Toxicity of A-cypermethrin, Emamectin Benzoate and Imidacloprid on AChE and Antioxidant Enzymes in The Honey Bee (Apis mellifera) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of the Advances in Agricultural Researches | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Article 18, Volume 21, Issue 3 - Serial Number 80, September 2016, Page 484-495 PDF (171.3 K) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Document Type: Research papers | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DOI: 10.21608/jalexu.2016.237287 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Authors | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Hesham Zaki Ibrahim1; Abd-Allah Hamed2; Mona Abdou* 3; Reda Abdel-Razik4; Nadia Hamed4 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Environmental Chemistry and Toxicology, Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2Pesticide Chemistry and Plant Protection, Plant Protection Institute, Agricultural Research Center, Ministry of Agriculture (Dokki, Cairo) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3Plant Protection Institute, Agricultural Research Center, Ministry of Agriculture, Alexandria. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
4Central Agricultural Pesticides Laboratory (CAPL), Agricultural Research Center (ARC), Alexandria, Egypt. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abstract | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The comparative toxic effects of α-cypermethrin, emamectin benzoate andimidacloprid were examined on honey bees (Apis mellifera L.). Separate groups of bee workers (A. mellifera) were left for 24 and 96hr to feed on 50% sucrose solution containing different concentrations of the tested insecticides, and the lethal concentration of each insecticide that caused 50% mortality (LC50) was estimated. The inhibitory effects of different selected concentrations (¼ LC50, ½ LC50 and LC50) on AChE, antioxidant enzymes activities (as environmental biomarkers) and the level of Malondialdehyde (MDA) (as an indicator for lipid peroxidation were determined in vivo after 24hr in head (for AChE) and in midgut (for antioxidant enzymes and MDA level) of surviving bees obtained after treatments with the insecticides to explore the possible effects of these compounds. Results indicated that exposure to emamectin benzoate showed toxicity to honey bees with LC50 value of 0.275mg/l, followed by α-cypermethrin (LC50= 5.4mg/l) while imidacloprid was the least toxic tested compound (LC50=24.97mg/l). Alpha (α) cypermethrin at the tested concentrations of 1.6, 2.7 and 5.4mg/l inhibited the activity of AChE in honey bees workers. Also, imidacloprid at 6.2, 12.5 and 24.97mg/l caused inhibition of AChE activity. On the other hand, emamectin benzoate at the concentrations of 0.07, 0.14 and 0.275mg/l caused high activation of AChE. In addition, the toxic effects of the tested compounds on antioxidant enzymes activities indicated that all compounds caused activation of these enzymes. Moreover, there was a decrease in the level of Malondialdehyde in the case of α-cypermethrin and imidacloprid, while emamectin benzoate elevated the level of MDA in honey bees. This suggests the use of AChE and antioxidant enzymes as environmental biomarkers for the assessment of the toxic effect of the tested insecticides on ecosystem health. It might allow the detection of early biological change, which may result in long-term physiological disturbances. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
: Apis mellifera; toxicity; α-cypermethrin; imidacloprid; emamectin benzoate; AChE; antioxidant enzymes; MDA | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Introduction Honey bees are considered to be good pollinators of many vegetable and fruit crops. Without adequate populations of bees, the production of these and other crops would be impossible. In addition, bee colonies are maintained for their honey and wax production. Many insecticides used for pest control are toxic to honey bees and therefore they can be used as indicator organisms for environmental pollution with pesticides.
