Chemosphere 132 (2015) 120–126
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Biological response of earthworm, Eisenia fetida, to five neonicotinoid insecticides Kai Wang 1, Sen Pang 1, Xiyan Mu, Suzhen Qi, Dongzhi Li, Feng Cui, Chengju Wang ⇑ College of Sciences, China Agricultural University, China
h i g h l i g h t s Investigated neonicotinoid insecticides significantly reduced fecundity of E. fetida. Neonicotinoid insecticides could damage the epidermal and midgut cells of E. fetida. The neonicotinoid insecticides pose a serious threat to E. fetida in the soil.
a r t i c l e
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Article history: Received 22 January 2015 Received in revised form 3 March 2015 Accepted 4 March 2015
Handling Editor: David Volz Keywords: Neonicotinoid insecticides Eisenia fetida Reproduction Cellulase activity Histophathology
a b s t r a c t Earthworms (Eisenia fetida) are one of the most abundant terrestrial species, and play an important role in maintaining the ecological function of soil. Neonicotinoids are some of the most widely used insecticides applied to crops. Studies on the effect of neonicotinoids on E. fetida are limited. In the present work, we evaluated the effects of five neonicotinoid insecticides on reproduction, cellulase activity and the tissues of E. fetida. The results showed that, the LC50 of imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid was 3.05, 2.69, 4.34, 0.93 and 2.68 mg kg1, respectively. They also could seriously affect the reproduction of E. fetida, reducing the fecundity by 84.0%, 39.5%, 54.3%, 45.7% and 39.5% at the sub-lethal concentrations of 2.0, 1.5, 0.80, 2.0 and 1.5 mg kg1, respectively. The cellulase activity of E. fetida was most sensitive to clothianidin. Significant disruption of the epidermal and midgut tissue was observed after 14 d exposure. In summary, we demonstrate that imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid have high toxic to earthworm, and can significantly inhibited fecundity and cellulase activity of E. fetida, and they also damage the epidermal and midgut cells of earthworm. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Neonicotinoid insecticides are among some of the most important insecticides applied to crops, which has been the world’s largest selling insecticide for many years (Jeschke and Nauen, 2008; Jeschke et al., 2011). Neonicotinoid insecticides are utilized throughout the world and account for one-fourth of the entire insecticide market because of their excellent insecticidal activity (Jeschke et al., 2011; Kagabu, 2011), the neonicotinoids are particularly suited for controlling many insects with biting and sucking mouth parts especially if swallowed (Laurino et al., 2011). They are also used in seed dressing for protection from soil insects, ⇑ Corresponding author at: No. 2 Yuan Mingyuan West Road, Haidian District, China Agriculture University, China. Tel.: +86 (0)10 62733824. E-mail address: [email protected] (C. Wang). 1 These authors contributed equally to this work and should be considered co-first authors. http://dx.doi.org/10.1016/j.chemosphere.2015.03.002 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
meanwhile, most of neonicotinoids insecticides have a short halflife in soil (Mohapatra et al., 2012; Wu et al., 2012; Yang et al., 2012; Dong et al., 2014), however, it was unclear whether neonicotinoids insecticides would affect the earthworm after long time exposed to neonicotinoids insecticides, so it is important to evaluate its toxicity to earthworm. Earthworms (Eisenia fetida) are one of the most abundant terrestrial species and play an important role in maintaining the ecological function of soil (Lee, 1985; Edwards and Bohlen, 1996; Edwards, 2004). They are often used in studies as important model organisms to assess the ecological hazards of toxic substances on the soil ecosystems (lanno et al., 2004; Römbke et al., 2005; Zhang et al., 2006; Schreck et al., 2008; Xu et al., 2010). Due to its short life cycle, high reproductive rate, ease of culture and sensitivity to chemicals, E. fetida has been used in toxicity studies (OECD, 1984), this species is also recommended for use by the Organization for Economic Cooperation and Development (OECD) in reproduction toxicity tests (OECD, 2004; Lowe and Butt, 2007).
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For more than three decades, E. fetida has played a major role in toxicity testing, with a strong emphasis placed on determining lethal endpoints. However, it is now widely recognized that toxicity tests designed to measure sub-lethal endpoints provide a more rapid diagnosis of potential contaminant effects (Lowe and Butt, 2007). The effects of toxic substances on reproduction, growth rates and behavioral traits of E. fetida have been reported (Khalil et al., 1996; Lukkari et al., 2005). Neonicotinoid insecticides, such as acetamiprid, imidacloprid, nitenpyram have high toxicity to E. fetida with the 14 d-LC50 for the acute toxicity in artificial soils were 1.52 mg kg1, 2.82 mg kg1, 3.91 mg kg1 respectively (Wang et al., 2012). Gomez-Eyles suggested that imidacloprid and thiacloprid caused a significant decrease in cocoon production with 2 mm sieved clay loam soil from Broughton Loam, Kettering, UK (Gomez-Eyles et al., 2009). Sublethal effects such as sperm deformities, have been documented in E. fetida at imidacloprid concentrations above 0.50 mg kg1 (Luo et al., 1999; Capowiez et al., 2006). Other significant sublethal effects such as weight loss and reduced burrowing activity have been observed in Allolobophora ictericia and Aporrectodea nocturna exposed to 0.5–1.0 mg kg1 imidacloprid (Capowiez et al., 2005, 2006). Nils also found imidacloprid could affect the midgut, chloragocytes, and epidermis of E. fetida (Dittbrenner et al., 2011). With the emergence of increased insect resistance to insecticides the application of higher doses of neonicotinoid insecticides may increase adverse effects to non-target organisms (Song et al., 1997; Tomizawa and Casida, 2003; Wang et al., 2012; Fairbrother et al., 2014). Therefore it is important to assess the ecological risks of neonicotinoid insecticides to ecosystems. Alothger imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid act as agonists on nicotinic acetylcholine receptors (nAChRs), they have different toxic to different organism. As we known, a trivial change in the structure of a pesticide may lead to great changes in the chemical properties and activities (Su et al., 2012), so the differences about the structure leads to the difference toxic to earthworm. In the present paper, the effects of these five neonicotinoids insecticides on acute toxicity, reproductive activity, histology and cellulase activity of E. fetida were tested. This study also could be considered as important investigation of sublethal effects of different neonicotinoids and can provide insight into mode of action. All theses are of great significance to develop new effective and safe neonicotinoids insecticides.
