the OTM value slowly increased on days 21 and 28. Cooke et al. (2003) reported that ROS cause DNA damage. Our results suggest that pyraclostrobin and excess ROS are closely linked to DNA damage in the earthworm coelomocyte. Pyraclostrobin induced ROS generation, causing DNA damage. After the 14th day, the degree of DNA damage was effectively controlled. Zhang et al. (2018) also reported a similar result when researching fluoxastrobin-induced oxidative damage in earthworms. These effects can be attributed to cellular SOD, CAT, POD, and GST activities and self-repair. Indeed, DNA repair mechanisms exist in cells (Collins et al., 1995). Biomarkers can be used in early warning tests to evaluate the toxicological effects of contamination (Livingstone, 1993). We can better understand the physiological and biochemical basis of adaptation of earthworms exposed to pyraclostrobin. These biomarkers are useful for people to determine the resistance of earthworms on pollutant and to evaluate the potential effects of pollutant on food chain. In others words, these biomarkers are also useful for ecological risk assessment. If the concentration of contaminants in the environment is too high and the earthworms themselves cannot effectively reduce oxidative damage, soil function and nutrient cycling will be affected. Then, we must consider ecological risk assessment and take measures to deal with it. 5. Conclusion Based on initial soil deposits of pyraclostrobin residues, this research was designed to investigate the subchronic toxicity of pyraclostrobin in earthworms, with oxidative damage occurring even at 0.1 mg/kg treatment. Antioxidant enzymes (SOD, CAT, and POD) and a detoxification enzyme (GST) remove excessive ROS to protect organisms. The MDA contents in the experimental groups reached the control level on day 28. On days 21 and 28, DNA damage in earthworms treated with 0.1 mg/kg pyraclostrobin decreased. However, DNA damage in earthworms treated with 1.0 and 2.5 mg/kg pyraclostrobin rose slowly after the 14th day. This information may be useful for evaluating pyraclostrobin risk in soil ecosystems. 6. Compliance with ethical standards Declarations of interest: None. 115

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