The use of environmental biomarkers in ecotoxicology is becoming a useful routine, and various endpoints have been proposed as valuable tools to assess the effects of environmental chemical contamination. Among the most biomarkers, acetylcholinesterase (AChE) inhibition which are being frequently used both in environmental monitoring and laboratory assays. AChE is an important enzyme for the maintenance of normal nerve function. Inhibition of this enzyme resulting from the irreversible binding at the AChE active site (ester-forming site) leads to the accumulation of acetylcholine in the synapse, resulting in the disruption of normal function (Ibrahim et al., 1998). Other biomarkers are also of great interest such as that related to detoxification potential of the studied organisms. In recent years, several reports have suggested that, insecticides cause oxidative stress, characterized by exposure to excessive reactive oxygen species (ROS). The body has developed several defense mechanisms against oxidative damage. These mechanisms are composed of enzymatic and non-enzymatic systems. The enzymatic mechanism is made of free radical scavengers like catalase (CAT) and the glutathione-depend enzymes such as glutathione-s-transferase (GST) and glutathione peroxidase (GPX)(Durak et al., 2010). Malondialdehyde (MDA) is one of the indicators of lipid peroxidation, and it also react with DNA, protein, enzyme and other biomolecules, leading to oxidative damage. Any variability in MDAdeterminations may arise from variability in non-enzymatic chemical events yielding lipid peroxide products. Some of these products may increase or decrease the activities of specific antioxidant enzymes (Janero, 1990). Therefore, the aim of the present research is to test the in vivo toxicity of α-cypermethrin, emamectin benzoate andimidacloprid against the honey bee (A. mellifera) workers. MATERIALS AND METHODS Honey bee (Apis mellifera) First cross (Carnilolan* Egyptian) honey bee colonies reared in the apiary of El-Sabhia Research Station, Agriculture Research Center, Ministry of Agriculture, Alexandria, Egypt were used in the present investigation. Bee workers were stored in a temperature controlled chamber in the dark at 25±1.5°C and 65±5% RH where they remained quiet and protected from stress-induced biochemical changes. Bees of age of 3-4 weeks were fed on 50% (W/V) sucrose solution ad libitum according to Sharaf El-Din)1982(.
Chemicals Acetylthiocholine iodide (ATCI), 5.5'-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB); Triton x-100, Folin Ciocalteau phenol reagent and bovine serum albumin (BSA) were purchased from sigma – Aldrich chemical company (Fancy Road, Poole, Dorest, BH12 4QH, England). Other chemicals used either for hatching procedure, toxicity tests or biochemical assays were obtained from BDH Laboratory supplies (Ltd. Limited, Poole, UK).
Tested Insecticides The insecticides tested were commercial formulations of α-cypermethrin (α-Zed 10% EC) as a, emamectin benzoate (Elector 2% EC) and imidacloprid (Best 25% EC) and these formulations were supplied by Arab Company for the Manufacture of Pesticides and Veterinary Medicines, Egypt. Toxicity of the tested insecticides Adult honeybee workers were exposed to a range of concentrations of each of the test compounds (α-cypermethrin, emamectin benzoate and imidacloprid) dispersed directly in 50% sucrose solution. Three replicate test groups, each of ten bees were used in each experiment. Three control batches, each of ten bees, were run in addition to the tested series (control insects were fed on 50% sucrose solution only). The bees were held in an experimental room at a temperature of 25 ± 2ºC and a relative humidity of 50-70%.The mortality was recorded after 24 and 96hrs and compared with control values. If the untreated check (control) group recorded mortality, correction for control mortality is made using Abbott’s formula (Abbott, 1925). Analysis of the mortality data was achieved by appropriate statistical methods (probit analysis) (Finney, 1971). Median lethal concentrations (LC50) and associated 95% confidence limits were calculated.
Half and quarter of LC50 were also calculated for each insecticide from the propit (mortality) / log dose regression equation (y= a + bx), where y= 4.3255 and 3.8497, respectively. AChE assay Laboratory strains of nursery honey bee workers were fed on α-cypermethrin, emamectin benzoate and imidacloprid dispersed in 50% sucrose solutions at ¼ LC50 (12.5% mortality), ½ LC50 ( 25% mortality) and LC50 ( 50% mortality) plus an untreated group as a control. AChE activity was assessed (in vivo) in head of adult honey bee workers. The heads were cut from surviving bees at time end points of 24 hr, placed in eppendorf tubes, snap frozen in liquid nitrogen and stored at 20ºC for AChE assay. AChE activity was assayed according to Ellman et al.)1961(.
