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Toxic effects of imidacloprid on adult loach (Misgurnus anguillicaudatus)
Accepted Manuscript Title: Toxic effects of imidacloprid on adult loach (Misgurnus anguillicaudatus) Author: Xiaohua Xia Xiaopei Xia Weiran Huo Hui Dong Linxia Zhang Zhongjie Chang PII: DOI: Reference:
S1382-6689(16)30136-3 http://dx.doi.org/doi:10.1016/j.etap.2016.05.030 ENVTOX 2532
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
10-12-2015 23-5-2016 29-5-2016
Please cite this article as: Xia, Xiaohua, Xia, Xiaopei, Huo, Weiran, Dong, Hui, Zhang, Linxia, Chang, Zhongjie, Toxic effects of imidacloprid on adult loach (Misgurnus anguillicaudatus).Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toxic
effects
of
imidacloprid
on
adult
loach
(Misgurnus
anguillicaudatus)
Xiaohua Xia, Xiaopei Xia, Weiran Huo, Hui Dong, Linxia Zhang, Zhongjie Chang* (College of Life Science, Henan Normal University, Xinxiang, Henan 453007, People’s Republic of China)
*For correspondence. Molecular and Genetic Laboratory, College of Life Science, Henan Normal University, 46# East of Construction Road, Xinxiang, Henan 453007, P. R. China Telephone number: +86-373-3326553 E-mail: [email protected]
Highlights 1. The values of LC50 (24、48、72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, safety concentration was 42.55 mg/L. The erythrocyte micronuclei assays and the comet assay results showed that imidacloprid has genetic toxic effect on the loach erythrocytes. 2. To assess the physiological and biochemical damage by imidacloprid, the activity values of hepatic glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) declined in treatment groups. 3. Histological examination of testis revealed that imidacloprid treatment resulted in disorganized lobules and cysts structures. 4. In the present work, we also investigated the joint toxicity of pesticides commonly used in paddy fields (imidacloprid and lambda-cyhalothrin) on Misgurnus anguillicaudatus, and confirmed that a synergistic effect existing in the binary mixtures.
Abstract： The present investigation was aimed to assess the effects of imidacloprid on the survival, genetic materials, hepatic transaminase activity and histopathology of loach (Misgurnus anguillicaudatus). The values of LC50 (24, 48, 72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, and the safety concentration was 42.55 mg/L. The erythrocyte micronuclei assays and the comet assay results showed that imidacloprid had genetic toxic effect on the loach erythrocytes. To assess the physiological and biochemical damage caused by imidacloprid, the activities of hepatic glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) were measured and their values declined in treatment groups. Histological examination of testis revealed that imidacloprid treatment resulted in disorganized lobules and cysts structures. In the present work, we also investigated the joint toxicity of pesticides commonly used in paddy fields (imidacloprid and lambda-cyhalothrin) on Misgurnus anguillicaudatus, and confirmed that a synergistic effect existing in the binary mixtures. The results of our study provide relevant and comparable toxicity information that are useful for safety application of pesticides. Keywords： Imidacloprid; Misgurnus anguillicaudatus; Toxicity testing; Comet assay; Joint toxicity
1. Introduction Pesticide application is an effective measure to control pests and increase crop yields. However, studies showed that in the process of pesticide application, only about 1% of the pesticide acted on target organisms, while the remaining pesticides remained in the soil or evaporated into the air or run off into the waters through surface. Pesticides in water cause massive aquatic animals killed and water pollution, which would ultimately be consumed and enter into human body. In recent years, effects of pesticides on non-target organisms in ecological environment have been the subject of worldwide concern (Dutra et al., 2009). The development of modern eco-agriculture is not only the objective requirement to establish the Scientific Concept of Development, but also the inevitable choice of implementing the strategy of sustainable development in agriculture (Cao et al., 2011). With the development of modern eco-agriculture model, fish, shrimp, crab and paddy cultivation in same field is lucrative and becomes more and more prevalent. In the process of practical application, the farmers often spray different pesticides depending on the type of crops and insect pests occurrence. Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new type of neonicotinoid insecticides with high activity. Neonicotinoid is the most important class of new synthetic insecticides used in crop protection and animal health care (Jeschke and Nauen, 2008; Thany, 2010). Imidacloprid has been widely used for controlling rice plant-hopper, leafhopper, aphids, and thrips on a variety of paddy, vegetables and other crops (Liu et al., 2005). Some researches regarding imidacloprid affected non-target organisms such as Porcellio scaber (Drobne et al., 2008), ramshorn snail (Sawasdee and Köhler, 2009), insect Chironomus riparius (Azevedo-Pereira et al., 2011), Solenopsis invicta(Wang L et al., 2015), rats (Vohra P. and Khera KS, 2015), and Wista rat (Duzguner and Erdogan, 2012) have been reported. In addition to rice plant-hoppers, stem borers are harmful to rice production, leading to decreased rice yield and inferior grain quality. Lambda-cyhalothrin can effectively control the rice borers. Lambda-cyhalothrin is a new generation type II synthetic pyrethroid, which acts as a neuropoison interfering in the ionic conductance of nerve membranes by prolonging the sodium current (Clark, 1997). There are some researches about the toxicity of lambda-cyhalothrin (Yousef, 2010; Ansari et al., 2012; Piner and Üner, 2012). And recently, it is reported Lambda-cyhalothrin affects non-target organisms such as Culex quinquefasciatus (Muthusamy R. and Shivakumar
M.S., 2015) and Amblyomma americanum (Hughes T.H. et al., 2014). Also our previous works (Xia et al., 2013; Zhang and Xia, 2013) were used to determine the environmental toxicity of lambda-cyhalothrin. Due to the overlapped occurring stage of rice borers and plant-hoppers in the process of practical production, farmers usually sprayed two or more pesticides at the same time, thus it is essential to evaluate the joint toxicity of pesticides. As we know, rice production in China plays an important role for the Chinese people and also affects the world rice market. Yuanyang County in Henan Province of China, is located in the Yellow River Basin, and is a veritable hometown of rice. Loach and rice mixed aquaculture in paddy is very popular in the local agriculture. In order to assess the toxicity of pesticides in paddy on aquatic animal, we utilized the micronucleus, comet, hepatic transaminase activity and histopathological assays to determine the toxicity of imidacloprid on Misgurnus anguillicaudatus (M. anguillicaudatus), and we also used Marking additive index (Marking L.L, 1977) to analyze the joint toxicity of imidacloprid and lambda-cyhalothrin on M. anguillicaudatus. The aim of our work is to provide scientific basis for rational application of pesticides over paddy fields, protection of the water environment and biosecurity. 2. Materials and Methods 2.1. Tested fish and major chemicals Adult M. anguillicaudatus with 9 to 12 g weight and 10 to 12 cm length were collected from wetlands in the old course of the Yellow River, Yuanyang County (Henan, China). The loaches were domesticated for 7 days and the water was changed with the sun bathed water once everyday, then the lively and healthy organisms were chosen to conduct experiment. During the test the water temperature was 20~25 ℃, pH 6.5~7.5, the quality of dissolved oxygen concentration was 5.5~6.5 mg/L. Imidacloprid was effective composition content 70% water dispersing agent, and lambda-cyhalothrin was 2.5% of water emulsion. Transaminase kits were purchased from the Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Acute toxicity on M. anguillicaudatus Acute toxicity test was carried out by using the Spearman-Kärber (Kärber, 1931) method with some modification to obtain the medium lethal concentration (LC50) of imidacloprid in M. anguillicaudatus after 24, 48, 72 and 96 hours of exposure. A total of sixty loaches were distributed into six groups, out of which 5 groups served as
