Ecotoxicology and Environmental Safety 114 (2015) 17–22

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Toxicity of a neonicotinoid insecticide, guadipyr, in earthworm (Eisenia fetida) Kai Wang, Xiyan Mu, Suzhen Qi, Tingting Chai, Sen Pang, Yang Yang, Chengju Wang, Jiazhen Jiang n,1 College of Sciences, China Agricultural University, Beijing, People’s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 1 August 2014 Received in revised form 21 December 2014 Accepted 22 December 2014

Neonicotinoid insecticides are new class of pesticides and it is very meaningful to evaluate the toxicity of guadipyr to earthworm (Eisenia fetida). In the present study, effects of guadipyr on reproduction, growth, catalase(CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE) and DNA damage in earthworm were assessed using an artificial soil medium. Guadipyr showed low toxicity to earthworms and did not elicit an effect on earthworm reproduction or growth in artificial soils at concentrations o 100 mg/kg. However, after exposure to guadipyr, the activity of SOD and CAT in earthworm increased and then decreased to control level. AChE activity decreased at day 3 at 50 and 100 mg/kg and then increased to control level. Our data indicate that guadipyr did not induce DNA damage in earthworms at concentration of o100 mg/kg. & 2014 Elsevier Inc. All rights reserved.

Keywords: Guadipyr Earthworm Toxicity evaluation Antioxidase DNA damage

1. Introduction Guadipyr [1-nitro-3-(6′-chloro-3-pyridylmethyl)-4-(E)-amyl aldehyde aminoguanidine] (Fig. 1) is a novel neonicotinoid insecticide which was developed by China Agricultural University in 2008. Guadipyr has a promise future in Chinese pesticide market because of its high effective activity on aphids and rice plant hoppers as well as its low toxicity to mammals, like mice, rats and rabbit (Su et al., 2012). Neonicotinoids were introduced to market about 50 years ago and are the first new class of synthetic insecticides (Tomizawa and Casida, 2003), which act as agonists on nicotinic acetylcholine receptors (nAChRs) (Matsuda et al., 2001). Due to their broadspectrum target in organism, many neonicotinoids have high toxicity to earthworm such as acetamiprid, imidacloprid, nitenpyram, clothianidin and thiacloprid (Wang et al., 2012). Studies reported that thiacloprid and imidacloprid can negatively affected the reproduction and growth of earthworm, a significant decrease in cocoon production were found at 0.291 and 1.91 mg/kg respectively (Gomez-Eyles et al., 2009), while deltamethrin and imidacloprid could significant inhibited the growth of earthworm n

Corresponding authors. Fax: þ 86 10 62733924. E-mail addresses: [email protected] (C. Wang), [email protected] (J. Jiang). 1 Address: China Agriculture University, No. 2 Yuan Mingyuan West Road, Haidian District, People's Republic of China. http://dx.doi.org/10.1016/j.ecoenv.2014.12.037 0147-6513/& 2014 Elsevier Inc. All rights reserved.

at 0.50 and 2.00 mg/kg respectively (Capowiez et al., 2005; Shi et al., 2007). Earthworm is common animal species in soil (Bartlett et al., 2010), and suitable indicator species for the ecotoxicological and ecological risks assessment of soil pollution due to pesticides or other toxic substances (Fent, 2003; Schreck et al., 2008; Xu et al., 2010). Among a lot of biomarkers, enzymatic activities like catalase (CAT), superoxide dismutase (SOD) and acetylcholinesterase (AChE) are used for environmental pollution assessment of pesticides for their rapid response, easy determination and important of action mechanisms in organism (Cao et al., 2014; Cortet et al., 1999; Scott-Fordsmand and Weeks, 2000a; Scott-Fordsmand and Weeks, 2000b; Song et al., 2009b; Xiong et al., 2014). There are some interactions with SOD, CAT, and AChE at the molecular level, Ben-Ari reported that enhanced expression of AChE variants may influence the expression of genes, including those involved in apoptosis (Ben-Ari et al., 2006), Zhang found that imidacloprid could significantly stimulated the activity of SOD and CAT in earthworm (Zhang et al., 2014). However, imidacloprid and acetamiprid can also induce significant DNA damage in earthworms (Kocaman and Topaktas, 2007; Zang et al., 2000), our earlier study showed that guadipyr could enhance the activity of AChE, CAT and SOD in Daphnia magna (Qi et al., 2013). Thus we speculated that guadipyr could also have some effect o AChE, CAT and SOD activities in earthworm. Guadipyr is not intended for use in soil, but it may still leach