2. Material and methods 2.1. Test ingredients E. fetida were purchased from the Beijing Dahuan Earthworm Factory (Beijing, China). After acclimation for 7 d to the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%), 6 mon age old earthworms (300–400 mg with a clitellum) were selected and deprived of food
for 4 h and then tested for the following experiments under the above artificial climate. 2.2. Insecticides Insecticides were obtained as follows: Imidacloprid (96% active ingredients; from Zhejiang Pesticide Factory), acetamiprid (96% active ingredients; from Beijing Pesticide Company), clothianidin (96% active ingredients; from Sumitomo Chemical Corp), nitenpyram (94% active ingredients; from Lianyungang Pesticide Company), and thiacloprid (95% active ingredients; from Xi’an Meibang Pesticide Co., Ltd., China). 2.3. Test soils Artificial soil composed of 10% (w/w) sphagnum peat moss, 20% (w/w) kaolin clay, and 70% (w/w) sand with 20% (w/w) water content was used in the present study according to published methods (OECD, 1984). For the following testes, the desired amount of insecticide in acetone was added to 10 g quartz sand. After the acetone had evaporated, 490 g dry constituents were blended in the correct proportions and mixed thoroughly. Deionized water was added to give an overall moisture content of 35% of the dry weight. Five hundred gram artificial soil with insecticide was thoroughly mixed and put into 1-L glass containers respectively. For the control treatment distilled water was used instead of insecticides. Ten earthworms were then placed into each container and covered with a polythene sheet with 1 mm integrated gauze to ensure sufficient ventilation. During the acute toxicity, reproduction cellulase activity and histology experiment, the glass container were kept in the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%). 2.4. Acute toxicity The acute toxicity test was performed using the OECD method (OECD, 1984). According to preliminary tests, each insecticide was dissolved in acetone and diluted to five concentrations (Table 1). Five replicates of each treatment were performed. 2.5. Reproduction According to the results of acute toxicity tests, five different concentrations (Table 1) were designed for study of the effect of neonicotinoids on reproduction. Ten six months age-old earthworms about (300–400 mg with a clitellum) were tested according to OECD 222 guideline for the testing of chemicals on reproduction (OECD, 2004). Sterile cow manure at 0.50 g per earthworm per week was used as food. At day 28, ten earthworms 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 d 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 were
Table 1 Test concentrations of neonicotinoid insecticides in experiments. Pesticides
Test concentration (mg kg1) Acute toxicity test
Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
1.0 0.9 1.8 0.125 0.5
2.0 1.4 2.7 0.25 1.0
Reproduction 3.0 2.0 4.0 0.5 2.0
4.0 3.0 6.0 1.0 3.0
5.0 4.5 7.5 2.0 4.0
0.1 0.05 0.025 0.01 0.05
0.2 0.125 0.05 0.025 0.125
Cellulase activity 0.5 0.25 0.1 0.05 0.25
1.0 0.75 0.5 0.2 0.75
2.0 1.5 2.0 0.8 1.5
0.005 0.005 0.005 0.005 0.005
0.02 0.02 0.02 0.02 0.02
Histology 0.1 0.1 0.1 0.1 0.1
0.4 0.4 0.4 0.2 0.4
0.9 0.9 0.9 0.4 0.9
1.0 0.02 0.1 0.1 0.25
3.0 0.25 0.25 0.5 1.0
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determined. Five replicates of each treatment were performed. Hatchability (number of juveniles hatched as% of control), cocoons per earthworm, hatchlings per cocoon, and cocoon weight were determined as described.
given in Supplementary Material: Table A and calibrated with an external standard.
2.6. Measurement of cellulase activity
All data are presented as the mean ± standard deviation (SD). Differences between the treatment and control were determined by one-way ANOVA, followed by a post hoc Dunnett test at p = 0.05. LC50 (median lethal concentration) and EC50 (concentration for 50% of hatch success) were calculated by probit regression analysis using SPSS 17.0 software.
According to the results of acute toxicity tests, five different concentrations about different insecticide (Table 1) were designed for study of the effect of neonicotinoids on cellulase activity. Adult earthworms about six months age old (300–400 mg with a clitellum) were selected for this test. Three replicates of each treatment were kept in the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%). The earthworms were collected after 1, 3, 7, 14, and 21 d of exposure. After they excreted their gut contents for 6 h, the earthworms were weighed. Six live earthworms from three parallel experiments of each treatment were homogenized with ice-cold deionized water weighing four times the body weight of the earthworms. The homogenates were centrifuged for 10 min at 2500 rpm, and the supernatant re-centrifuged for 5 min at 3000 rpm. The supernatant was collected and stored briefly at 0–4 °C, until cellulase activity was assayed using the carboxymethyl cellulose (CMC) assay as described by Ghose (Ghose, 1987). Total protein content was measured by the method of Bradford calibrated with bovine serum albumin (Bradford, 1976).
2.7. Histology According to the results of acute toxicity tests and the maximum residues in soil, two different concentrations of neonicotinoids (Table 1) were selected for study of the effect on intestinal and epidermal histology. After 14 d exposure, the earthworms were kept in filter paper for 6 h at 22 °C to clear the intestines of soil particles. Earthworms were then fixed with 10% (w/v) formalin for 24 h, and embedded into paraffin and 4 lm cross sections, directly behind the clitellum region produced with a freezing microtome (LeLCA RM 2016, Germany). Finally, the sections were stained with hematoxylin–eosin (HE) as well as a combined method using Periodic acid-Schiff–Alcianblue. A light microscope (OLYMPUS CX31) was used to examine the samples. Histology of the midgut and epidermis was qualitatively described, and histopathological changes were semi-quantitatively assessed for each earthworm with the procedure described for molluscs by Triebskorn and Köhler (Triebskorn, 2003). 2.8. Analyzing neonicotinoid insecticides in artificial soil Ten grams of each soil sample were collected from separate experiment with separate concentrations at the indicated observation time. Each sample was added to 50 mL tube, and then 10 mL distilled water added. Five replicates were used for each time point. The mixture was homogenized for 3 min and allowed to stand for 10 min. Next, 10 mL acetonitrile was added and the mixture was vigorously shaken for 3 min. Ten grams of a mixture of MgSO4:NaCl mixture (4:1 w/w) was added and shaken for an additional 3 min, and then the sample 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 and the supernatant injected into a high performance liquid chromatography (HPLC) system. The following chromatographic conditions were used: PAD detector; C18 stainless steel column (250 mm 46 mm with 5 lm packing); 25 °C column temperature; 1.0 mL min1 flow rate; 20 lL injection volume; mobile phase and wavelength were as
2.9. Data modeling and statistical analysis
3. Results 3.1. Neonicotinoid insecticide analysis in artificial soils The deviation between nominal and actual concentrations of neonicotinoid insecticides was less than 20% for the first time. The concentration then decreased with time (Supplementary Material: Tables B.1–15). Some concentrations were below the HPLC detection limit and in these cases the nominal dosage is used in the discussion and calculations. 3.2. Acute toxicity of neonicotinoids on E. fetida The results of the acute toxicity tests showed significant differences in LC50 values among the tested compounds (Table 2). The acute toxicity of clothianidin to E. fetida after 14 d exposure was significantly higher than the toxicity of the other four insecticides. The LC50 of imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid to E. fetida were 3.05 mg kg1, 2.69 mg kg1, 4.34 mg kg1, 0.93 mg kg1 and 2.68 mg kg1. The order of these five neonicotinoid insecticides in terms of acute toxicity was clothianidin > thiacloprid acetamiprid imidacloprid > nintenpyramm. 3.3. Effects neonicotinoids on reproduction of E. fetida The results of reproduction parameters measured for E. fetida exposed to five neonicotinoid insecticides are shown in Table 3. Increasing concentrations of five neonicotinoid insecticides caused significant decreases in 35.71%, 24.93%, 43.27%, 13.46% and 18.33% hatchability at 2.0, 1.5, 0.80, 2.0 and 1.5 mg kg1, respectively, mean cocoons per worm, mean hatchlings per cocoon, and mean cocoon weight compared with controls. At the highest concentrations, all compounds had a highly significant effect on cocoon production (p < 0.001). However acetamiprid and clothianidin were most toxic, and exhibited highly significant toxic effects on all parameters at absolute concentrations of 0.75 and 0.80 mg kg1, respectively. Table 2 also shows the effects of five neonicotinoid insecticides on reproduction of E. fetida. The reproduction of E. fetida exposed to neonicotinoid insecticides in soil was significantly inhibited by
Table 2 Acute and reproduction toxicity of neonicotinoid insecticides on E. fetida. Insecticides
Acute toxicity
Reproduction toxicity
1
LC50 (mg kg Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
)
3.05 2.69 4.34 0.93 2.68
Note: 95% CL: 95% confidence limit.