imidacloprid treatment groups and another as a control. On the basis of preliminary experiments, 5 treatment groups were exposed to the imidacloprid solution at the concentration of 115.00, 132.25, 152.09, 174.90, 201.14 mg/L for 96 hours, respectively. And the control group was placed in aerated tap water for the same period. Each test group exchanged with its same concentration imidacloprid solution after exposure of 24 hours and no food was provided during the test. Experiment was repeated three times. The dead and surviving loaches were recorded in each group during the exposure period. The LC50 of imidacloprid on M. anguillicaudatus after 24, 48, 72 and 96 hours of exposure were calculated using the amended Spearman-Kärber method. 2.3. Erythrocyte micronuclei assays According to the result of acute toxicity test, sixty adult loaches were randomly divided into 5 groups (2 parallels in each group, 6 fish in each parallel), out of which 4 groups served as treatment group that were exposed respectively to the imidacloprid solution at the concentration of 43.0, 67.0, 91.0 and 115.0 mg/L for 6 days, and another as a control group. After 2, 4 and 6 days of exposure, the blood samples were obtained and smeared on slides for the micronuclei assays according to Hoshina et al (Hoshina et al, 2008). Six thousand polychromatic erythrocytes were analyzed per loach. Slides were observed using a light microscope with a ×100 immersion objective. The frequencies of micronuclei and nuclear anomalies were calculated as follows: Micronuclei‰=Number of cells containing micronucleus / Total number of cells counted ×1000, Nuclear anomalies‰=Number of cells containing nuclear anomalies / Total number of cells counted ×1000. 2.4. Comet assays The experimental design of enzymes activity assays was the same to the micronuclei test. The comet assays was performed as described by Singh et al. and Piperakis et al. (Singh et al., 1988; Piperakis et al., 2006) with minor modification. Low melting agarose gel was prepared, and added the cells samples to the high melting agarose gel, covered coverslips. Then cell lysis after removed the cover glass, 4 °C cracking 1.5-2h. DNA was placed in an alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA; pH13) for 10 min to allow the DNA to unwind, followed by electrophoresis in the same buffer for 20 min at 15 V, 150 mA. The slides with 0.1M
Tris-HCl (pH=7.5) for 15min (Hannah V.Pie et al., 2015). After neutralization is completed, add EB on slides, observed use fluorescence microscope. The image processing by using an image analysis package (CASP). 2.5. Hepatic transaminase activity assays The experimental design of enzymes activity assays was the same to the micronuclei test. After 2, 4, and 6 days of exposure, two fishes of each group were dissected and the samples in liver were collected for GPT and GOT activity assays. All the steps are carried out in accordance with the specifications. The results of these enzymatic assays were given in units of enzymatic activity per gram of protein (U/g prot). The method of coomassie brilliant blue (G250) was used to determine the protein content. 2.6. Histopathological studies The concentrations of the experimental groups of histopathological test were the same to the micronuclei assays. After 6 days of exposure, for the histopathological observations, fresh liver and testis of control and treated groups were immersion fixed in neutral buffered 10% formalin. Following an overnight fixation, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Blocks were made and 5 µm thick sections were double stained with hematoxylin and eosin (HE) and observed under light microscope. 2.7. Combined toxicity of imidacloprid and lambda-cyhalothrin The joint toxicity of binary mixtures consisted of imidacloprid and lambda-cyhalothrin were conducted at an equal-toxic ratio (1:1) based on observed LC50 values (96 hours). The acute toxicity results of lambda-cyhalothrin on M. anguillicaudatus saw our previous work (Xia et al., 2013). Test method and the calculation method of LC50 value were same to the above single acute toxicity test. 2.8. Evaluation method for joint toxicity The joint effect of aquatic toxicology Marking additive index (AI) method was used in this paper to analyze and quantify the joint effects of mixtures. The toxic summation of combined compound (S) was calculated using the following equation S=Am/A+Bm/B, Where A and B are medium lethal concentration (LC50) values of imidacloprid and lambda-cyhalothrin alone, respectively. Am and Bm are LC50 values of mixtures.
The additive index (AI) can be calculated as follows: If S>1.0, AI=–S+1, and if S≤1.0, AI=1/S–1. Joint toxicity was characterized as the following: The joint effect is additive when AI=0, antagonistic when AI<0, and synergistic when AI>0(Marking L.L., 1977). 2.9. Statistical analysis All data were processed and analyzed by Excel and SPSS 13.0 software (SPSS, Chicago, USA) then shown as mean ± standard deviation (SD). According to data of all groups analyzed by LSD in One Way ANOVA program at same time, the difference was considered significant when p was less than 0.05, if p was less than 0.01, the difference was extremely significant. 3. Results 3.1. Acute toxicity of imidacloprid on M. anguillicaudatus Loaches showed hyperactivity such as moving and writhing quickly when exposed to imidacloprid. In high-dose group, loaches jumped, curved their body, lost balance gradually, until death. The acute toxicity result of different doses of imidacloprid on M. anguillicaudatus was shown in Table 1. The LC50 (24, 48, 72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, and the safety concentration was calculated as 42.55 mg/L through the formula. 3.2. Genetic materials damage induced by imidacloprid in loach erythrocytes Normal loach erythrocytes were oval or round, and cell nucleus usually located in the center of cell with clear nuclear membrane. Micronuclei was induced by imidacloprid in the cytoplasm, separated from the main nuclear and its size was one third of the main nuclear or less, the color of micronuclei stained by Giemsa was blue-violet or purple. From Table 2, significant differences between the control group and each treated group (P < 0.01) for all imidacloprid treatment times were observed. From the results we found the micronuclei frequencies ascended with increasing imidacloprid concentration and treatment time. The highest frequency of micronuclei (7.333‰) was observed on the sixth day in the highest dose group. In addition to the formation of micronucleus, loach erythrocytes induced by imidacloprid also has some nuclear anomalies, such as nucleoplasm deflected inward or outward, nuclear deformation and so on. The result of nuclear anomalies was similar to micronuclei frequencies. The highest frequency of nuclear anomalies (73.875‰) was observed on the sixth day in the highest dose group (Table 3). The comet assay used tail DNA content as the parameters to describe the loach