Antioxidant enzymes assay After 24 hrs of feeding on the tested insecticides (α-cypermethrin, emamectin benzoate and imidacloprid) at the concentration of ¼ LC50, ½ LC50 and LC50 plus untreated group (as a control), GST, CAT, and GPx were assessed (in vivo) in midgut homogenate of the adult honey bee workers. Catalase (CAT) was determined according to Aebi (1984), Glutathione-s-transferase (GST) was determined according to Habig et al. (1974), Glutathione peroxidase (GPX) was measured according to Paglia and Valentine (1967) and Malondialdehyde level (MDA) was estimated according to Sotoh (1978) and Ohkawa et al. ( 1979). All data were expressed as mean ± SD
RESULS and DISCUSIONS Toxicity of α-cypermethrin, emamectin benzoate and imidacloprid on the honey bee workers (A. mellifera) Results inTable (1) presented the calculated LC50 and the associated parameters (confidence limits and slope) of each of the tested insecticides against the workers of the honey bee. It is evident from LC50 values of the tested insecticides that emamectin benzoate was the most toxic tested compound tohoney bee workers with LC50 values of 0.275 and 0.184mg/l after 24 and 96 hr time exposure, respectively, followed by α-cypermethrin with LC50 values of 5.411 and 3.267mg/l after 24 and 96 hr of treatment, respectively. Imidacloprid was proved to be the least toxic insecticide with LC50 values of 24.97and 10.98mg/l after 24 and 96 hrs of treatment, respectively. It was noticed that, the toxicity of the tested insecticides against A. mellifera increased asthe exposure time increased. Yan-Xia et al. (2008) reported that methylamino abamectin benzoate 1% EC was extremely toxic to bees with LC50 value of 0.490 mg/l after 48-hrs. The presented results are also in accordance with Laurino et al. (2013). Who found that LD50 value of imidacloprid at 24 hours was 118 ng/bee, at 48 hours was 90.09 ng/bee and at 72 hours was 69.68ng/bee and they also reported that the toxicity of imidacloprid increased with the increase of the exposure time. Moreover, Hassona and Kordy (2015)proved that certain pesticides (Vertimec® as a bio-insecticide) was toxic against honey bee forgers. Rasuli et al. (2015)showed that fenpropathrin as a synthetic pyrethroid insecticide had high acute oral toxicity (LC50-24h and LC50-48hrs were 0.54 and 0.30 ppm, respectively).
Table (1). Toxicity of α-cypermethrin, emamectin benzoate and imidacloprid against the honey bees (A. mellifera) workers after 24 and 96hrs of treatment
)a: lethal concentration causing 50% mortality after 24hr and b: lethal concentration causing 50% mortality after 96hr)
In vivo AChE activity in A. mellifera exposed to α-cypermethrin, emamectin benzoate and imidacloprid for 24hr The results in Figure (1) show the in vivo effects of the tested insecticides on AChE activityof A. mellifera. The results revealed that imidacloprid at the concentrations of 6.2, 12.5, and 24.97 mg/l (¼ LC50, ½ LC50 and LC50) caused 6.2 16.5, and 23.4% inhibition, respectively. Also, α-cypermethrin caused inhibition percentages of 2.1, 5.1, and 7.5% when it was tested at the concentrations of 1.6, 2.7, and 5.4mg/l, subsequently. On the other hand, emamectin benzoate at the concentrations of 0.07, 0.15, and 0.275mg/l caused 60, 73.2, 82.8% increase of A. mellifera AChE activity, respectively. These present results are in agreement with those of Bendahau et al. (1999)who reported that cypermethrin slightly inhibit acetylcholinesterase activity on honey bees and this because AChE is not a specific target or site to synthetic pyrethroids. Also, Badiou et al. (2008) used acetylcholinesterase (AChE) as a biomarker (or indicator) of exposure to deltamethrin insecticide in the honey bee, A. mellifera.Moreover, Menessy (2011)revealed that avermectin insecticide (milbemectin) cause in vivo activation of AChE of honey bee head. In addition, Badiou et al. (2012)observed that there were no response for AChE on honey bees treated with the neonicotinoid insecticide thiamethoxam. Moreover, these results are in accordance with those obtained by Boily et al. (2013) who reported that AChE activity on foraging bees was inhibited in response to a neonicotinoid (imidocloprid).
Figure (1). In vivo effects of α-cypermethrin, emamectin benzoate and imidacloprid on AChE activity of A. mellifera.