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400 kg, 2000 kg water consumption, so corresponding concentration of guadipyr at soil, 10.0, 50.0 and 100 mg/kg were used for the 21 days subchronic toxicity testing and enzyme measurements. 2.3.1. Subchronic toxicity testing Five replicates of ten earthworms were used for each treatment in this test. For growth inhibition, the bodyweight of each earthworm was measured at 0, 3, 7, 14, and 21 days after 4 h fasting. During the test, no food supplied for each earthworms. Growth inhibition rates were determined using the following equation: Fig. 1. Structure of guadipyr.

into the soil by rain or other means following application. The residue chemicals in soil can accumulate in and effect the development of soil animals and finally may impact human health through food chain. Thus, the goal of this study is to assess the risk of guadipyr on soil organism, Eisenia fetida thoroughly using artificial soil. The contents of this research include the following: (1) 14 days acute toxicity of guadipyr to earthworms; (2) 56 days reproduction toxicity of of guadipyr to earthworms; and (3) effect of guadipyr on AChE, CAT, SOD activities and DNA damage after 21 days exposed. Hoping these information could help the registration and application of guadipyr in future.

2. Materials and Methods 2.1. Chemicals and earthworms Guadipyr (95% pure) was obtained from Hefei Xingyu Co., Ltd. (Anhui, China,). Eisenia fetida were purchased from the Beijing Dahuan Earthworm Factory (Beijing, China). After acclimation for 7 days to the artificial climate (207 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lx), adult earthworms (300– 400 mg with a clitellum) were selected and deprived of food for 4 h until further experimentation under the above artificial climate through this whole study. 2.2. Artificial soil and toxicity testing 2.2.1. Artificial soil Artificial soil composed of 10% sphagnum peat moss, 20% kaolin clay, and 70% sand with 20% water content (OECD, 1984) was used in the present study. 2.2.2. Acute toxicity testing A 14 day acute toxicity test of guadipyr to earthworm were proceeded according to OECD guideline 207 (OECD, 1984). Based on the preliminary experiment, there were no dead earthworm founded at the dosage below 100 mg/kg, so 100, 200, 400, 600 and 800 mg/kg by five replicates  10 earthworms were chosen for formal testing. All signs of harm or damage to the earthworms such as coelomic fluid extrusion, body swelling and decay were recorded at day 7 and 14 after exposure. 2.3. Subchronic toxicity testing and enzyme measurements According to the result of acute toxicity and trial test, there were no significant differences (p o 0.05) between control and treatment at the concentrations o10 mg/kg, considering the recommending field usage of guadipyr was 20.0 g a.i./ha at 200 kg,

In = (W0 − Wt)/W0 × 100% In this equation, In is the growth inhibition rate, W0 is the average weight of earthworm at the beginning of the growth test, and Wt is the average weight after exposure for t days (Wu et al., 2012). Same growth test, another set of earthworms was proceeding reproduction test according to OECD 222 guideline for the testing of chemicals (OECD, 2004). Concentrations of guadipyr were 5.0, 10.0, 50.0 and 100 mg/kg and the duration was 4 weeks. Sterile cow manure at 0.50 g per earthworm per week was used as food. At day 28, all adult worms were counted and removed from each glass container; while cocoons and juveniles in each container were counted and preserved to incubate in test vessels for another 28 days without feeding. At the end of the second 4 weeks period, the number of juveniles hatched form the cocoons in the test soil and the cocoon numbers are determined. 2.3.2. Enzyme activity measurements The same experiment as the growth inhibit test was repeated have for enzyme activity measurements. Five live earthworms from five replications were deprived of food for 4 h. To each tube, phosphate buffer solution (PBS) buffer (pH 7.4) was added (weight: volume ratio ¼ 1:4) and then the mixture was homogenized and centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was kept at o  80 °C after protein concentration determination (Bradford, 1976).until enzymatic activity analysis. AChE activity was determined according to the protocol developed by Ellman et al. (Ellman et al., 1961), one AChE activity union was described as the amount of AChE that hydrolyzes 1.0 nmol of acetylthiocholine per min. CAT activity was determined as described by Song et al. (2009a), and one CAT activity union was described as the amount of CAT that degrades 1.0 μmol of H2O2 per min. SOD activity was determined by measuring its ability to inhibit the photochemical reduction of pyrogallol, as described by Marklund and Marklund (1974). 2.4. Comet assay Five replicates of ten earthworms for treatment of guadipyr (10.0, 50.0, and 100 mg/kg) were prepared, and the controls were exposed to the same volume of deionized water. Earthworm coelomocytes were obtained using the method described by Verschaeve and Gilles (1995) and the alkaline comet assay was performed as originally described by Singh et al. (1988) and Xu et al. (2013). The comet assay was observed through an OLYMPUS fluorescence microscope equipped with a CCD camera. Fifty random non-overlapping cells were imaged on each parallel set of slides (400  magnification). The captured images were analyzed using CASP (Końca et al., 2003). Olive tail moment (OTM), tail moment (TM), and tail DNA percentage were used to quantify DNA damage after exposed to guadipyr.