erythrocytes DNA damage case (Fig. 1, Table 4). The results showed that, on the second day, the 43.0 mg/L and 67.0 mg/L treatment groups showed no significant differences compared with the control group. On the fourth day, the 43.0 mg/L treatment group was not significantly different from the control group but the 67.0 mg/L treatment group showed significant difference with the control group, while other treatment groups showed much significant differences with the control group. On the sixth day, all treatment groups showed much significant differences with the control group. Loach erythrocytes tail DNA content increased gradually and reached 60.668% in the 91.0 mg/L treatment group. 3.3. Effects of imidacloprid on GPT and GOT activities of loach hepatocyte Effects of imidacloprid on GPT and GOT activities of loach hepatocyte were given in Fig. 2 and 3. Exposed to imidacloprid decreased the liver GPT and GOT activities in tested fish compared with control. At the same time with the increase of concentration of imidacloprid, GPT and GOT activities in the liver decreased; and at the same concentration with the extension of time, GPT and GOT activities also declined, exhibiting a negative correlation. There was extremely significant difference (p<0.01) between each treatment group and its control group, except that the GPT activities in treatment groups of 2 days exposure (p<0.05). 3.4. Histopathology Histological examinations were performed and no significant histopathological changes were revealed in liver at any dose level (Fig. 4). But histopathological effects were observed mainly in the exposed testis. Representative pictures of histopathological examination of testes in control and treated groups were shown in Fig. 5. In imidacloprid (91.0 mg/L) treatment, disorganization of cysts and increase of interstitial tissue were found in Fig. 5b. We also could see disorganized lobules, obviously expanded gaps, reduced ratios of spermatids and spermatozoa in imidacloprid (115.0 mg/L) treated loaches (Fig. 5c). 3.5. Analysis of joint toxicity effect The tests of the joint toxicity of imidacloprid and lambda-cyhalothrin were conducted at an equal-toxic ratio (1:1), and the results were shown in Table 5. It turned out that in the binary mixtures, the LC50 (24, 48, 72 and 96 hours) of imidacloprid were 75.74, 49.57, 39.66 and 36.27 mg/L, and the LC50 (24, 48, 72 and 96 hours) of lambda-cyhalothrin were 9.54, 6.24, 4.99 and 4.57 µg/l, respectively. The
AI value (0.34, 0.76, 0.85, 1.00) indicated a synergistic effect existing between imidacloprid and lambda-cyhalothrin, and this effect increased with time. These results suggested the existence of imidacloprid increased the toxicity of lambda-cyhalothrin, at the same time, lambda-cyhalothrin also strengthened the toxicity of imidacloprid. 4. Discussion LC50 can generally represent the degree of toxicity of toxicants. It is an important index and can be confirmed by an acute toxicity test (Li et al., 2012). According to the Chinese evaluation criteria of the chemical pesticide environmental safety evaluation test guidelines (GB/T 31270.1-2014GB/T 31270.21-2014), pesticide is low toxic to fish when the LC50 value (96 hours) is more than 10 mg/L. Our acute result showed that the 96 hours LC50 of imidacloprid on loach was 145.85 mg/L, thus imidacloprid belonged to low toxicity pesticide to loach. Fish absorb poisonous water in the environment, which can lead to blood stem cell chromosome breakage, thus affecting the peripheral blood of environment (Jiang G. C., 2009). Micronuclei is formed when the chromosome or chromosome fragment induced by various harmful factors fail to be incorporated into the new nucleus during cell division (Fenech, 2000). At present, the micronuclei assay is the preferred testing method in genetic toxicity testing, especially when detecting the mutagenic effects in fish cells exposed to toxicant (Ail et al., 2008; Nwani et al., 2010; Anbumani and Mohankumar, 2012). In recent years, the simultaneous change of morphological nuclear abnormalities together with micronuclei has attracted considerable attention (Ayllon and Garcia-Vazquez, 2000; Bolognesi et al., 2006). In this research, we detected the frequencies of micronuclei and nuclear abnormalities, demonstrated that imidacloprid had some damage in genetic materials of loach. Anbumani and Mohankumar have observed a correlation between the frequencies of micronuclei and nuclear abnormalities, suggesting the significance of nuclear abnormalities as a prospective biomarker (Anbumani and Mohankumar, 2012). Comet assay is another method to examine the genetic toxicity of cells. Tail DNA content is an important issue to reflect the injured degree of DNA. If the tail DNA content is high, it will reveal that DNA broke seriously and get much genetic toxicity of cells. In this study, tail DNA content increased gradually from the low dose treatment group to the medium dose treatment group, which revealed that
imidacloprid has obvious effect on loach DNA in a dose-dependent manner. The cell tail DNA content decreased in the highest dose treatment group compared with the other groups. It can be explained as follows: the DNA encountered severe injury, DNA fragments disappeared when it was through the electrophoresis, the measurement value decreased finally. The cell tail DNA content increased gradually with the time extending, which revealed that imidacloprid has the obvious effect on loach DNA in a time-dependent manner (Risso-deFavemey C et a1., 2001; Vanzella T P et a1., 2007).
The liver is the main site of urea synthesis and has the detoxification function. Changing in the glutamic-pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activity is the main sensitive index reflecting the damaged liver tissue, which can be evaluated to the toxicity and side effects of drugs (Lin L. et a1., 2004.). In the assessment of physiological and biochemical damage by imidacloprid, GPT and GOT were used as indexes to measure hepatotoxicity induced by imidacloprid in M. anguillicaudatus. Result obtained from the present study showed that the liver GPT and GOT activities in tested fish were declined compared with control. The reason might be: under normal circumstances, the activities of GPT and GOT in liver were relatively high; but if the liver was injured, the cytomembrane permeability of hepatic cell would change, and then a lot of GPT and GOT in liver would transport into blood plasma, resulting the decrease of the activation of liver aminotransferases. Histopathology has received more attention as an endpoint in aquatic organisms, because histopathological changes are often the result of the integration of a substantial number of interactive physiological processes (Mlambo et al., 2009). In our work, no obvious histopathological effects were observed in the exposed livers. This result may be due to the low toxicity of imidacloprid on M. anguillicaudatus. In the short-term and sub-lethal exposure of imidacloprid, many physiological and biochemical indexes (GPT and GOT, Fig. 2 and 3) changed, but did not cause the pathological changes of the liver. A lot of researches showed that many pesticides having endocrine disruptors properties to disturb the endogenous hormonal function of organisms, affecting behaviour and secondary sexual characteristics as well as the gonads (Yousef, 2010; Matsuda et al., 2001). Kapoor et al. have proved imidacloprid was capable of generating an adverse effect on reproductive system (Kapoor et al., 2011). So we chose the testis as our histomorphological studies’ material. According
to the experimental results, disorganization of cysts and increase of interstitial tissue were found in imidacloprid treatment (91.0 mg/L). We also could see disorganized lobules, obviously expanded gaps, reduced ratios of spermatids and spermatozoa in imidacloprid treated (115.0 mg/L). High dose imidacloprid has strong influence on loaches testis development. We should pay more attention on these estrogenic pesticides, some further studies must be taken. In order to improve the yield and quality of the crops, in the process of practical application, farmers often spray two or more pesticides at the same time. Lambda-cyhalothrin and imidacloprid belong to pyrethroid insecticides and neonicotinoid insecticides, respectively, and they have been widely used as effective pesticides
in
agriculture.