95% CL
EC50 (mg kg1)
95% CL
2.84–3.27 2.54–2.85 4.16–4.54 0.77–1.11 2.48–2.87
0.954 0.318 0.261 0.368 0.202
0.463–2.783 0.119–0.98 0.104–1.009 0.080–2.76 0.055–1.748
K. Wang et al. / Chemosphere 132 (2015) 120–126 Table 3 Reproduction parameters measured for E. fetida exposed to difference concentrations of five neonicotinoid insecticides. Concentrations (mg kg1)
Mean cocoons per E. fetida
Mean hatchlings per cocoon
Mean cocoon weight (mg)
Control 0.00
4.05 ± 0.05
3.97 ± 0.22
13.75 ± 1.61
Imidacloprid 0.10 0.20 0.50 1.00 2.00
4.07 ± 0.32 4.15 ± 0.15 3.09 ± 0.33* 2.35 ± 0.15*** 0.65 ± 0.05***
3.67 ± 0.33 2.57 ± 0.01 2.37 ± 0.30 2.69 ± 0.69** 2.25 ± 0.25***
13.36 ± 1.06 12.46 ± 1.96* 10.96 ± 0.38 *** 8.00 ± 2.43*** 6.79 ± 0.13***
Acetamiprid 0.05 0.125 0.25 0.75 1.50
3.99 ± 0.33 4.03 ± 0.67 3.55 ± 0.45 2.05 ± 0.15*** 2.45 ± 0.15*
3.96 ± 0.57 3.07 ± 0.56 2.47 ± 0.68** 2.79 ± 0.21* 0.99 ± 0.26***
12.75 ± 0.93 10.50 ± 1.06*** 10.00 ± 1.57*** 8.33 ± 0.94*** 7.05 ± 0.61***
Clothianidin 0.01 0.025 0.05 0.20 0.80
4.16 ± 0.78 4.43 ± 0.25 3.50 ± 0.10 3.10 ± 0.50* 1.85 ± 0.05***
4.09 ± 0.65 3.98 ± 0.59 4.75 ± 0.46 3.70 ± 0.46 1.81 ± 0.06***
13.58 ± 1.50 12.66 ± 1.30* 11.30 ± 2.89*** 11.07 ± 0.84*** 9.55 ± 1.57***
Nitenpyram 0.025 0.05 0.10 0.50 2.00
4.02 ± 0.07 4.32 ± 0.33 3.70 ± 0.10 2.80 ± 0.10* 2.20 ± 0.10***
4.00 ± 0.40 3.49 ± 0.68 3.51 ± 0.17 1.89 ± 1.11*** 1.88 ± 0.63***
12.75 ± 0.95 9.04 ± 0.50*** 10.81 ± 1.39*** 10.57 ± 1.07*** 9.80 ± 1.43***
Thiacloprid 0.05 0.125 0.25 0.75 1.50
4.16 ± 0.40 4.05 ± 0.35 3.75 ± 0.05 2.85 ± 0.75* 2.45 ± 0.35***
3.74 ± 0.70 2.98 ± 0.05 2.50 ± 0.08** 2.61 ± 0.09* 1.25 ± 0.25***
14.05 ± 1.31 13.60 ± 1.48 12.52 ± 1.14* 11.42 ± 2.12*** 9.25 ± 0.86***
Note: Data are expressed as mean ± sd (n = 5). Statistical significance versus control group. * p < 0.05. ** p < 0.01. *** p < 0.001.
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concentrations less than 1.0 mg kg1, however there was no statistical significance in the individual EC50s, which varied from a low of 0.261 mg kg1 for nitenpyram to a high of 0.954 mg kg1 for imidacloprid. 3.4. Effects of neonicotinoids on cellulase activity of E. fetida The effect of the five neonicotinoids on E. fetida was further sought by studying cellulose activity. No significant effect on cellulase activity was observed after 21 d of exposure to five concentrations of nitenpyram (Fig. 1A) (p < 0.05). However the cellulase activity of E. fetida extracts increased significantly after 1 d of exposure to imidacloprid (Fig. 1C). However cellulase activity of E. fetida returned to normal levels in the following test period (Fig. 1B, C and E). A sharp increase in cellulase activity was only observed after 1 d of exposure to the highest concentrations of imidacloprid (Fig. 1C). The concentration of acetamiprid that caused an increase in cellulose activity was lower than the concentration of imidacloprid. Moreover, the cellulase activity of E. fetida displayed an increasing trend with an increase of acetamiprid concentration (Fig. 1B). Unlike the above three neonicotinoid insecticides, clothianidin significantly inhibited cellulase activity. The cellulase activity of E. fetida treated with clothianidin was significantly lower than that of the control group (Fig. 1D). 3.5. Histopathological examination In E. fetida, the condition of the epidermal cells deteriorated with increasing pesticide concentration. Many mucocytes of the epidermis were hypertrophic and showed an irregular cellular compartmentation. After exposure to higher pesticide concentrations, disintegration of epidermal cells was observed (Fig. 2). With increasing pesticide concentration, the cells of the midgut tissue often showed a degradation of the cellular compartmentation as well as a reduced density of the cytoplasm and irregularly shaped nuclei (Fig. 3). In general, compared with other four neonicotinoid insecticides, the effect of acetamiprid on the epidermal and midgut was more
Fig. 1. The cellulose activity of E. fetida after the exposure to soil with nitenpyram (A), acetamiprid (B), imidacloprid (C), clothianidin (D) and thiacloprid (E). Data are expressed as the mean ± SD, and are significant ⁄ for p < 0.05.
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Fig. 2. Effect of neonicotinoid insecticides on epidermal surface of the E. fetida after 14 d of exposure: I: smooth surface of epidermis; II: clear cellular compartmentation; III: Nucleus oval in shape; IV: irregular surface of epidermis; V: irregular cellular compartmentation; VI: irregular shape and altered size of nucleus; VII: cells disintegrated.
Fig. 3. Effect of neonicotinoid insecticides on midgut tissue of E. fetida after 14 d of exposure: I: columnar-shaped cells; II: clear cellular compartmentation; III: nucleus oval in shape; IV: medium-dense cytoplasm; V: irregular cell shape; VI: irregular cellular compartmentation; VII: irregular shape and altered size of nucleus; VIII: cytoplasm of sparse density, intercellular space enhanced; IX: cells disintegrated.