Effects of α-cypermethrin, emamectin benzoate and imidacloprid on antioxidant enzymes activities and MDA level in A. mellifera Theresults in Table (2) show the in vivo effects of the tested insecticides on certainantioxidant enzymes activities of A. mellifera. Data revealed that there was a significant increase in the catalase (CAT), glutatione-s-transferase (GST) and glutathione peroxidase (GPx) activities depend on the concentration of insecticides used as compared to the control. In addition, there was a decrease in the level of Malondialdehyde (MDA) in the case of α-cypermethrin and imidacloprid. On the other hand, emamectin benzoate caused slight elevation (11.1%) in the level of MDA in A. mellifera as compared with control (Table 3). These results demonstrate that enhanced activities of CAT, GST, and GPx can lead to elimination of ROS in the midgut of honey bees. Also, the increase in CAT, GST, and GPx activities may be an attempt to counteract the increase in MDA level as a defense mechanism by cells against free radicals generation. The oxidative destruction of lipids acts in a chain reaction to form lipid hydroperoxides, which can decompose to MDA as the end product. The decrease in lipid peroxidation during the treatment with α-cypermethrin and imidacloprid may be due to an increase in antioxidant defense. These results are in agreement with those of Yu et al. (1984)who found that the insecticidepermethrin significantly stimulated the glutathione-s-transferase in adult worker bees (Apis mellifera L.). Also, Johnson et al. (2006)reported that three pyrethroids (cyfluthrin, lambda-cyhalothrin and tau-fluvalinate) may activate GSTs enzyme in honey bee worker. Moreover, these finding are supported with those of Badiou et al. (2012). They evaluated the development of GST and CAT as enzyme biomarkers of exposure to neonicotinoid xenobiotics such as thiamethoxam in the honey bee, A. mellifera and they found an increase on GST and CAT activities due to such exposure.
Table (2). In vivo effects of α-cypermethrin, emamectin benzoate and imidacloprid on the activity of these three enzymes in treated A.mellifera workers
*mean ± SD
Table (3). In vivo toxic effect of α-cypermethrin, emamectin benzoate and imidacloprid on MDA level in A. mellifera
Conclusion The current study compared and evaluated the toxicity of three insecticides belonging to different chemical groups (α-cypermethrin, emamectin benzoate and imidacloprid) on honey bee (A. mellifera) workers. AChE and antioxidant enzymes activities were determined and quantified to investigate the possibility of using such enzymes as environmental biomarkers (indicators) of exposure to these insecticides. It is therefore suggested that these insecticides must be used only with greatest care as they may impact on honey bees. Further studies are needed to investigate the duration of behavioral effects of these compounds on bees, in relation to biomarker response, particularly at sublethal doses. Therefore, this profile of biomarker variation could represent a useful fingerprint to characterize the exposure to different groups of insecticides.
Acknowledgements First of all, I thank Allah, without his willing and support this work would have never been possible. I wish to express my deep gratitude to Prof. Dr. Hesham Zaki Ibrahim, Professor of Environmental Chemistry and Toxicology, Department of Environmental Studies, Institute of Graduate Studies and Research, IGSR, Alexandria University for his supervision, continuous encouragement, and sincere efforts throughout this work to make this research possible.
My sincere thank to Prof. Dr. Abd–Allah, M. Hamed, Head Reseracher, Plant Protection Research Institute, Agricultural Research Center, Dokki, Cairo, for his supervision and encouragement. I wish to thank Dr. Nadia Ali Hamed, Researcher, Central Insecticides Laboratory, Agricultural Research Center, Alex. and Dr. Reda Khamis Abd-Elrazik, Researcher, Central Insecticides Laboratory, Agricultural Research Center, Alex. for their advice, and valuable experience in honeybee field. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
References | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abbott, W. S. (1925). A method for computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265 – 267.
Aebi, H. (1984). Catalase in vivo. Methods Enzymol., 105: 121 – 126.
Badiou, A., M. Meleda and N. Belzuncesa (2008). Honeybee, Apis mellifera acetylcholinesterase - A biomarker to detect deltamethrin exposure. Ecotoxicol. and Environ. Safety, 96(2): 246-253.
