Environ Sci Pollut Res DOI 10.1007/s11356-015-4122-6

RESEARCH ARTICLE

Inhibition and recovery of biomarkers of earthworm Eisenia fetida after exposure to thiacloprid Lei Feng & Lan Zhang & Yanning Zhang & Pei Zhang & Hongyun Jiang

Received: 19 August 2014 / Accepted: 11 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Thiacloprid, a neonicotinoid insecticide, has been used widely in agriculture worldwide. In recent years, the adverse effects of neonicotinoid insecticides on non-target organisms have attracted more and more attention. In the present study, effects of thiacloprid on molecular biomarkers (GST, CarE, CAT, SOD, POD, and DNA damage) of earthworm Eisenia fetida were investigated using the artificial OECD soil for the first time. Earthworms were exposed to thiacloprid (1 and 3 mg/kg) for 7, 14, and 28 days and then transferred to the clean OECD soil for 35, 42, and 56 days. Results showed that activities of GST, CarE, CAT, SOD, and POD are inhibited following the exposure to thiacloprid at one or more sample times and then increased during the recovery course compared with the control. Significant DNA damage to E. fetida was also observed by olive tail moments in comet assay. These results suggested that thiacloprid could have harmful effect on earthworms, and these studied biomarkers might be used in the assessment of the risk of thiacloprid to the soil ecosystem environment. Keywords Thiacloprid . Eisenia fetida . Biomarkers . Inhibition . Recovery . Artificial OECD soil

Introduction In modern agriculture, the soil contamination due to the widespread use of pesticides attracts more and more attention. Responsible editor: Henner Hollert Lei Feng and Lan Zhang contributed equally to this work. L. Feng : L. Zhang : Y. Zhang : P. Zhang : H. Jiang (*) State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China e-mail: [email protected]

Although pesticides are designed for specific targets, they do affect non-target organisms when they are used (Damalas and Eleftherohorinos 2011). The earthworm Eisenia fetida is a soil-dwelling organism belonging to the Lumbricidae family. They are considered as soil ecosystem engineers and are one of the most suitable bioindicators to assess the ecological risks of toxic substances in the terrestrial environment (Römbke et al. 2005; Sanchez-Hernández 2006; Pelosi et al. 2014; Bernard et al. 2014). Therefore, understanding the adverse effects of pesticides on earthworms is essential to predict the potential effects of pesticides on soil environment. It has been reported that biochemical responses in organisms against environmental stress are regarded as early warning indices of pollution in the environment (Pelosi et al. 2014; Bernard et al. 2014). Many enzymatic activities have been considered as biomarkers of environmental contaminants (Booth and O’Halloran 2001; Sanchez-Hernández 2006; Song et al. 2009; Lin et al. 2012a, b; Velki et al. 2013, 2014). Carboxylesterase (CarE) is one of esterases that are used as biomarkers of pesticide exposure (Wheelock et al. 2008). Reactive oxygen species (ROS) can be generated in living organisms exposed to environmental pollution, and accumulation of ROS does damage to cellular components such as proteins, nucleic acids, and lipids (Halliwell and Gutteridge 1999; Kammenga et al. 2000; Zhang et al. 2014). Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST) play a crucial role in scavenging ROS generated during aerobic metabolism. Therefore, changes of these enzymatic activities are good indicators of the toxic effects of contaminants on living organisms (Viswanathan 1997; Aly and Schröder 2008; Jemec et al. 2010; Novais et al. 2014). Alkaline single-cell gel electrophoresis (SCGE, comet assay), a simple, reliable, and straightforward method, is a widely used to detect DNA damage caused by environmental stress in a wide variety of eukaryotic cells (Cotelle and Férard 1999).