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dramatic, suggesting acetamiprid could affect epidermal and midgut cells at 0.025 mg kg1, while the other neonicotinoids damaged epidermal and midgut cells at higher concentrations. 4. Discussion In this paper, the acute toxicity of five neonicotinoid insecticides to E. fetida was evaluated using the artificial soil method. The results indicated that neonicotinoid insecticides had high toxicity to E. fetida, especially clothianidin. Further tests on reproductive activity indicated these insecticides could seriously affect the reproduction on E. fetida (Gomez-Eyles et al., 2009). Excessive use of these insecticides might result in harm. The toxicity to E. fetida may help determine the appropriate dosage during agricultural usage. Sub-lethal concentrations of pollutants may affect the behavior, growth, reproduction and organizational structure of earthworm, which are more close to long-term toxic effects in the actual ecological system (Capowiez et al., 2006; Reinecke and Reinecke, 2007). Studies reveal that measurement of toxic effects on reproduction may be more sensitive than the survival index for environmental risk assessment (Kuperman et al., 2004; Capowiez et al., 2006; Reinecke and Reinecke, 2007)). Acetochlor has been reported to inhibit E. fetida growth and rate of cocoon production at concentrations greater than 20 mg kg1 (Xiao et al., 2006). Furthermore, studies related to the effects of heavy metal pollution and reproductive toxicity on E. fetida has also been reported (Lukkari et al., 2005; Nahmani et al., 2007). Our results demonstrate that these five neonicotinoid insecticides significantly affected E. fetida cocoon production and hatching in a concentration-dependent manner. Cocoon production was found to be very sensitive to neonicotinoid insecticides during the experiment. Capowiez has reported a similar result, suggesting that imidacloprid and thiacloprid cause a significant decrease in cocoon production at 1.91 mg kg1 and 0.51 mg kg1 respectively (Capowiez et al., 2006). Sublethal effects (e.g. sperm deformities) on E. fetida have been documented at imidacloprid concentrations above 0.5 mg kg1 (Luo et al., 1999; Capowiez et al., 2006). Other significant effects of imidacloprid such as weight loss and reduced burrowing activity have been found at levels of 0.5–1.0 mg kg1. The effect on reproductive capacity of E. fetida was a more sensitive parameter for studying the adverse effects of neonicotinoid insecticides. The reproduction of E. fetida exposed to soil with low concentrations of neonicotinoid insecticides, was significantly inhibited. Earthworm is the largest biomass on land, which is very important to soil environment (Bartlett et al., 2010), it is at the bottom of the land food chain, once the earthworm population was affected by pesticides, then the whole food chain will be affected, at last ruin the whole ecological system. Cellulase is a very important enzyme for E. fetida. The change of cellulase activity can directly influence the ability of E. fetida to decompose plant litter and other cellulosic material (Luo et al., 1999; Shi et al., 2007). Imidacloprid and acetochlor were found to inhibit the cellulase activity of E. fetida (Xiao et al., 2006; Luo et al., 2009). In the present study, the opposite effects on the cellulase activity in E. fetida were observed after the treatment with neonicotinoid insecticides. There were no significant short-term effects on the cellulase activity in E. fetida after treatment with nitenpyram or acetamiprid and imidacloprid, respectively. However, during the test period, the cellulase activity of E. fetida treated with clothianidin was significantly lower than that of the control group (Fig. 1D). This might be the reason that clothianidin has higher acute toxicity to E. fetida. Losing the ability to decompose plant litter and other cellulosic materials can be one of the reasons for the higher acute toxicity of clothianidin to E. fetida than the other four neonicotinoid insecticides.
Histopathology is now increasingly being used as a biomarker of environmental stress (Stentiford and Feist, 2005; Eseigbe et al., 2013). In the present study, five neonicotinoid insecticides injured midgut and epidermal tissues. Various discrete pathological changes were observed viz; altered shape and size of cells and nuclei, irregular cellular compartmentation, and an irregular surface of the epidermis. Epidermal and midgut cells disintegrated with higher concentrations (e.g. at 1.0 mg kg1 acetamiprid and 3.0 mg kg1 imidacloprid). This result agrees with the report of Dittbrenner et al. (2011) who reported that the midgut tissue was damaged by 14 d exposure to 0.2 mg kg1 imidacloprid. Epidermal cells and midgut cells were very sensitive to neonicotinoid insecticides, midgut and epidermal tissues were damaged at low concentrations, so the change of epidermal cells and midgut cells can evaluate the toxic of neonicotinoid insecticides. Similar effects on cellular integrity were reported for E. fetida exposed to an organophosphorus insecticide, profenofos (Chakra Reddy and Venkateswara Rao, 2008). From the histopathological examination, we found increased mucus and cytothesis of midgut tissue cells at lower test concentrations, but some cells was disintegrated at higher test concentrations. Günther reported that environmental stressors could induce hypertrophy and hyperplasia of E. fetida mucocytes leading to mucus increase as protective reaction (Guenther and Greven, 1990). The effect of neonicotinoid insecticides on tissues and reproduction may be the reasons that these five neonicotinoid insecticides have high toxic to E. fetida. The changes of reproduction, cellulase activity and histology were more sensitive, which can be monitored at sub-lethal concentrations, it has important value for protection population and ecosystem, moreover it can use for pollutant monitoring and early warning. The sub-lethal concentrations for difference neonicotinoid insecticides of reproduction, cellulase activity and histology were shown in Table 4, the sub-lethal concentrations about the reproduction were lower than which about cellulase and histology, but all the sub-lethal concentrations are much higher than the detected residual dosage in soils (Wu et al., 2012; Yang et al., 2012; Ramasubramanian, 2013; Dong et al., 2014; Sharma and Singh, 2014) Imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid affected reproduction at 0.2 mg kg1, 1 1 1 0.125 mg kg , 0.05 mg kg , 0.05 mg kg and 0.25 mg kg1 after 56 d exposure. Imidacloprid and thiacloprid, within the levels found in soils could cause epidermal and midgut cell damage. Therefore, the application of neonicotinoid insecticides poses a serious threat to E. fetida in soils. In summary, we demonstrate that imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid have high acute toxicity to E. fetida. The inhibition of cellulase activity may result in higher acute toxicity of clothianidin to E. fetida. The significant adverse effects of neonicotinoid insecticides to reproduction of E. fetida were confirmed by studying epidermal cells and midgut cells which were very sensitive to neonicotinoid insecticides. This maybe the reason that the neonicotinoid insecticides were toxic to E. fetida. These results suggest that the five neonicotinoid insecticides have adverse effects on E. fetida. The sub-lethal
Table 4 The sub-lethal concentrations used in different experiments. Pesticides
Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
Sub-lethal concentrations (mg kg1) Histology
Reproduction
1.0 0.25 0.25 0.50 1.0
0.5 0.25 0.1 0.05 0.25
1.0 0.75 0.5 0.2 0.75
Cellulase activity 2.0 1.5 2.0 0.8 1.5
0.4 0.4 0.4 0.2 0.4
0.9 0.9 0.9 0.4 0.9
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concentrations of reproduction, cellulase activity and histology were lower than residual dosage in soil, much lower than the recommend high dosage, so the toxicity and appropriate field dosage need to be considered during usage. Furthermore, the reproduction of E. fetida and histopathology are more appropriate parameters to assess the potential adverse effects than acute toxicity. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.03.002. References Bartlett, M.D., Briones, M.J., Neilson, R., Schmidt, O., Spurgeon, D., Creamer, R.E., 2010. A critical review of current methods in earthworm ecology: from individuals to populations. Eur. J. Soil Biol. 46, 67–73. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Capowiez, Y., Bastardie, F., Costagliola, G., 2006. Sublethal effects of imidacloprid on the burrowing behaviour of two earthworm species: Modifications of the 3D burrow systems in artificial cores and consequences on gas diffusion in soil. Soil Biol. Biochem. 38, 285–293. Capowiez, Y., Rault, M., Costagliola, G., Mazzia, C., 2005. Lethal and sublethal effects of imidacloprid on two earthworm species (Aporrectodea nocturna and Allolobophora icterica). Biol. Fert. Soil 41, 135–143. Chakra Reddy, N., Venkateswara Rao, J., 2008. Biological response of earthworm, Eisenia foetida (Savigny) to an organophosphorous pesticide, profenofos. Ecotoxicol. Environ. Saf. 71, 574–582. Dittbrenner, N., Schmitt, H., Capowiez, Y., Triebskorn, R., 2011. Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Body mass change and histopathology. J. Soil Sediment. 11, 1000–1010. Dong, S., Qiao, K., Wang, H., Zhu, Y., Xia, X., Wang, K., 2014. Dissipation rate of thiacloprid and its control effect against Bemisia tabaci in greenhouse tomato after soil application. Pest Manag. Sci. 70, 1267–1273. Edwards, C.A., 2004. Earthworm Ecology. CRC Press. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms. Springer Science & Business Media. Eseigbe, F.J., Doherty, V.F., Sogbanmu, T.O., Otitoloju, A.A., 2013. Histopathology alterations and lipid peroxidation as biomarkers of hydrocarbon-induced stress in earthworm, Eudrilus eugeniae. Environ. Monit. Assess. 185, 2189–2196. Fairbrother, A., Purdy, J., Anderson, T., Fell, R., 2014. Risks of neonicotinoid insecticides to honeybees. Environ. Toxicol. Chem. 33, 719–731. Ghose, T.K., 1987. Measurement of cellulase activity. Pure Appl. Chem. 59, 257–268. Gomez-Eyles, J.L., Svendsen, C., Lister, L., Martin, H., Hodson, M.E., Spurgeon, D.J., 2009. Measuring and modelling mixture toxicity of imidacloprid and thiacloprid on Caenorhabditis elegans and Eisenia fetida. Ecotoxicol. Environ. Saf. 72, 71–79. Guenther, A., Greven, H., 1990. Increase of the number of epidermal gland-cells: an unspecific response of Lumbricus terrestris L (Lumbricidae: Oligochaeta) to different environmental stressors. Zool. Anz. – J. Comp. Zool. 225, 278–286. Jeschke, P., Nauen, R., 2008. Neonicotinoids – from zero to hero in insecticide chemistry. Pest Manag. Sci. 64, 1084–1098. Jeschke, P., Nauen, R., Schindler, M., Elbert, A., 2011. Overview of the status and global strategy for neonicotinoids. J. Agric. Food Chem. 59, 2897–2908. Kagabu, S., 2011. Discovery of imidacloprid and further developments from strategic molecular designs. J. Agric. Food Chem. 59, 2887–2896. Khalil, M.A., Abdel-Lateif, H.M., Bayoumi, B.M., Straalen, N.M.v., 1996. Analysis of separate and combined effects of heavy metals on the growth of Aporrectodea caliginosa (Oligochaeta; Annelida), using the toxic unit approach. Appl. Soil Ecol. 4, 213–219. Kuperman, R.G., Checkai, R.T., Simini, M., Phillips, C.T., 2004. Manganese toxicity in soil for Eisenia fetida, Enchytraeus crypticus (Oligochaeta), and Folsomia candida (Collembola). Ecotoxicol. Environ. Saf. 57, 48–53.