Badiou, A., S. M. Carvalho, J. L. Brunet, G. A. Carvalho, A. Bulete, B. Giroud and L. P. Belzunces (2012). Development of biomarkers of exposure to xenobiotics in the honey bees Apis mellifera: Application to the systemic insecticide thiamethoxam. Ecotoxicol. and Enviro. Safety, 82: 22-31.
Bendahou, N., C. Flechea and M. Bouniash (1999). Biological and biochemical effects of chronic exposure to very low levels of dietary cypermethrin (Cymbush®) on honey bee colonies (Hymenoptera: Apidae). Ecotoxicol. and Environ. Safety, 44 (2): 147 – 153.
Boily, M., B. Sarrasin, C. DeBlois, P. Aras and M. Chagnon (2013). Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments. Envion. Sci. Pollut. Res., 1568-1588.
Durak, D., S. Kalender, G. F. Uzun, F. Demir and Y. Kalender (2010). Mercury chloride-induced oxidative stress in human erythrocytes and the effect of vitamins C and E in vitro. Africa J. Biotech., 9(4): 488-495.
Ellman, G. L., K. D. Courtiney, V. Andres and R. M. Featherstones (1961). Anew and rapid colorimetric determination of aceylcholinesterase activity. Biochem. Pharmacol., 7: 88 – 95.
Finney, D. J. (1971). Probit analysis. 3rd ed., Cambridge, London and New York. Cambridge Univ. Press, Cambridge, pp 333.
Habig, W., M. Pabst and W. Jakoby (1974). Glutathione-s-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249: 7130 – 7139.
Hassona, Nadia M. and A. M. Kordy (2015). Relationship between toxicity of certain pesticides to the honey bee, Apis mellifera L. (Hymenoptera: Apidae) foragers and their haemolymph amino acids. Middle East App. Sci., 5 (1): 19 – 25.
Ibrahim, H., R. Kheir, S. Helmi, J. Lewis, and M. Crane (1998). Effects of organophosphorus, Carbamate, pyrethroid and organochlorine pesticides and a heavy metal on survival and chlorinesterase activity of Chironomus riparius Meigen. Bull. Environ. Contam. Toxicol.,60: 448 - 455.
Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid – reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biol. Med., 9: 515 – 540.
Johnson, R. M., Z. Wen, M. A. Schuler and M. R. Berenbaum (2006). Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases. J. Econ. Entomol., 99(4): 1046-1050.
Laurino, D., A. Manino, A. Patetta and M. Porporato (2013). Toxicity of neonicotiniod insecticides on different honey bee genotypes. Bull. Insectol., 66 (1): 119 – 126.
Menessy, Abeer A. M. (2011). New approaches for control some insect pests and mites. Ph.D Thesis. Faculty of Agric. Damanhour Univ., Egypt.
Ohkawa, H., W. Ohishi and K. Yagi (1979). Lipid peroxide in vivo. Anal. Biochem., 95: 351.
Paglia, D. E. and W. N. Valentine (1967). Glutathione peroxidase in vivo. J. Lab. Clin. Med., 70: 158 – 169.
Rasuli, F., J. N. Rafie and A. Sadeghi (2015). The acute oral toxicity of commonly used pesticides in Iran to honey bees (Apis mellifera meda.). J. Apic. Sci., 59(1): 17 – 26.
Sharaf El – Din, M. (1982). Laboratory and field studies on toxicity of insecticides to honey bees (Apis mellifera L.). M. Sc. Thesis, Fac. of Agric. Shebin El-kom- Menoufia, Univ., Egypt.
Sotoh, K. (1978). Lipid Peroxide in vivo. Clinica Chimica Acta, 90: 37.
Yan – Xia Y., J.Shao – Qiang, W.Fang – Lin and Z.Gou – Nian (2008). Oral toxicity and Risk of fine insecticides to honey bees. Chinese J. of Pest. Sci., 5: 15 – 28.
Yu, S. J., F. A. Robinson and J. L. Nation (1984). Detoxification capacity in the honeybees, Apis mellifera L. Pestic. Biochem. Physiol., San Diiego, 22(3): 360 - 368.
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