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Thiacloprid[(Z)-3-(6-chloro-3-pyridylmethyl)-1,3thiazolidin-2-ylidenecyanami-de] is a second-generation neonicotinoid pesticide. It is the first chloronicotinyl insecticide to have activity not only against sucking insects such as aphids, whiteflies, and some jassids but also against weevils, leafminers, and various species of beetles, and it shows good plant compatibility in all relevant crops. Therefore, thiacloprid is considered to be an excellent insecticide for combating difficult-to-control insect pests encountered in fruit or vegetable cultivation (Elbert et al. 2000; Jeschke et al. 2001). However, little is known about thiacloprid toxicity to nontarget organisms and potential effects on the environment (Beketov and Liess 2008; Langer-Jaesrich et al. 2010). Due to the concern over the widespread use of imidacloprid, the first neonicotinoid insecticide, effects of this insecticide to earthworm have been reported in several earlier studies. Luo et al. (1999) and Zang et al. (2000) found that 0.5 mg/kg imidacloprid induce sperm deformities in E. fetida (Zhang et al. 2014). Under field conditions, a decrease in the production of earthworm casts induced by imidacloprid was observed during a period of 120 days (Lal et al. 2001). The behavior of two earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) was significantly altered at concentrations of imidacloprid below 4 mg/kg in dry soil (Capowiez et al. 2003, 2006; Dittbrenner et al. 2011). Zhang et al. (2014) reported that the activity of biomarkers, such as SOD, CAT, POD, and ROS, in the earthworm E. fetida was closely related to the dose and duration of the exposure to imidacloprid, which indicated imidacloprid exhibit a potentially harmful effect on E. fetida at the biochemical level. In the present study, we used earthworm E. fetida to investigate the effects of thiacloprid on detoxification enzymes glutathione S-transferase (GST) and carboxylesterase (CarE), antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (POD), as well as DNA damage. Results of these studies will provide fundamental information and comprehensive understanding in earthworms exposed to thiacloprid under standard laboratory conditions.

Materials and methods Earthworms and chemicals The earthworm E. fetida, one of OECD-recommended earthworm test species, was purchased from Shangxinbao Earthworm Farm Co., Ltd., China (OECD 1984). After 7 days of cultivation in a mixture of 50 % cattle manure and 50 % peat moss under the condition of 20 °C and 35 % moisture content, healthy earthworms weighing 250–300 mg with clitella were selected for further experiment. Thiacloprid (98.3 %) was obtained from Tianjin Xingguang Chemical Co. Ltd. All other chemicals were of

reagent grade and purchased from Sigma Chemical Co. and Beijing Chemical Co. Exposure procedure OECD artificial soil was used in this study, and sample composition strictly followed the OECD guideline: 10 % ground sphagnum peat (50 % kaolinite), and 70 % fine sand (OECD 1984). The soil pH was adjusted to 6.0±0.5 by addition of calcium carbonate and the moisture was adjusted to 35 % using distilled water. Before the exposure procedure, the selected earthworms were cultured for 24 h in this artificial soil. Effects of thiacloprid on selected biomarkers were examined using two concentrations 1 and 3 mg a.i.kg−1 dry soil, which are selected based upon the sublethal effects of this neonicotinoid insect on earthworm (Vejares et al. 2010). Our results showed that values of LC50 and LC5 were 8.43 and 3.19 mg a.i.kg−1 dry soil tested by artificial soil tests according to the OECD method (OECD 1984). No mortality was observed during the exposure procedure. The control group was mixed with the same volume of acetone. Three replicates were set for each treatment and control groups. The exposure tests were conducted in pre-cleaned 1000-mL glass jars. Each jar was filled with 500 g of OECD artificial soil and ten earthworms. Following 28 days of thiacloprid exposure, individuals (thiacloprid-exposed and control earthworms) were transferred to the clean OECD artificial soil and kept there to the 56th days (OECD 2004). Earthworms were collected randomly from each replicate on the 7th, 14th, 21st, 28th, 35th, 42nd, and 56th day. Periodically, earthworms were maintained at 20 °C and photoperiol 12 h dark:12 h light. The final moisture contents of the soil were adjusted to 35 % of the dry weight by addition of distilled water. The wetted dried cow dung was added to the soil surface for feeding the earthworm at a rate of 0.5 g per earthworm weekly (Liu et al. 2009). Enzyme extraction The earthworms collected as samples were placed in a glass homogenizer and homogenized in 0.1 M, pH 7.2, cold phosphate buffer (1:9, w/v) under ice-cold conditions with a glass homogenizer. The homogenates were then centrifuged at 10, 000 rpm and 4 °C for 30 min. After centrifugation, the resulting supernatant was stored at −80 °C for further enzyme analysis (Song et al. 2009). Enzyme assays The activity of glutathione GST toward 1-chloro-2,4-dinitrobenzene (CDNB) was determined according to the method of Habig et al. (1974). Reaction mixtures contained CDNB (1 mM), glutathione (GSH, 25 mM), and the enzyme in a total