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Biological response of earthworm, Eisenia fetida, to five neonicotinoid insecticides Kai Wang 1, Sen Pang 1, Xiyan Mu, Suzhen Qi, Dongzhi Li, Feng Cui, Chengju Wang ⇑ College of Sciences, China Agricultural University, China
h i g h l i g h t s Investigated neonicotinoid insecticides significantly reduced fecundity of E. fetida. Neonicotinoid insecticides could damage the epidermal and midgut cells of E. fetida. The neonicotinoid insecticides pose a serious threat to E. fetida in the soil.
a r t i c l e
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Article history: Received 22 January 2015 Received in revised form 3 March 2015 Accepted 4 March 2015
Handling Editor: David Volz Keywords: Neonicotinoid insecticides Eisenia fetida Reproduction Cellulase activity Histophathology
a b s t r a c t Earthworms (Eisenia fetida) are one of the most abundant terrestrial species, and play an important role in maintaining the ecological function of soil. Neonicotinoids are some of the most widely used insecticides applied to crops. Studies on the effect of neonicotinoids on E. fetida are limited. In the present work, we evaluated the effects of five neonicotinoid insecticides on reproduction, cellulase activity and the tissues of E. fetida. The results showed that, the LC50 of imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid was 3.05, 2.69, 4.34, 0.93 and 2.68 mg kg1, respectively. They also could seriously affect the reproduction of E. fetida, reducing the fecundity by 84.0%, 39.5%, 54.3%, 45.7% and 39.5% at the sub-lethal concentrations of 2.0, 1.5, 0.80, 2.0 and 1.5 mg kg1, respectively. The cellulase activity of E. fetida was most sensitive to clothianidin. Significant disruption of the epidermal and midgut tissue was observed after 14 d exposure. In summary, we demonstrate that imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid have high toxic to earthworm, and can significantly inhibited fecundity and cellulase activity of E. fetida, and they also damage the epidermal and midgut cells of earthworm. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Neonicotinoid insecticides are among some of the most important insecticides applied to crops, which has been the world’s largest selling insecticide for many years (Jeschke and Nauen, 2008; Jeschke et al., 2011). Neonicotinoid insecticides are utilized throughout the world and account for one-fourth of the entire insecticide market because of their excellent insecticidal activity (Jeschke et al., 2011; Kagabu, 2011), the neonicotinoids are particularly suited for controlling many insects with biting and sucking mouth parts especially if swallowed (Laurino et al., 2011). They are also used in seed dressing for protection from soil insects, ⇑ Corresponding author at: No. 2 Yuan Mingyuan West Road, Haidian District, China Agriculture University, China. Tel.: +86 (0)10 62733824. E-mail address: [email protected] (C. Wang). 1 These authors contributed equally to this work and should be considered co-first authors. http://dx.doi.org/10.1016/j.chemosphere.2015.03.002 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
meanwhile, most of neonicotinoids insecticides have a short halflife in soil (Mohapatra et al., 2012; Wu et al., 2012; Yang et al., 2012; Dong et al., 2014), however, it was unclear whether neonicotinoids insecticides would affect the earthworm after long time exposed to neonicotinoids insecticides, so it is important to evaluate its toxicity to earthworm. Earthworms (Eisenia fetida) are one of the most abundant terrestrial species and play an important role in maintaining the ecological function of soil (Lee, 1985; Edwards and Bohlen, 1996; Edwards, 2004). They are often used in studies as important model organisms to assess the ecological hazards of toxic substances on the soil ecosystems (lanno et al., 2004; Römbke et al., 2005; Zhang et al., 2006; Schreck et al., 2008; Xu et al., 2010). Due to its short life cycle, high reproductive rate, ease of culture and sensitivity to chemicals, E. fetida has been used in toxicity studies (OECD, 1984), this species is also recommended for use by the Organization for Economic Cooperation and Development (OECD) in reproduction toxicity tests (OECD, 2004; Lowe and Butt, 2007).
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For more than three decades, E. fetida has played a major role in toxicity testing, with a strong emphasis placed on determining lethal endpoints. However, it is now widely recognized that toxicity tests designed to measure sub-lethal endpoints provide a more rapid diagnosis of potential contaminant effects (Lowe and Butt, 2007). The effects of toxic substances on reproduction, growth rates and behavioral traits of E. fetida have been reported (Khalil et al., 1996; Lukkari et al., 2005). Neonicotinoid insecticides, such as acetamiprid, imidacloprid, nitenpyram have high toxicity to E. fetida with the 14 d-LC50 for the acute toxicity in artificial soils were 1.52 mg kg1, 2.82 mg kg1, 3.91 mg kg1 respectively (Wang et al., 2012). Gomez-Eyles suggested that imidacloprid and thiacloprid caused a significant decrease in cocoon production with 2 mm sieved clay loam soil from Broughton Loam, Kettering, UK (Gomez-Eyles et al., 2009). Sublethal effects such as sperm deformities, have been documented in E. fetida at imidacloprid concentrations above 0.50 mg kg1 (Luo et al., 1999; Capowiez et al., 2006). Other significant sublethal effects such as weight loss and reduced burrowing activity have been observed in Allolobophora ictericia and Aporrectodea nocturna exposed to 0.5–1.0 mg kg1 imidacloprid (Capowiez et al., 2005, 2006). Nils also found imidacloprid could affect the midgut, chloragocytes, and epidermis of E. fetida (Dittbrenner et al., 2011). With the emergence of increased insect resistance to insecticides the application of higher doses of neonicotinoid insecticides may increase adverse effects to non-target organisms (Song et al., 1997; Tomizawa and Casida, 2003; Wang et al., 2012; Fairbrother et al., 2014). Therefore it is important to assess the ecological risks of neonicotinoid insecticides to ecosystems. Alothger imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid act as agonists on nicotinic acetylcholine receptors (nAChRs), they have different toxic to different organism. As we known, a trivial change in the structure of a pesticide may lead to great changes in the chemical properties and activities (Su et al., 2012), so the differences about the structure leads to the difference toxic to earthworm. In the present paper, the effects of these five neonicotinoids insecticides on acute toxicity, reproductive activity, histology and cellulase activity of E. fetida were tested. This study also could be considered as important investigation of sublethal effects of different neonicotinoids and can provide insight into mode of action. All theses are of great significance to develop new effective and safe neonicotinoids insecticides.