The
chemical
structures
of
imidacloprid
and
lambda-cyhalothrin are shown in Fig. 6. In the present study, we examined the joint toxicity of imidacloprid and lambda-cyhalothrin on the loach, M. anguillicaudatus. Results showed that a synergistic effect existing in the binary mixtures. This could be explained by their effective components having superimposed effect. For the traditional pyrethroid insecticides, the cyano can influence cytochrome c and the electronic transmit system, extend the spinal nerve membrane depolarization time, thus interfere with the normal conduction of the central nervous signals (Soderlund and Bloomquist,1989). Neonicotinoid insecticides, in which the oxygen on the nitro or the nitrogen on the pyridine ring can form the hydrogen bond with the nicotin acetylcholine receptors (nAChRs). These receptors form a ion channels complex to regulate the nervous system, mainly responsible for fast synaptic transmission. Neonicotinoid insecticides mainly interfere with nervous system of the insect through selective control insect nervous system nAChRs (Tomizawa and Casida, 2003). When pyrethroid and neonicotinoid insecticides existed simultaneously, they were both acting on the nervous system, so their combination could enhance the toxicity. As Chenchen have proved that, the imidacloprid and lambda-cyhalothrin have synergistic effect on zebrafish toxicity (Chenchen, 2014). The growth and breeding of loaches would suffer a greater threat when the paddy fields, or water areas were polluted by these pesticides at the same time. In conclusion, the results of our study provided theoretical and scientific significances for safety application of imidacloprid and lambda-cyhalothrin. In addition, our work also offered certain realistic guiding meanings in applying paddy pesticides to farmers especially the local farmers in YuanYang country, China, and
promoted the fast and healthy development of modern eco-agriculture model.
Acknowledgments This work is supported by grants from the National Natural Science Foundation of China (no.31200923), Young Core Instructor Foundation of Henan Normal University (no. 5101049470610) Doctor Subject Foundation of Henan Normal University (no. 5101049170109).
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Fig. 1. The comet results of different concentrations of imidacloprid in loach erythrocytes. (a) A view of control M. anguillicaudatus, (b) in imidacloprid (43.0 mg/L) treated M. anguillicaudatus, (c) in imidacloprid (67.0 mg/L) treated M. anguillicaudatus. (d) in imidacloprid (91.0 mg/L) treated M. anguillicaudatus, (e) in imidacloprid (115.0 mg/L) treated M. anguillicaudatus.
Fig. 2. The activity of GPT induced by imidacloprid in M. anguillicaudatus hepar. Values are expressed as means ±SD, vertical lines represent the standard deviation (SD); asterisks above the bars indicate values significant differences between the control and the exposure groups at the same time. * indicates significant difference at 0.05 level; ** indicates significant difference at 0.01 level.
Fig. 3. The activity of GOT induced by imidacloprid in M. anguillicaudatus hepar. Values are expressed as means ±SD, vertical lines represent the standard deviation(SD); asterisks above the bars indicate values significant differences between the control and the exposure groups at the same time. ** indicates significant difference at 0.01 level.
Fig. 4. No significant histopathological changes were observed in liver (200×). (a) A view of liver of control M. anguillicaudatus, (b) the highest dose (115.0 mg/L) treated liver. H&E stain.
Fig. 5. Representative pictures of histopathological examination of testes in control and treated groups (200×). (a) A view of testis of control M. anguillicaudatus, (b) in imidacloprid (91.0 mg/L) treated testis, (c) in imidacloprid (115.0 mg/L) treated testis. H&E stain.
Fig. 6. The chemical structures of imidacloprid and lambda-cyhalothrin.
Table 1 Acute toxicity of imidacloprid to M. Anguillicaudatus.
Average percent mortality ± SD
Doses
LC50(mg/l)
24h
48h
72h
96h
Control
0.0±0.0
0.0±0.0
0.0±0.0
0.0±0.0
115.00 132.25 152.09 174.90 201.14
0.0±0.0 6.7±5.8 20.0±10.0** 73.3±5.8** 80.0±0.0**
10.0±0.0 13.3±5.8* 30.0±10.0** 73.3±5.8** 93.3±5.8**
10.0±0.0** 23.3±5.8** 56.7±5.8** 80.0±0.0** 100.0±0.0**
13.3±5.8** 26.7±5.8** 56.7±5.8** 83.3±5.8** 100.0±0.0**
mg/l
24h
48h
72h
96h
167.7
158.6
147.9
145.8
Note: Values followed by * mean significant difference at p<0.05, Values followed by ** mean significant difference at p<0.01.
Table.2 Results of micronuclei in M. anguillicaudatus erythrocytes induced by imidacloprid
Concentration (mg/l) Control 43.0 67.0 91.0 115.0
Number of cells observed 6000 6000 6000 6000 6000
Micronuclei rate ± SD（‰） 2day 0.708±0.160 1.500±0.430** 2.333±0.360** 2.958±0.344** 3.500±0.430**
4day 0.708±0.160 2.583±0.096** 3.042±0.160** 3.750±0.518** 4.417±0.833**
Note: Values followed by ** mean significant difference at p<0.01.
6day 0.708±0.160 3.125±0.438** 3.833±0.491** 4.208±0.832** 7.333±0.782**
Table 3 Results of nuclear anomalies in M. anguillicaudatus erythrocytes induced by imidacloprid
Concentration (mg/l)
Number of cells observed
Control 43.0 67.0 91.0 115.0
6000 6000 6000 6000 6000
nuclear anomalies rate ± SD（‰） 2day 1.458±0.285 14.125±2.910** 21.167±2.117** 30.250±3.143** 36.083±3.573**
4day 1.458±0.285 26.125±0.896** 30.708±1.022** 37.042±2.926** 42.083±10.308**
Note: Values followed by ** mean significant difference at p<0.01.