K. Wang et al. / Ecotoxicology and Environmental Safety 114 (2015) 17–22

2.5. Analyzing guadipyr in artificial soil For five replicates in each toxicity test, ten grams of each soil sample were collected at the established observation time. Each of them was added to 50 mL tube, followed by 10.0 mL distilled water addition. The mixture was homogenized for 3 min and stand for 10 min. Next, 10.0 mL acetonitrile was added and the mixture was vigorously shaken for 3 min. Then 10.0 g mixture of MgSO4: NaCl mixture (4:1 w/w) was added into the tube and the tube was shaken for an additional 3 min, last step the guadipyr was extracted in an ultrasonic water bath for 10 min at room temperature. After extraction, the sample was centrifuged at 8000 rpm for 5 min. Finally, the upper extraction contains guadipyr was injected into high performance liquid chromatography (HPLC) system. The following chromatographic conditions were used: PAD detector; C18 stainless steel column (250 mm  46 mm  5 μm); 25 °C column temperature; mobile phase consisting of 7:3 (v: v) acetonitrile: formic acid (0.1%); 1.0 mL/min flow rate; 286 nm wavelength; 20.0 μL injection volume; and the external standard method was quantitatively used to determine the concentration of guadipyr in soil. 2.6. Statistical analysis All data are presented as the mean 7standard deviation (SD) in the present day. Differences between the treatment and control were determined by one-way ANOVA, followed by a post hoc Dunnett test at p ¼0.05.

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day 3, 7, 14 and 21 respectively. For reproduction test of guadipyr, the number of cocoons for per earthworm is 3.82, and its hatchability is 76.7%. After continuously exposed to guadipyr at various concentrations for 56 days, no significant effect was observed on cocoons’ reproduction. However, the hatchability of cocoons decreased along with the increasing concentrations of guadipyr, but the difference was not significant due to the high standard deviation. A: Hatchability; B: The number of cocoons per earthworm 3.3. Effects of guadipyr on AChE, CAT, SOD activities and Dna DAMAGE Seen form Fig. 4A, significant decrease in AChE activity was calutated from control at day 3 after exposure to guadipyr at 50.0 and 100 mg/kg (po 0.05). But in the following test period, the AChE activity recovered to control level without significant difference. Differently form AChE, significant increase of CAT activity in earthworm was observed at day 7, 14 and 21 after exposed to guadipyr at 50.0 (excluded day 21) and 100 mg/kg. SOD activity had a different change form both AChE and CAT. The first significant increase of SOD activity was found as early as day 3 under 10.0 mg/kg guadipyr exposure, while day 7 for CAT and SOD increase peaks were founded at day 7 under 50.0 and 100 mg/kg and at day 14 under 100 mg/kg. At day 21, SOD activity recovered to original level of control. Data are expressed as the mean 7SD. Statistical significance vs the control group is shown. np o0.05; nnp o0.01. A: Effects of guadipyr on AChE activity; B: Effects of guadipyr on CAT activity; C: Effects of guadipyr on SOD activity.