2. Material and methods 2.1. Test ingredients E. fetida were purchased from the Beijing Dahuan Earthworm Factory (Beijing, China). After acclimation for 7 d to the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%), 6 mon age old earthworms (300–400 mg with a clitellum) were selected and deprived of food
for 4 h and then tested for the following experiments under the above artificial climate. 2.2. Insecticides Insecticides were obtained as follows: Imidacloprid (96% active ingredients; from Zhejiang Pesticide Factory), acetamiprid (96% active ingredients; from Beijing Pesticide Company), clothianidin (96% active ingredients; from Sumitomo Chemical Corp), nitenpyram (94% active ingredients; from Lianyungang Pesticide Company), and thiacloprid (95% active ingredients; from Xi’an Meibang Pesticide Co., Ltd., China). 2.3. Test soils Artificial soil composed of 10% (w/w) sphagnum peat moss, 20% (w/w) kaolin clay, and 70% (w/w) sand with 20% (w/w) water content was used in the present study according to published methods (OECD, 1984). For the following testes, the desired amount of insecticide in acetone was added to 10 g quartz sand. After the acetone had evaporated, 490 g dry constituents were blended in the correct proportions and mixed thoroughly. Deionized water was added to give an overall moisture content of 35% of the dry weight. Five hundred gram artificial soil with insecticide was thoroughly mixed and put into 1-L glass containers respectively. For the control treatment distilled water was used instead of insecticides. Ten earthworms were then placed into each container and covered with a polythene sheet with 1 mm integrated gauze to ensure sufficient ventilation. During the acute toxicity, reproduction cellulase activity and histology experiment, the glass container were kept in the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%). 2.4. Acute toxicity The acute toxicity test was performed using the OECD method (OECD, 1984). According to preliminary tests, each insecticide was dissolved in acetone and diluted to five concentrations (Table 1). Five replicates of each treatment were performed. 2.5. Reproduction According to the results of acute toxicity tests, five different concentrations (Table 1) were designed for study of the effect of neonicotinoids on reproduction. Ten six months age-old earthworms about (300–400 mg with a clitellum) were tested according to OECD 222 guideline for the testing of chemicals on reproduction (OECD, 2004). Sterile cow manure at 0.50 g per earthworm per week was used as food. At day 28, ten earthworms 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 d 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 were
Table 1 Test concentrations of neonicotinoid insecticides in experiments. Pesticides
Test concentration (mg kg1) Acute toxicity test
Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
1.0 0.9 1.8 0.125 0.5
2.0 1.4 2.7 0.25 1.0
Reproduction 3.0 2.0 4.0 0.5 2.0
4.0 3.0 6.0 1.0 3.0
5.0 4.5 7.5 2.0 4.0
0.1 0.05 0.025 0.01 0.05
0.2 0.125 0.05 0.025 0.125
Cellulase activity 0.5 0.25 0.1 0.05 0.25
1.0 0.75 0.5 0.2 0.75
2.0 1.5 2.0 0.8 1.5
0.005 0.005 0.005 0.005 0.005
0.02 0.02 0.02 0.02 0.02
Histology 0.1 0.1 0.1 0.1 0.1
0.4 0.4 0.4 0.2 0.4
0.9 0.9 0.9 0.4 0.9
1.0 0.02 0.1 0.1 0.25
3.0 0.25 0.25 0.5 1.0
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determined. Five replicates of each treatment were performed. Hatchability (number of juveniles hatched as% of control), cocoons per earthworm, hatchlings per cocoon, and cocoon weight were determined as described.
given in Supplementary Material: Table A and calibrated with an external standard.
2.6. Measurement of cellulase activity
All data are presented as the mean ± standard deviation (SD). Differences between the treatment and control were determined by one-way ANOVA, followed by a post hoc Dunnett test at p = 0.05. LC50 (median lethal concentration) and EC50 (concentration for 50% of hatch success) were calculated by probit regression analysis using SPSS 17.0 software.
According to the results of acute toxicity tests, five different concentrations about different insecticide (Table 1) were designed for study of the effect of neonicotinoids on cellulase activity. Adult earthworms about six months age old (300–400 mg with a clitellum) were selected for this test. Three replicates of each treatment were kept in the artificial climate (20 ± 1 °C with a dark: light ratio of 8:16 h with illumination of 600 lux and, humidity: 80–85%). The earthworms were collected after 1, 3, 7, 14, and 21 d of exposure. After they excreted their gut contents for 6 h, the earthworms were weighed. Six live earthworms from three parallel experiments of each treatment were homogenized with ice-cold deionized water weighing four times the body weight of the earthworms. The homogenates were centrifuged for 10 min at 2500 rpm, and the supernatant re-centrifuged for 5 min at 3000 rpm. The supernatant was collected and stored briefly at 0–4 °C, until cellulase activity was assayed using the carboxymethyl cellulose (CMC) assay as described by Ghose (Ghose, 1987). Total protein content was measured by the method of Bradford calibrated with bovine serum albumin (Bradford, 1976).
2.7. Histology According to the results of acute toxicity tests and the maximum residues in soil, two different concentrations of neonicotinoids (Table 1) were selected for study of the effect on intestinal and epidermal histology. After 14 d exposure, the earthworms were kept in filter paper for 6 h at 22 °C to clear the intestines of soil particles. Earthworms were then fixed with 10% (w/v) formalin for 24 h, and embedded into paraffin and 4 lm cross sections, directly behind the clitellum region produced with a freezing microtome (LeLCA RM 2016, Germany). Finally, the sections were stained with hematoxylin–eosin (HE) as well as a combined method using Periodic acid-Schiff–Alcianblue. A light microscope (OLYMPUS CX31) was used to examine the samples. Histology of the midgut and epidermis was qualitatively described, and histopathological changes were semi-quantitatively assessed for each earthworm with the procedure described for molluscs by Triebskorn and Köhler (Triebskorn, 2003). 2.8. Analyzing neonicotinoid insecticides in artificial soil Ten grams of each soil sample were collected from separate experiment with separate concentrations at the indicated observation time. Each sample was added to 50 mL tube, and then 10 mL distilled water added. Five replicates were used for each time point. The mixture was homogenized for 3 min and allowed to stand for 10 min. Next, 10 mL acetonitrile was added and the mixture was vigorously shaken for 3 min. Ten grams of a mixture of MgSO4:NaCl mixture (4:1 w/w) was added and shaken for an additional 3 min, and then the sample 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 and the supernatant injected into a high performance liquid chromatography (HPLC) system. The following chromatographic conditions were used: PAD detector; C18 stainless steel column (250 mm 46 mm with 5 lm packing); 25 °C column temperature; 1.0 mL min1 flow rate; 20 lL injection volume; mobile phase and wavelength were as
2.9. Data modeling and statistical analysis
3. Results 3.1. Neonicotinoid insecticide analysis in artificial soils The deviation between nominal and actual concentrations of neonicotinoid insecticides was less than 20% for the first time. The concentration then decreased with time (Supplementary Material: Tables B.1–15). Some concentrations were below the HPLC detection limit and in these cases the nominal dosage is used in the discussion and calculations. 3.2. Acute toxicity of neonicotinoids on E. fetida The results of the acute toxicity tests showed significant differences in LC50 values among the tested compounds (Table 2). The acute toxicity of clothianidin to E. fetida after 14 d exposure was significantly higher than the toxicity of the other four insecticides. The LC50 of imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid to E. fetida were 3.05 mg kg1, 2.69 mg kg1, 4.34 mg kg1, 0.93 mg kg1 and 2.68 mg kg1. The order of these five neonicotinoid insecticides in terms of acute toxicity was clothianidin > thiacloprid acetamiprid imidacloprid > nintenpyramm. 3.3. Effects neonicotinoids on reproduction of E. fetida The results of reproduction parameters measured for E. fetida exposed to five neonicotinoid insecticides are shown in Table 3. Increasing concentrations of five neonicotinoid insecticides caused significant decreases in 35.71%, 24.93%, 43.27%, 13.46% and 18.33% hatchability at 2.0, 1.5, 0.80, 2.0 and 1.5 mg kg1, respectively, mean cocoons per worm, mean hatchlings per cocoon, and mean cocoon weight compared with controls. At the highest concentrations, all compounds had a highly significant effect on cocoon production (p < 0.001). However acetamiprid and clothianidin were most toxic, and exhibited highly significant toxic effects on all parameters at absolute concentrations of 0.75 and 0.80 mg kg1, respectively. Table 2 also shows the effects of five neonicotinoid insecticides on reproduction of E. fetida. The reproduction of E. fetida exposed to neonicotinoid insecticides in soil was significantly inhibited by
Table 2 Acute and reproduction toxicity of neonicotinoid insecticides on E. fetida. Insecticides
Acute toxicity
Reproduction toxicity
1
LC50 (mg kg Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
)
3.05 2.69 4.34 0.93 2.68
Note: 95% CL: 95% confidence limit.