6day 1.458±0.285 33.750±1.076** 39.792±3.360** 45.542±2.522** 73.875±3.023**
Table 4 Results of comet in M. anguillicaudatus erythrocytes by imidacloprid Concentration (mg/l) Control 43.0 67.0 91.0 115.0
Tail DNA ± SD（%） 2day 0.230±0.071 1.610±0.397 2.957±0.008 31.366±1.366** 20.749±0.456**
4day 0.230±0.071 5.387±0.445 8.294±0.443* 56.136±5.721** 38.180±2.008**
6day 0.230±0.071 8.145±2.142** 10.409±2.819** 60.668±5.497** 39.025±5.393**
Note: Values followed by * mean significant difference at p<0.05, Values followed by ** mean significant difference at p<0.01
Table 5 Joint toxic action of imidacloprid and lambda-cyhalothrin at the toxicity of 1:1 on M. anguillicaudatus Exposure time
LC50 Imidacloprid (mg/l)
lambda-cyhalothrin (µg/l)
S
AI
Combined action
24h
75.74
9.54
0.75
0.34
48h 72h 96h
49.57 39.66 36.27
6.24 4.99 4.57
0.57 0.54 0.50
0.76 0.85 1.00
synergistic synergistic synergistic synergistic
S1382-6689(16)30136-3 http://dx.doi.org/doi:10.1016/j.etap.2016.05.030 ENVTOX 2532
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
10-12-2015 23-5-2016 29-5-2016
Please cite this article as: Xia, Xiaohua, Xia, Xiaopei, Huo, Weiran, Dong, Hui, Zhang, Linxia, Chang, Zhongjie, Toxic effects of imidacloprid on adult loach (Misgurnus anguillicaudatus).Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2016.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toxic
effects
of
imidacloprid
on
adult
loach
(Misgurnus
anguillicaudatus)
Xiaohua Xia, Xiaopei Xia, Weiran Huo, Hui Dong, Linxia Zhang, Zhongjie Chang* (College of Life Science, Henan Normal University, Xinxiang, Henan 453007, People’s Republic of China)
*For correspondence. Molecular and Genetic Laboratory, College of Life Science, Henan Normal University, 46# East of Construction Road, Xinxiang, Henan 453007, P. R. China Telephone number: +86-373-3326553 E-mail: [email protected]
Highlights 1. The values of LC50 (24、48、72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, safety concentration was 42.55 mg/L. The erythrocyte micronuclei assays and the comet assay results showed that imidacloprid has genetic toxic effect on the loach erythrocytes. 2. To assess the physiological and biochemical damage by imidacloprid, the activity values of hepatic glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) declined in treatment groups. 3. Histological examination of testis revealed that imidacloprid treatment resulted in disorganized lobules and cysts structures. 4. In the present work, we also investigated the joint toxicity of pesticides commonly used in paddy fields (imidacloprid and lambda-cyhalothrin) on Misgurnus anguillicaudatus, and confirmed that a synergistic effect existing in the binary mixtures.
Abstract： The present investigation was aimed to assess the effects of imidacloprid on the survival, genetic materials, hepatic transaminase activity and histopathology of loach (Misgurnus anguillicaudatus). The values of LC50 (24, 48, 72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, and the safety concentration was 42.55 mg/L. The erythrocyte micronuclei assays and the comet assay results showed that imidacloprid had genetic toxic effect on the loach erythrocytes. To assess the physiological and biochemical damage caused by imidacloprid, the activities of hepatic glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) were measured and their values declined in treatment groups. Histological examination of testis revealed that imidacloprid treatment resulted in disorganized lobules and cysts structures. In the present work, we also investigated the joint toxicity of pesticides commonly used in paddy fields (imidacloprid and lambda-cyhalothrin) on Misgurnus anguillicaudatus, and confirmed that a synergistic effect existing in the binary mixtures. The results of our study provide relevant and comparable toxicity information that are useful for safety application of pesticides. Keywords： Imidacloprid; Misgurnus anguillicaudatus; Toxicity testing; Comet assay; Joint toxicity
1. Introduction Pesticide application is an effective measure to control pests and increase crop yields. However, studies showed that in the process of pesticide application, only about 1% of the pesticide acted on target organisms, while the remaining pesticides remained in the soil or evaporated into the air or run off into the waters through surface. Pesticides in water cause massive aquatic animals killed and water pollution, which would ultimately be consumed and enter into human body. In recent years, effects of pesticides on non-target organisms in ecological environment have been the subject of worldwide concern (Dutra et al., 2009). The development of modern eco-agriculture is not only the objective requirement to establish the Scientific Concept of Development, but also the inevitable choice of implementing the strategy of sustainable development in agriculture (Cao et al., 2011). With the development of modern eco-agriculture model, fish, shrimp, crab and paddy cultivation in same field is lucrative and becomes more and more prevalent. In the process of practical application, the farmers often spray different pesticides depending on the type of crops and insect pests occurrence. Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new type of neonicotinoid insecticides with high activity. Neonicotinoid is the most important class of new synthetic insecticides used in crop protection and animal health care (Jeschke and Nauen, 2008; Thany, 2010). Imidacloprid has been widely used for controlling rice plant-hopper, leafhopper, aphids, and thrips on a variety of paddy, vegetables and other crops (Liu et al., 2005). Some researches regarding imidacloprid affected non-target organisms such as Porcellio scaber (Drobne et al., 2008), ramshorn snail (Sawasdee and Köhler, 2009), insect Chironomus riparius (Azevedo-Pereira et al., 2011), Solenopsis invicta(Wang L et al., 2015), rats (Vohra P. and Khera KS, 2015), and Wista rat (Duzguner and Erdogan, 2012) have been reported. In addition to rice plant-hoppers, stem borers are harmful to rice production, leading to decreased rice yield and inferior grain quality. Lambda-cyhalothrin can effectively control the rice borers. Lambda-cyhalothrin is a new generation type II synthetic pyrethroid, which acts as a neuropoison interfering in the ionic conductance of nerve membranes by prolonging the sodium current (Clark, 1997). There are some researches about the toxicity of lambda-cyhalothrin (Yousef, 2010; Ansari et al., 2012; Piner and Üner, 2012). And recently, it is reported Lambda-cyhalothrin affects non-target organisms such as Culex quinquefasciatus (Muthusamy R. and Shivakumar
M.S., 2015) and Amblyomma americanum (Hughes T.H. et al., 2014). Also our previous works (Xia et al., 2013; Zhang and Xia, 2013) were used to determine the environmental toxicity of lambda-cyhalothrin. Due to the overlapped occurring stage of rice borers and plant-hoppers in the process of practical production, farmers usually sprayed two or more pesticides at the same time, thus it is essential to evaluate the joint toxicity of pesticides. As we know, rice production in China plays an important role for the Chinese people and also affects the world rice market. Yuanyang County in Henan Province of China, is located in the Yellow River Basin, and is a veritable hometown of rice. Loach and rice mixed aquaculture in paddy is very popular in the local agriculture. In order to assess the toxicity of pesticides in paddy on aquatic animal, we utilized the micronucleus, comet, hepatic transaminase activity and histopathological assays to determine the toxicity of imidacloprid on Misgurnus anguillicaudatus (M. anguillicaudatus), and we also used Marking additive index (Marking L.L, 1977) to analyze the joint toxicity of imidacloprid and lambda-cyhalothrin on M. anguillicaudatus. The aim of our work is to provide scientific basis for rational application of pesticides over paddy fields, protection of the water environment and biosecurity. 2. Materials and Methods 2.1. Tested fish and major chemicals Adult M. anguillicaudatus with 9 to 12 g weight and 10 to 12 cm length were collected from wetlands in the old course of the Yellow River, Yuanyang County (Henan, China). The loaches were domesticated for 7 days and the water was changed with the sun bathed water once everyday, then the lively and healthy organisms were chosen to conduct experiment. During the test the water temperature was 20~25 ℃, pH 6.5~7.5, the quality of dissolved oxygen concentration was 5.5~6.5 mg/L. Imidacloprid was effective composition content 70% water dispersing agent, and lambda-cyhalothrin was 2.5% of water emulsion. Transaminase kits were purchased from the Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Acute toxicity on M. anguillicaudatus Acute toxicity test was carried out by using the Spearman-Kärber (Kärber, 1931) method with some modification to obtain the medium lethal concentration (LC50) of imidacloprid in M. anguillicaudatus after 24, 48, 72 and 96 hours of exposure. A total of sixty loaches were distributed into six groups, out of which 5 groups served as