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volume of 0.9 mL. The reaction was started by adding CDNB. Subsequently, the rate of change in optical density (OD) at 340 nm during the initial 2 min was measured and converted to activity using the extinction coefficient of 9.6 mM−1 cm−1. Carboxylesterase activity using α-naphthyl acetate (α-NA) was determined according to Bunyan et al. (1968). The reaction medium (200 μL, final volume) contained 25 mM Tris–HCl (pH 7.6), 1 mM CaCl2, and 2 mM α-NA, and it was incubated for 10 min at 25 °C with the enzyme. The reaction was stopped by the addition of 50 μL of 2.5 % SDS in 0.1 % Fast Red ITR/ 2.5 % Triton X-100. Solutions were allowed to stand for 30 min at 25 °C and dark. The absorbance of the naphthol–Fast Red ITR complex was read at 530 nm, and the concentration of αnaphthol was determined using an α-naphthol standard curve which was measured under the same conditions. Absorbance values were converted to nmol naphthol/min/mg protein using naphthol standard curves and protein contents. Total SOD activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT), as described by Giannopolitis and Ries (1977) with slight modification. The reaction mixture (1 mL, total volume,) contained 50 mM phosphate buffer (pH 7.8), 100 mM ethyl-enediaminetetraacetic acid (EDTA), 130 mM methionine, 750 mM NBT, 20 mM riboflavin, and 30 μL enzyme extract. Riboflavin was added last, and the tubes were shaken and illuminated with 4000 Lx fluorescent tubes. The reaction was allowed to proceed for 15 min, following which the lights were switched off, and the tubes were covered with a black cloth. Absorbance of the reaction mixture was read at 560 nm. One unit of SOD activity (U) was defined as the amount of enzyme required to cause 50 % inhibition of the NBT photoreduction rate, and the result was expressed as U/mg protein. Catalase (CAT) activity was determined as described by Xu et al. (1997). The enzyme activity was calculated from the decrease in ultraviolet absorption with time, following degradation of H2O2 by CAT present in the sample. One unit of CAT activity was defined as the enzyme quantity required to consume half of H2O2 in 100 s at 25 °C. POD activity was determined according to the method of Kochba et al. (1977) with slight modification. The reaction process was measured by recording absorbance at 470 nm as soon as 0.02 mL of the supernatant was added to 0.98 mL of reaction mixture, which contained 50 mL potassium phosphate buffer (0.1 M, pH 6.0), 19 μL 30 % H2O2, and 28 μL guaiacol. One activity unit of POD was defined as the amount of enzyme that caused an increase of 0.01 absorbance units per minute, and the result was expressed as units per milligram protein. Protein content Protein concentration was assayed according to the method of Bradford (Bradford 1976) using crystalline bovine serum albumin (Sigma, China) as standard.

Comet assay According to Eyambe et al. (1991), earthworm coelomocytes were obtained using the noninvasive extrusion method. Individual earthworms were rinsed in the extrusion medium composed of 5 % ethanol, 95 % saline, 2.5 mg/mL EDTA, and 10 mg/mL guaiacol glyceryl ether (pH 7.3). Coelomocytes were spontaneously secreted in the medium and washed with phosphate-buffered saline (PBS) prior to the comet assay. The cells were collected by centrifugation (3000g, 10 min) and placed on ice prior to the comet assay. The comet assay was performed according to Singh et al. (1988). The cell suspension was mixed with 100 mL of 0.7 % low melting agar (LMA) in PBS at 37 °C and pipette onto fully frosted slides pre-coated with a layer of 100 mL 0.8 % normal melting agar (NMA). After solidification on ice, the slides were immersed into a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA (pH 10.0), 1 % Na-sarcosinate, 10 % dimethyl sulfoxide (DMSO), and 1 % Triton X-100). Slides were then incubated in an electrophoresis tank containing 300 mM NaOH with 1 mM Na2-EDTA for 20 min prior to electrophoresis for 15 min at 25 V (300 mA). The slides were then neutralized (0.4 M Tris, pH 7.5) thrice at 5-min intervals and stained with ethidium bromide (2.0 μg/mL) for fluorescence microscopy analysis (Olympus BX51 fluorescence microscope) using a digital imaging system. The images of the SCGE were analyzed using the Comet Assay Software Project (CASP). The olive tail moment (OTM, arbitrary unit), product of the distance between the center of gravity of the head and the center of gravity of the hail and percent tail DNA, was used to quantify the extent of DNA damage induced by thiacloprid exposure (Końca et al. 2003). Statistical analysis All results were expressed as means with the corresponding standard errors. One-way analysis of variance (ANOVA) followed by post hoc comparisons were carried out to test for significant differences among the treated and control groups. A significant difference was indicated as p