95% CL
EC50 (mg kg1)
95% CL
2.84–3.27 2.54–2.85 4.16–4.54 0.77–1.11 2.48–2.87
0.954 0.318 0.261 0.368 0.202
0.463–2.783 0.119–0.98 0.104–1.009 0.080–2.76 0.055–1.748
K. Wang et al. / Chemosphere 132 (2015) 120–126 Table 3 Reproduction parameters measured for E. fetida exposed to difference concentrations of five neonicotinoid insecticides. Concentrations (mg kg1)
Mean cocoons per E. fetida
Mean hatchlings per cocoon
Mean cocoon weight (mg)
Control 0.00
4.05 ± 0.05
3.97 ± 0.22
13.75 ± 1.61
Imidacloprid 0.10 0.20 0.50 1.00 2.00
4.07 ± 0.32 4.15 ± 0.15 3.09 ± 0.33* 2.35 ± 0.15*** 0.65 ± 0.05***
3.67 ± 0.33 2.57 ± 0.01 2.37 ± 0.30 2.69 ± 0.69** 2.25 ± 0.25***
13.36 ± 1.06 12.46 ± 1.96* 10.96 ± 0.38 *** 8.00 ± 2.43*** 6.79 ± 0.13***
Acetamiprid 0.05 0.125 0.25 0.75 1.50
3.99 ± 0.33 4.03 ± 0.67 3.55 ± 0.45 2.05 ± 0.15*** 2.45 ± 0.15*
3.96 ± 0.57 3.07 ± 0.56 2.47 ± 0.68** 2.79 ± 0.21* 0.99 ± 0.26***
12.75 ± 0.93 10.50 ± 1.06*** 10.00 ± 1.57*** 8.33 ± 0.94*** 7.05 ± 0.61***
Clothianidin 0.01 0.025 0.05 0.20 0.80
4.16 ± 0.78 4.43 ± 0.25 3.50 ± 0.10 3.10 ± 0.50* 1.85 ± 0.05***
4.09 ± 0.65 3.98 ± 0.59 4.75 ± 0.46 3.70 ± 0.46 1.81 ± 0.06***
13.58 ± 1.50 12.66 ± 1.30* 11.30 ± 2.89*** 11.07 ± 0.84*** 9.55 ± 1.57***
Nitenpyram 0.025 0.05 0.10 0.50 2.00
4.02 ± 0.07 4.32 ± 0.33 3.70 ± 0.10 2.80 ± 0.10* 2.20 ± 0.10***
4.00 ± 0.40 3.49 ± 0.68 3.51 ± 0.17 1.89 ± 1.11*** 1.88 ± 0.63***
12.75 ± 0.95 9.04 ± 0.50*** 10.81 ± 1.39*** 10.57 ± 1.07*** 9.80 ± 1.43***
Thiacloprid 0.05 0.125 0.25 0.75 1.50
4.16 ± 0.40 4.05 ± 0.35 3.75 ± 0.05 2.85 ± 0.75* 2.45 ± 0.35***
3.74 ± 0.70 2.98 ± 0.05 2.50 ± 0.08** 2.61 ± 0.09* 1.25 ± 0.25***
14.05 ± 1.31 13.60 ± 1.48 12.52 ± 1.14* 11.42 ± 2.12*** 9.25 ± 0.86***
Note: Data are expressed as mean ± sd (n = 5). Statistical significance versus control group. * p < 0.05. ** p < 0.01. *** p < 0.001.
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concentrations less than 1.0 mg kg1, however there was no statistical significance in the individual EC50s, which varied from a low of 0.261 mg kg1 for nitenpyram to a high of 0.954 mg kg1 for imidacloprid. 3.4. Effects of neonicotinoids on cellulase activity of E. fetida The effect of the five neonicotinoids on E. fetida was further sought by studying cellulose activity. No significant effect on cellulase activity was observed after 21 d of exposure to five concentrations of nitenpyram (Fig. 1A) (p < 0.05). However the cellulase activity of E. fetida extracts increased significantly after 1 d of exposure to imidacloprid (Fig. 1C). However cellulase activity of E. fetida returned to normal levels in the following test period (Fig. 1B, C and E). A sharp increase in cellulase activity was only observed after 1 d of exposure to the highest concentrations of imidacloprid (Fig. 1C). The concentration of acetamiprid that caused an increase in cellulose activity was lower than the concentration of imidacloprid. Moreover, the cellulase activity of E. fetida displayed an increasing trend with an increase of acetamiprid concentration (Fig. 1B). Unlike the above three neonicotinoid insecticides, clothianidin significantly inhibited cellulase activity. The cellulase activity of E. fetida treated with clothianidin was significantly lower than that of the control group (Fig. 1D). 3.5. Histopathological examination In E. fetida, the condition of the epidermal cells deteriorated with increasing pesticide concentration. Many mucocytes of the epidermis were hypertrophic and showed an irregular cellular compartmentation. After exposure to higher pesticide concentrations, disintegration of epidermal cells was observed (Fig. 2). With increasing pesticide concentration, the cells of the midgut tissue often showed a degradation of the cellular compartmentation as well as a reduced density of the cytoplasm and irregularly shaped nuclei (Fig. 3). In general, compared with other four neonicotinoid insecticides, the effect of acetamiprid on the epidermal and midgut was more
Fig. 1. The cellulose activity of E. fetida after the exposure to soil with nitenpyram (A), acetamiprid (B), imidacloprid (C), clothianidin (D) and thiacloprid (E). Data are expressed as the mean ± SD, and are significant ⁄ for p < 0.05.
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Fig. 2. Effect of neonicotinoid insecticides on epidermal surface of the E. fetida after 14 d of exposure: I: smooth surface of epidermis; II: clear cellular compartmentation; III: Nucleus oval in shape; IV: irregular surface of epidermis; V: irregular cellular compartmentation; VI: irregular shape and altered size of nucleus; VII: cells disintegrated.