imidacloprid treatment groups and another as a control. On the basis of preliminary experiments, 5 treatment groups were exposed to the imidacloprid solution at the concentration of 115.00, 132.25, 152.09, 174.90, 201.14 mg/L for 96 hours, respectively. And the control group was placed in aerated tap water for the same period. Each test group exchanged with its same concentration imidacloprid solution after exposure of 24 hours and no food was provided during the test. Experiment was repeated three times. The dead and surviving loaches were recorded in each group during the exposure period. The LC50 of imidacloprid on M. anguillicaudatus after 24, 48, 72 and 96 hours of exposure were calculated using the amended Spearman-Kärber method. 2.3. Erythrocyte micronuclei assays According to the result of acute toxicity test, sixty adult loaches were randomly divided into 5 groups (2 parallels in each group, 6 fish in each parallel), out of which 4 groups served as treatment group that were exposed respectively to the imidacloprid solution at the concentration of 43.0, 67.0, 91.0 and 115.0 mg/L for 6 days, and another as a control group. After 2, 4 and 6 days of exposure, the blood samples were obtained and smeared on slides for the micronuclei assays according to Hoshina et al (Hoshina et al, 2008). Six thousand polychromatic erythrocytes were analyzed per loach. Slides were observed using a light microscope with a ×100 immersion objective. The frequencies of micronuclei and nuclear anomalies were calculated as follows: Micronuclei‰=Number of cells containing micronucleus / Total number of cells counted ×1000, Nuclear anomalies‰=Number of cells containing nuclear anomalies / Total number of cells counted ×1000. 2.4. Comet assays The experimental design of enzymes activity assays was the same to the micronuclei test. The comet assays was performed as described by Singh et al. and Piperakis et al. (Singh et al., 1988; Piperakis et al., 2006) with minor modification. Low melting agarose gel was prepared, and added the cells samples to the high melting agarose gel, covered coverslips. Then cell lysis after removed the cover glass, 4 °C cracking 1.5-2h. DNA was placed in an alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA; pH13) for 10 min to allow the DNA to unwind, followed by electrophoresis in the same buffer for 20 min at 15 V, 150 mA. The slides with 0.1M
Tris-HCl (pH=7.5) for 15min (Hannah V.Pie et al., 2015). After neutralization is completed, add EB on slides, observed use fluorescence microscope. The image processing by using an image analysis package (CASP). 2.5. Hepatic transaminase activity assays The experimental design of enzymes activity assays was the same to the micronuclei test. After 2, 4, and 6 days of exposure, two fishes of each group were dissected and the samples in liver were collected for GPT and GOT activity assays. All the steps are carried out in accordance with the specifications. The results of these enzymatic assays were given in units of enzymatic activity per gram of protein (U/g prot). The method of coomassie brilliant blue (G250) was used to determine the protein content. 2.6. Histopathological studies The concentrations of the experimental groups of histopathological test were the same to the micronuclei assays. After 6 days of exposure, for the histopathological observations, fresh liver and testis of control and treated groups were immersion fixed in neutral buffered 10% formalin. Following an overnight fixation, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Blocks were made and 5 µm thick sections were double stained with hematoxylin and eosin (HE) and observed under light microscope. 2.7. Combined toxicity of imidacloprid and lambda-cyhalothrin The joint toxicity of binary mixtures consisted of imidacloprid and lambda-cyhalothrin were conducted at an equal-toxic ratio (1:1) based on observed LC50 values (96 hours). The acute toxicity results of lambda-cyhalothrin on M. anguillicaudatus saw our previous work (Xia et al., 2013). Test method and the calculation method of LC50 value were same to the above single acute toxicity test. 2.8. Evaluation method for joint toxicity The joint effect of aquatic toxicology Marking additive index (AI) method was used in this paper to analyze and quantify the joint effects of mixtures. The toxic summation of combined compound (S) was calculated using the following equation S=Am/A+Bm/B, Where A and B are medium lethal concentration (LC50) values of imidacloprid and lambda-cyhalothrin alone, respectively. Am and Bm are LC50 values of mixtures.
The additive index (AI) can be calculated as follows: If S>1.0, AI=–S+1, and if S≤1.0, AI=1/S–1. Joint toxicity was characterized as the following: The joint effect is additive when AI=0, antagonistic when AI<0, and synergistic when AI>0(Marking L.L., 1977). 2.9. Statistical analysis All data were processed and analyzed by Excel and SPSS 13.0 software (SPSS, Chicago, USA) then shown as mean ± standard deviation (SD). According to data of all groups analyzed by LSD in One Way ANOVA program at same time, the difference was considered significant when p was less than 0.05, if p was less than 0.01, the difference was extremely significant. 3. Results 3.1. Acute toxicity of imidacloprid on M. anguillicaudatus Loaches showed hyperactivity such as moving and writhing quickly when exposed to imidacloprid. In high-dose group, loaches jumped, curved their body, lost balance gradually, until death. The acute toxicity result of different doses of imidacloprid on M. anguillicaudatus was shown in Table 1. The LC50 (24, 48, 72 and 96 hours) of imidacloprid were 167.7, 158.6, 147.9 and 145.8 mg/L, respectively, and the safety concentration was calculated as 42.55 mg/L through the formula. 3.2. Genetic materials damage induced by imidacloprid in loach erythrocytes Normal loach erythrocytes were oval or round, and cell nucleus usually located in the center of cell with clear nuclear membrane. Micronuclei was induced by imidacloprid in the cytoplasm, separated from the main nuclear and its size was one third of the main nuclear or less, the color of micronuclei stained by Giemsa was blue-violet or purple. From Table 2, significant differences between the control group and each treated group (P < 0.01) for all imidacloprid treatment times were observed. From the results we found the micronuclei frequencies ascended with increasing imidacloprid concentration and treatment time. The highest frequency of micronuclei (7.333‰) was observed on the sixth day in the highest dose group. In addition to the formation of micronucleus, loach erythrocytes induced by imidacloprid also has some nuclear anomalies, such as nucleoplasm deflected inward or outward, nuclear deformation and so on. The result of nuclear anomalies was similar to micronuclei frequencies. The highest frequency of nuclear anomalies (73.875‰) was observed on the sixth day in the highest dose group (Table 3). The comet assay used tail DNA content as the parameters to describe the loach