3. Results 3.1. Actual concentrations of guadipyr in test soil Analysis results indicated that the deviations between nominal and actual concentrations of guadipyr were less than 20% within the first day and then the concentration decreed along with exposure time increase (Table 1), some of them even can’t be monitored with HPLC at the last days of test, so nominal dosage were used for all the results expression and discussion to avoid ambiguity. 3.2. Effect of guadipyr on mortality, growth and reproduction of earthworm During 14 days acute test, no death and symptoms of poisoning in any treatment group was observed. During the 21 days subchronic exposure, the growth ofEisenia fetida was significantly inhibited by guadipyr only at 100 mg/kg, the inhibition rate was as high as 33.7%, 46.8%, 51.5% and 66.3% at

For DNA damage after exposed for 21 days under 10.0, 50.0 and 100 mg/kg guadipyr, the change in OTM, TM and tail DNA was not insignificant. A: Olive Tail Moment; B: Tail DNA%; C: Tail Moment. Error bars indicate standard deviation (SD).

4. Discussion As mentioned earlier, guadipyr is a new neonicotinoid pesticide that high effective to pests but safe to mammals (Su et al., 2012). The present study proved that guadipyr is also safe to earthworm. 100 mg/kg is the upper limit of toxicity test of OECD for chemicals in artificial soil. If the morality of test organisms is less than 50%, then the test chemicals is classified as low toxic. Thus, guadipyr is low toxic to earthworm and its LC50 is 4100 mg/kg, which is much higher than that of acetamiprid (8.93 mg/kg), imidacloprid

Table 1 Nominal and actual concentrations of guadipyr during the test at difference exposure period.

0d 3d 7d 14 d 21 d 28 d 56 d

10 mg/kg

50 mg/kg

100 mg/kg

200 mg/kg

400 mg/kg

600 mg/kg

800 mg/kg

8.79 7 0.74 6.29 7 0.14 4.25 7 0.08 2.46 7 0.12 1.09 7 0.07 0.40 7 0.01 ND

47.2 7 1.08 46.5 7 0.50 21.17 0.05 11.2 7 0.06 5.23 7 0.22 2.20 7 0.05 0.86 7 0.01

93.5 7 2.09 62.2 7 0.59 42.17 0.93 13.3 7 0.54 6.187 0.39 4.56 7 0.09 0.197 0.02

189 71.17 NA 108 71.03 67.5 70.95 NA NA NA

3617 1.19 NA 2117 1.12 1727 0.65 NA NA NA

582 7 1.04 NA 3777 1.22 2157 0.91 NA NA NA

781 7 1.48 NA 4577 1.03 378 7 0.83 NA NA NA

Note: Results are expressed as means 7 SD. ND: Not detected by the HPLC. NA: not applicable.

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Fig. 2. Inhibition rate of guadipyr on growth ofEisenia fetida. Data are expressed as the mean 7SD. Statistical significance vs the control group is shown. *po 0.05; **p o 0.01.

(10.0 mg/kg), clothianidin (9.25 mg/kg), nitenpyram (3.91 mg/kg) and thiacloprid (8.55 mg/kg) (Wang et al., 2012). Another strong evidence for guadipyr is safe to earthworm is that guadipyr at 100 mg/kg had no significant effect on earthworms’ reproduction of cocoons and its hatchability after exposed for 56 days, better than imidacloprid and thiacloprid which caused a significant decrease in cocoon production at 1.91 mg/kg and 0.291 mg/kg (p o0.05) respectively (Gomez-Eyles et al., 2009). However significant inhibition on earthworms’ growth was calculated at 100 mg/kg, which is still much lower than the 2.0 mg/kg imidacloprid on Lumbricus terrestris (Dittbrenner et al., 2011). One considering factor for low toxicity of guadipyr is that artificial soil was used instead of fields soil in this study. Artificial soil itself has some bad effect on earthworms’ growth (Figs. 2–5), it just reflect the toxicity of guadipyr to earthworms at a level, but can’t show the real conditions in the field. As the behavior and ecotoxicity of chemicals would be different in different soil, further studies of guadipyr toxicity in real field will be needed performed. Another more important factor might be the fast degradation of guadipyr in soil. Seen from the Table 1, about 50% guadipyr were degradation at day 7, and about 90% degradation at day 14. Degradation of chemicals in soil is complicated, as a novel neonicotinoid insecticide, soil behavior of guadipyr would be another research topic in future. Oxidative stress plays an important role in the toxicity of