Fig. 3. Effect of neonicotinoid insecticides on midgut tissue of E. fetida after 14 d of exposure: I: columnar-shaped cells; II: clear cellular compartmentation; III: nucleus oval in shape; IV: medium-dense cytoplasm; V: irregular cell shape; VI: irregular cellular compartmentation; VII: irregular shape and altered size of nucleus; VIII: cytoplasm of sparse density, intercellular space enhanced; IX: cells disintegrated.
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dramatic, suggesting acetamiprid could affect epidermal and midgut cells at 0.025 mg kg1, while the other neonicotinoids damaged epidermal and midgut cells at higher concentrations. 4. Discussion In this paper, the acute toxicity of five neonicotinoid insecticides to E. fetida was evaluated using the artificial soil method. The results indicated that neonicotinoid insecticides had high toxicity to E. fetida, especially clothianidin. Further tests on reproductive activity indicated these insecticides could seriously affect the reproduction on E. fetida (Gomez-Eyles et al., 2009). Excessive use of these insecticides might result in harm. The toxicity to E. fetida may help determine the appropriate dosage during agricultural usage. Sub-lethal concentrations of pollutants may affect the behavior, growth, reproduction and organizational structure of earthworm, which are more close to long-term toxic effects in the actual ecological system (Capowiez et al., 2006; Reinecke and Reinecke, 2007). Studies reveal that measurement of toxic effects on reproduction may be more sensitive than the survival index for environmental risk assessment (Kuperman et al., 2004; Capowiez et al., 2006; Reinecke and Reinecke, 2007)). Acetochlor has been reported to inhibit E. fetida growth and rate of cocoon production at concentrations greater than 20 mg kg1 (Xiao et al., 2006). Furthermore, studies related to the effects of heavy metal pollution and reproductive toxicity on E. fetida has also been reported (Lukkari et al., 2005; Nahmani et al., 2007). Our results demonstrate that these five neonicotinoid insecticides significantly affected E. fetida cocoon production and hatching in a concentration-dependent manner. Cocoon production was found to be very sensitive to neonicotinoid insecticides during the experiment. Capowiez has reported a similar result, suggesting that imidacloprid and thiacloprid cause a significant decrease in cocoon production at 1.91 mg kg1 and 0.51 mg kg1 respectively (Capowiez et al., 2006). Sublethal effects (e.g. sperm deformities) on E. fetida have been documented at imidacloprid concentrations above 0.5 mg kg1 (Luo et al., 1999; Capowiez et al., 2006). Other significant effects of imidacloprid such as weight loss and reduced burrowing activity have been found at levels of 0.5–1.0 mg kg1. The effect on reproductive capacity of E. fetida was a more sensitive parameter for studying the adverse effects of neonicotinoid insecticides. The reproduction of E. fetida exposed to soil with low concentrations of neonicotinoid insecticides, was significantly inhibited. Earthworm is the largest biomass on land, which is very important to soil environment (Bartlett et al., 2010), it is at the bottom of the land food chain, once the earthworm population was affected by pesticides, then the whole food chain will be affected, at last ruin the whole ecological system. Cellulase is a very important enzyme for E. fetida. The change of cellulase activity can directly influence the ability of E. fetida to decompose plant litter and other cellulosic material (Luo et al., 1999; Shi et al., 2007). Imidacloprid and acetochlor were found to inhibit the cellulase activity of E. fetida (Xiao et al., 2006; Luo et al., 2009). In the present study, the opposite effects on the cellulase activity in E. fetida were observed after the treatment with neonicotinoid insecticides. There were no significant short-term effects on the cellulase activity in E. fetida after treatment with nitenpyram or acetamiprid and imidacloprid, respectively. However, during the test period, the cellulase activity of E. fetida treated with clothianidin was significantly lower than that of the control group (Fig. 1D). This might be the reason that clothianidin has higher acute toxicity to E. fetida. Losing the ability to decompose plant litter and other cellulosic materials can be one of the reasons for the higher acute toxicity of clothianidin to E. fetida than the other four neonicotinoid insecticides.
Histopathology is now increasingly being used as a biomarker of environmental stress (Stentiford and Feist, 2005; Eseigbe et al., 2013). In the present study, five neonicotinoid insecticides injured midgut and epidermal tissues. Various discrete pathological changes were observed viz; altered shape and size of cells and nuclei, irregular cellular compartmentation, and an irregular surface of the epidermis. Epidermal and midgut cells disintegrated with higher concentrations (e.g. at 1.0 mg kg1 acetamiprid and 3.0 mg kg1 imidacloprid). This result agrees with the report of Dittbrenner et al. (2011) who reported that the midgut tissue was damaged by 14 d exposure to 0.2 mg kg1 imidacloprid. Epidermal cells and midgut cells were very sensitive to neonicotinoid insecticides, midgut and epidermal tissues were damaged at low concentrations, so the change of epidermal cells and midgut cells can evaluate the toxic of neonicotinoid insecticides. Similar effects on cellular integrity were reported for E. fetida exposed to an organophosphorus insecticide, profenofos (Chakra Reddy and Venkateswara Rao, 2008). From the histopathological examination, we found increased mucus and cytothesis of midgut tissue cells at lower test concentrations, but some cells was disintegrated at higher test concentrations. Günther reported that environmental stressors could induce hypertrophy and hyperplasia of E. fetida mucocytes leading to mucus increase as protective reaction (Guenther and Greven, 1990). The effect of neonicotinoid insecticides on tissues and reproduction may be the reasons that these five neonicotinoid insecticides have high toxic to E. fetida. The changes of reproduction, cellulase activity and histology were more sensitive, which can be monitored at sub-lethal concentrations, it has important value for protection population and ecosystem, moreover it can use for pollutant monitoring and early warning. The sub-lethal concentrations for difference neonicotinoid insecticides of reproduction, cellulase activity and histology were shown in Table 4, the sub-lethal concentrations about the reproduction were lower than which about cellulase and histology, but all the sub-lethal concentrations are much higher than the detected residual dosage in soils (Wu et al., 2012; Yang et al., 2012; Ramasubramanian, 2013; Dong et al., 2014; Sharma and Singh, 2014) Imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid affected reproduction at 0.2 mg kg1, 1 1 1 0.125 mg kg , 0.05 mg kg , 0.05 mg kg and 0.25 mg kg1 after 56 d exposure. Imidacloprid and thiacloprid, within the levels found in soils could cause epidermal and midgut cell damage. Therefore, the application of neonicotinoid insecticides poses a serious threat to E. fetida in soils. In summary, we demonstrate that imidacloprid, acetamiprid, nitenpyram, clothianidin and thiacloprid have high acute toxicity to E. fetida. The inhibition of cellulase activity may result in higher acute toxicity of clothianidin to E. fetida. The significant adverse effects of neonicotinoid insecticides to reproduction of E. fetida were confirmed by studying epidermal cells and midgut cells which were very sensitive to neonicotinoid insecticides. This maybe the reason that the neonicotinoid insecticides were toxic to E. fetida. These results suggest that the five neonicotinoid insecticides have adverse effects on E. fetida. The sub-lethal
Table 4 The sub-lethal concentrations used in different experiments. Pesticides
Imidacloprid Acetamiprid Nitenpyram Clothianidin Thiacloprid
Sub-lethal concentrations (mg kg1) Histology
Reproduction
1.0 0.25 0.25 0.50 1.0
0.5 0.25 0.1 0.05 0.25
1.0 0.75 0.5 0.2 0.75
Cellulase activity 2.0 1.5 2.0 0.8 1.5
0.4 0.4 0.4 0.2 0.4
0.9 0.9 0.9 0.4 0.9
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