erythrocytes DNA damage case (Fig. 1, Table 4). The results showed that, on the second day, the 43.0 mg/L and 67.0 mg/L treatment groups showed no significant differences compared with the control group. On the fourth day, the 43.0 mg/L treatment group was not significantly different from the control group but the 67.0 mg/L treatment group showed significant difference with the control group, while other treatment groups showed much significant differences with the control group. On the sixth day, all treatment groups showed much significant differences with the control group. Loach erythrocytes tail DNA content increased gradually and reached 60.668% in the 91.0 mg/L treatment group. 3.3. Effects of imidacloprid on GPT and GOT activities of loach hepatocyte Effects of imidacloprid on GPT and GOT activities of loach hepatocyte were given in Fig. 2 and 3. Exposed to imidacloprid decreased the liver GPT and GOT activities in tested fish compared with control. At the same time with the increase of concentration of imidacloprid, GPT and GOT activities in the liver decreased; and at the same concentration with the extension of time, GPT and GOT activities also declined, exhibiting a negative correlation. There was extremely significant difference (p<0.01) between each treatment group and its control group, except that the GPT activities in treatment groups of 2 days exposure (p<0.05). 3.4. Histopathology Histological examinations were performed and no significant histopathological changes were revealed in liver at any dose level (Fig. 4). But histopathological effects were observed mainly in the exposed testis. Representative pictures of histopathological examination of testes in control and treated groups were shown in Fig. 5. In imidacloprid (91.0 mg/L) treatment, disorganization of cysts and increase of interstitial tissue were found in Fig. 5b. We also could see disorganized lobules, obviously expanded gaps, reduced ratios of spermatids and spermatozoa in imidacloprid (115.0 mg/L) treated loaches (Fig. 5c). 3.5. Analysis of joint toxicity effect The tests of the joint toxicity of imidacloprid and lambda-cyhalothrin were conducted at an equal-toxic ratio (1:1), and the results were shown in Table 5. It turned out that in the binary mixtures, the LC50 (24, 48, 72 and 96 hours) of imidacloprid were 75.74, 49.57, 39.66 and 36.27 mg/L, and the LC50 (24, 48, 72 and 96 hours) of lambda-cyhalothrin were 9.54, 6.24, 4.99 and 4.57 µg/l, respectively. The
AI value (0.34, 0.76, 0.85, 1.00) indicated a synergistic effect existing between imidacloprid and lambda-cyhalothrin, and this effect increased with time. These results suggested the existence of imidacloprid increased the toxicity of lambda-cyhalothrin, at the same time, lambda-cyhalothrin also strengthened the toxicity of imidacloprid. 4. Discussion LC50 can generally represent the degree of toxicity of toxicants. It is an important index and can be confirmed by an acute toxicity test (Li et al., 2012). According to the Chinese evaluation criteria of the chemical pesticide environmental safety evaluation test guidelines (GB/T 31270.1-2014GB/T 31270.21-2014), pesticide is low toxic to fish when the LC50 value (96 hours) is more than 10 mg/L. Our acute result showed that the 96 hours LC50 of imidacloprid on loach was 145.85 mg/L, thus imidacloprid belonged to low toxicity pesticide to loach. Fish absorb poisonous water in the environment, which can lead to blood stem cell chromosome breakage, thus affecting the peripheral blood of environment (Jiang G. C., 2009). Micronuclei is formed when the chromosome or chromosome fragment induced by various harmful factors fail to be incorporated into the new nucleus during cell division (Fenech, 2000). At present, the micronuclei assay is the preferred testing method in genetic toxicity testing, especially when detecting the mutagenic effects in fish cells exposed to toxicant (Ail et al., 2008; Nwani et al., 2010; Anbumani and Mohankumar, 2012). In recent years, the simultaneous change of morphological nuclear abnormalities together with micronuclei has attracted considerable attention (Ayllon and Garcia-Vazquez, 2000; Bolognesi et al., 2006). In this research, we detected the frequencies of micronuclei and nuclear abnormalities, demonstrated that imidacloprid had some damage in genetic materials of loach. Anbumani and Mohankumar have observed a correlation between the frequencies of micronuclei and nuclear abnormalities, suggesting the significance of nuclear abnormalities as a prospective biomarker (Anbumani and Mohankumar, 2012). Comet assay is another method to examine the genetic toxicity of cells. Tail DNA content is an important issue to reflect the injured degree of DNA. If the tail DNA content is high, it will reveal that DNA broke seriously and get much genetic toxicity of cells. In this study, tail DNA content increased gradually from the low dose treatment group to the medium dose treatment group, which revealed that
imidacloprid has obvious effect on loach DNA in a dose-dependent manner. The cell tail DNA content decreased in the highest dose treatment group compared with the other groups. It can be explained as follows: the DNA encountered severe injury, DNA fragments disappeared when it was through the electrophoresis, the measurement value decreased finally. The cell tail DNA content increased gradually with the time extending, which revealed that imidacloprid has the obvious effect on loach DNA in a time-dependent manner (Risso-deFavemey C et a1., 2001; Vanzella T P et a1., 2007).
The liver is the main site of urea synthesis and has the detoxification function. Changing in the glutamic-pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activity is the main sensitive index reflecting the damaged liver tissue, which can be evaluated to the toxicity and side effects of drugs (Lin L. et a1., 2004.). In the assessment of physiological and biochemical damage by imidacloprid, GPT and GOT were used as indexes to measure hepatotoxicity induced by imidacloprid in M. anguillicaudatus. Result obtained from the present study showed that the liver GPT and GOT activities in tested fish were declined compared with control. The reason might be: under normal circumstances, the activities of GPT and GOT in liver were relatively high; but if the liver was injured, the cytomembrane permeability of hepatic cell would change, and then a lot of GPT and GOT in liver would transport into blood plasma, resulting the decrease of the activation of liver aminotransferases. Histopathology has received more attention as an endpoint in aquatic organisms, because histopathological changes are often the result of the integration of a substantial number of interactive physiological processes (Mlambo et al., 2009). In our work, no obvious histopathological effects were observed in the exposed livers. This result may be due to the low toxicity of imidacloprid on M. anguillicaudatus. In the short-term and sub-lethal exposure of imidacloprid, many physiological and biochemical indexes (GPT and GOT, Fig. 2 and 3) changed, but did not cause the pathological changes of the liver. A lot of researches showed that many pesticides having endocrine disruptors properties to disturb the endogenous hormonal function of organisms, affecting behaviour and secondary sexual characteristics as well as the gonads (Yousef, 2010; Matsuda et al., 2001). Kapoor et al. have proved imidacloprid was capable of generating an adverse effect on reproductive system (Kapoor et al., 2011). So we chose the testis as our histomorphological studies’ material. According
to the experimental results, disorganization of cysts and increase of interstitial tissue were found in imidacloprid treatment (91.0 mg/L). We also could see disorganized lobules, obviously expanded gaps, reduced ratios of spermatids and spermatozoa in imidacloprid treated (115.0 mg/L). High dose imidacloprid has strong influence on loaches testis development. We should pay more attention on these estrogenic pesticides, some further studies must be taken. In order to improve the yield and quality of the crops, in the process of practical application, farmers often spray two or more pesticides at the same time. Lambda-cyhalothrin and imidacloprid belong to pyrethroid insecticides and neonicotinoid insecticides, respectively, and they have been widely used as effective pesticides
in
agriculture.