various pesticides (Ranjbar et al., 2002), as pesticides can induce oxidative stress after spreading into the environment and comprehensive antioxidant defense mechanisms and enzymatic antioxidant defenses (Banerjee et al., 2001; Valavanidis et al., 2006), guadipyr induced stress response in earthworms after spreading into the artificial soil, so it significant inhibited AChE activity at 50.0 mg/kg and 100 mg/kg (p o0.05) after 3 days exposure. Velki and Hackenberger also found 0.1 mg/kg deltamethrin inhibits earthworm’s AChE activity after 1 day exposure (Velki and Hackenberger, 2013). But with time increase and comprehensive low toxicity of guadipyr, the cells have developed repair mechanisms to resistance the stress, hence the AChE activity recovered at day 7, 14 and 21. SOD is the enzyme that uses O2- as the substrate to convert O2 and H2O2 (Barata et al., 2005; Singh et al., 2006). Studies report that SOD activities increase in earthworms during light and severe stress (Xue et al., 2009), this study demonstrates similar results the Fig. 4-C. As an important antioxidant enzyme, CAT effectively removes excess in vivo H2O2 (which causes oxidative damage in cells if accumulation is excessive) by transforming H2O2 into H2O and O2, thereby maintaining H2O2 balance (Zhang et al., 2007). Zhang found that imidacloprid could significantly stimulated the activity of SOD and CAT of earthworm after 1, 7, 14 days exposure at 2.0 mg/kg (Zhang et al., 2014) and the changes in SOD and CAT activities indicate that the synergistic effects of the antioxidant enzymes in earthworm remove excess O−2 and H2O2, which can used to explain the enhancement of CAT and SOD activities induced by guadipyr. In this study, the CAT activity is more sensitive to guadipyr, so the CAT activity of earthworm could be used as early, sensitive biomarkers of exposure to guadipyr to reflect the physiological impairment, which unlikely earlier report that guadipyr can significantly enhance AChE in Daphnia magna, but no obvious impact on CAT activity was noted within 48 h (Qi et al., 2013). Accumulation of ROS such as H2O2 and superoxide radical (O2  ) does damage to cellular components such as DNA, proteins, and lipids (Lopez et al., 2006). Atrazine exhibits genotoxicity by causing single and double-strand breaks in DNA through the formation of ROS (Song et al., 2009b). The comet assay is a simple and sensitive way to detect singlestrand DNA breakage as well as assess DNA repair in individual cells (Singh et al., 1988). As reported the imidacloprid, acetamiprid, endosulfan and atrazine can significant induce DNA damage in earthworms at 0.20 mg/kg, 25.0 μg/mL, 0.10 mg/kg and 2.50 mg/ kg, respectively (Kocaman and Topaktas, 2007; Liu et al., 2009; Song et al., 2009a; Zang et al., 2000) but not guadipyr in the

Fig. 3. Mean number of cocoons per earthworm and hatchability of cocoons after exposed to guadipyr for 56 days. Error bars indicate standard deviation (SD).

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Fig. 4. Effects of guadipyr on AChE, CAT, and SOD activities inEisenia fetida.

Fig. 5. DNA damage caused by guadipyr.

present study. Cells have developed repair mechanisms to correct naturally occurring changes in DNA (Song et al., 2009b), CAT and SOD clean out excessive ROS which can lead to a permanent change or damage of DNA. These indicated in another way that guadipyr is safe to earthworm under the conditions in this research. Refers to 20.0 g a.i./ha dosage in field, guadipyr may not harm to the earthworm in field.

5. Conclusion This study firstly fully highlights the toxicity of guadipyr to earthworm (Eisenia fetida) during acute and subchronic exposure. Guadipyr has low toxicity to earthworm with a 14 days LC50 4100 mg/kg and couldn’t impact the growth and reproduction of earthworm. In addition, guadipyr could modify the activity of AChE, CAT and SOD at a level, but didn’t induce significant DNA damage in coelomocytes with 100 mg/kg. The lower toxicity of guadipyr than other neonicotinoid insecticides (Wang et al., 2012) to earthworms and its low mammalian toxicity (Su et al., 2012) make guadipyr a very promising candidate for imidacloprid and future commercial pesticides.

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