The
chemical
structures
of
imidacloprid
and
lambda-cyhalothrin are shown in Fig. 6. In the present study, we examined the joint toxicity of imidacloprid and lambda-cyhalothrin on the loach, M. anguillicaudatus. Results showed that a synergistic effect existing in the binary mixtures. This could be explained by their effective components having superimposed effect. For the traditional pyrethroid insecticides, the cyano can influence cytochrome c and the electronic transmit system, extend the spinal nerve membrane depolarization time, thus interfere with the normal conduction of the central nervous signals (Soderlund and Bloomquist,1989). Neonicotinoid insecticides, in which the oxygen on the nitro or the nitrogen on the pyridine ring can form the hydrogen bond with the nicotin acetylcholine receptors (nAChRs). These receptors form a ion channels complex to regulate the nervous system, mainly responsible for fast synaptic transmission. Neonicotinoid insecticides mainly interfere with nervous system of the insect through selective control insect nervous system nAChRs (Tomizawa and Casida, 2003). When pyrethroid and neonicotinoid insecticides existed simultaneously, they were both acting on the nervous system, so their combination could enhance the toxicity. As Chenchen have proved that, the imidacloprid and lambda-cyhalothrin have synergistic effect on zebrafish toxicity (Chenchen, 2014). The growth and breeding of loaches would suffer a greater threat when the paddy fields, or water areas were polluted by these pesticides at the same time. In conclusion, the results of our study provided theoretical and scientific significances for safety application of imidacloprid and lambda-cyhalothrin. In addition, our work also offered certain realistic guiding meanings in applying paddy pesticides to farmers especially the local farmers in YuanYang country, China, and
promoted the fast and healthy development of modern eco-agriculture model.
Acknowledgments This work is supported by grants from the National Natural Science Foundation of China (no.31200923), Young Core Instructor Foundation of Henan Normal University (no. 5101049470610) Doctor Subject Foundation of Henan Normal University (no. 5101049170109).
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Fig. 1. The comet results of different concentrations of imidacloprid in loach erythrocytes. (a) A view of control M. anguillicaudatus, (b) in imidacloprid (43.0 mg/L) treated M. anguillicaudatus, (c) in imidacloprid (67.0 mg/L) treated M. anguillicaudatus. (d) in imidacloprid (91.0 mg/L) treated M. anguillicaudatus, (e) in imidacloprid (115.0 mg/L) treated M. anguillicaudatus.
Fig. 2. The activity of GPT induced by imidacloprid in M. anguillicaudatus hepar. Values are expressed as means ±SD, vertical lines represent the standard deviation (SD); asterisks above the bars indicate values significant differences between the control and the exposure groups at the same time. * indicates significant difference at 0.05 level; ** indicates significant difference at 0.01 level.
Fig. 3. The activity of GOT induced by imidacloprid in M. anguillicaudatus hepar. Values are expressed as means ±SD, vertical lines represent the standard deviation(SD); asterisks above the bars indicate values significant differences between the control and the exposure groups at the same time. ** indicates significant difference at 0.01 level.
Fig. 4. No significant histopathological changes were observed in liver (200×). (a) A view of liver of control M. anguillicaudatus, (b) the highest dose (115.0 mg/L) treated liver. H&E stain.
Fig. 5. Representative pictures of histopathological examination of testes in control and treated groups (200×). (a) A view of testis of control M. anguillicaudatus, (b) in imidacloprid (91.0 mg/L) treated testis, (c) in imidacloprid (115.0 mg/L) treated testis. H&E stain.
Fig. 6. The chemical structures of imidacloprid and lambda-cyhalothrin.
Table 1 Acute toxicity of imidacloprid to M. Anguillicaudatus.
Average percent mortality ± SD
Doses
LC50(mg/l)
24h
48h
72h
96h
Control
0.0±0.0
0.0±0.0
0.0±0.0
0.0±0.0
115.00 132.25 152.09 174.90 201.14
0.0±0.0 6.7±5.8 20.0±10.0** 73.3±5.8** 80.0±0.0**
10.0±0.0 13.3±5.8* 30.0±10.0** 73.3±5.8** 93.3±5.8**
10.0±0.0** 23.3±5.8** 56.7±5.8** 80.0±0.0** 100.0±0.0**
13.3±5.8** 26.7±5.8** 56.7±5.8** 83.3±5.8** 100.0±0.0**
mg/l
24h
48h
72h
96h
167.7
158.6
147.9
145.8
Note: Values followed by * mean significant difference at p<0.05, Values followed by ** mean significant difference at p<0.01.
Table.2 Results of micronuclei in M. anguillicaudatus erythrocytes induced by imidacloprid
Concentration (mg/l) Control 43.0 67.0 91.0 115.0
Number of cells observed 6000 6000 6000 6000 6000
Micronuclei rate ± SD（‰） 2day 0.708±0.160 1.500±0.430** 2.333±0.360** 2.958±0.344** 3.500±0.430**
4day 0.708±0.160 2.583±0.096** 3.042±0.160** 3.750±0.518** 4.417±0.833**
Note: Values followed by ** mean significant difference at p<0.01.
6day 0.708±0.160 3.125±0.438** 3.833±0.491** 4.208±0.832** 7.333±0.782**
Table 3 Results of nuclear anomalies in M. anguillicaudatus erythrocytes induced by imidacloprid
Concentration (mg/l)
Number of cells observed
Control 43.0 67.0 91.0 115.0
6000 6000 6000 6000 6000
nuclear anomalies rate ± SD（‰） 2day 1.458±0.285 14.125±2.910** 21.167±2.117** 30.250±3.143** 36.083±3.573**
4day 1.458±0.285 26.125±0.896** 30.708±1.022** 37.042±2.926** 42.083±10.308**
Note: Values followed by ** mean significant difference at p<0.01.
6day 1.458±0.285 33.750±1.076** 39.792±3.360** 45.542±2.522** 73.875±3.023**
Table 4 Results of comet in M. anguillicaudatus erythrocytes by imidacloprid Concentration (mg/l) Control 43.0 67.0 91.0 115.0
Tail DNA ± SD（%） 2day 0.230±0.071 1.610±0.397 2.957±0.008 31.366±1.366** 20.749±0.456**
4day 0.230±0.071 5.387±0.445 8.294±0.443* 56.136±5.721** 38.180±2.008**
6day 0.230±0.071 8.145±2.142** 10.409±2.819** 60.668±5.497** 39.025±5.393**
Note: Values followed by * mean significant difference at p<0.05, Values followed by ** mean significant difference at p<0.01
Table 5 Joint toxic action of imidacloprid and lambda-cyhalothrin at the toxicity of 1:1 on M. anguillicaudatus Exposure time
LC50 Imidacloprid (mg/l)
lambda-cyhalothrin (µg/l)
S
AI
Combined action
24h
75.74
9.54
0.75
0.34
48h 72h 96h
49.57 39.66 36.27
6.24 4.99 4.57
0.57 0.54 0.50
0.76 0.85 1.00
synergistic synergistic synergistic synergistic