Biomarkers of imidacloprid toxicity in Japanese quail, Coturnix coturnix japonica

The in vivo effect of the oral sublethal doses of 3.014 mg kg−1 of IMI (1/25 LD50) for 1, 7, 14, and 28 days every other day on Japanese quail was investigated. The results revealed that certain biomarkers in the selected tissues of the quail such as acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), aminotransaminases (alanine aminotransferase, ALT, and aspartate aminotransaminase, AST), phosphatases (acid phosphatase, ACP, and alkaline phosphatase, ALP), lactate dehydrogenase (LDH), adenosine-triphosphatase (ATPase), glutathione-S-transferase (GST), lipid peroxidation (LPO), and blood glucose showed significant inductions, while significant reductions in the levels of glutathione-reduced (GSH), deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) were noticed. In this study, the molecular mechanisms of the toxic effects of imidacloprid on quails were elucidated regarding neurotoxicity, hepatotoxicity, oxidative stress, lipid peroxidation, antioxidant activity, and genotoxicity. Because IMI induced alterations in the levels of these biomarkers in Japanese quail; therefore, Japanese quail as a wild avian can be used as a suite bioindicator to detect imidacloprid toxicity.


Introduction
Living organisms are usually exposed to a vast array of synthetic and naturally occurring chemicals. With the industrial revolution and the emergence of synthetic chemistry, a variety of new chemicals were synthesized such as fertilizers and pesticides (Wexler et al. 2014). The use of pesticides in agriculture has increased rapidly during the past decades with the discovery of extremely high insecticidal activities of nerve-active compounds such as organochlorines, organophosphates, carbamates, and pyrethroids that opened an era of synthetic insecticides (Casida 2009). However, the effectiveness of these insecticides regrettably has diminished over time due to the environmental and health problem and emergence of resistant insect strains with enhanced detoxification mechanisms; therefore, new chemicals of neuroactive insecticide with different sensitive targets were synthesized (Ffrench-Constant et al. 2004). Neonicotinoids are a relatively new and widely used class of synthetic insecticides (Englert et al. 2017) specifically acting on the central nervous system of invertebrates than vertebrates due to their greater affinity for insect nicotinic acetylcholine receptors (nAChRs); for example, imidacloprid (IMI) has 565 times greater affinity for insect nAChRs than for vertebrate nAChRs (Tomizawa and Casida 2005). There are several factors that make neonicotinoid success, like flexibility, usage on a variety of crop types, lack of resistance among pest species, and safety to consumers (Bass et al. 2015;Elbert et al. 2008). In 2013, the European Commission declared a moratorium on the use of three neonicotinoids, namely, IMI, thiamethoxam, and clothianidin, for seed coating, soil treatment, and foliar treatment due to their toxicity to pollinators, but have been approved to be used for seed treatment of winter cereals (Lopez-Antia et al. 2015). It is acknowledged that the application of neonicotinoids is considered as a potential inductor of adverse effects to birds (Wexler et al. 2014) and is responsible for population declines of several species of predatory birds through eggshell thinning (Hallmann et al. 2014).
Responsible Editor: Philippe Garrigues IMI (1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine) is a systemic insecticide belonging to the family of neonicotinoids and was launched in 1991 for the first time by Bayer Crop-Science (Elbert et al. 2007). It is the most widely used insecticide in the world (Jeschke et al. 2011;Goulson 2013) against piercing and sucking insects' pests as well as has high efficiency to control flea on cats and dogs (Tomizawa and Casida 2005). IMI has relatively low soil persistence with a half-life ranging from 40 to 997 days with high insecticidal activity at a very low application rate (Hopwood et al. 2012). It affects the central nervous system of pests (Rawi et al. 2019) and acts as agonist by binding to specific insect nAChRs (Wang et al. 2011;Jeschke et al. 2013), causing prolonged activation of the receptor, which lead to symptoms of neurotoxicity (USEPA 2003) similar to the attention-deficit hyperactivity disorder (ADHD) which is responsible for behavioral deficits in children (Janner et al. 2021). Unfortunately, the use of IMI as seed treatments on some crops poses risks to small birds, and the ingestion of even a few treated seeds could cause mortality or reproductive impairment to sensitive birds (Gibbons et al. 2015;Bean et al. 2019;Sibiya et al. 2019) affecting genomic, cellular, endocrine, immunological, growth, reproductive, and neurobehavioral endpoints and can cause lethality in birds (Matsuda et al. 2001;Lopez-Antia et al. 2012Eng et al. 2017). Also, IMI can successfully cross the blood-brain barrier and bioaccumulate in animals (Campbell et al. 2022) causing damage to other tissues and organs through oxidative stress and inflammation (Duzguner and Erdogan 2010;Emam et al. 2018), hepatotoxicity, and cytotoxicity (Wankhede et al. 2017) as well as induces liver fibrosis via activation of the transforming growth factor-β1/Smad proteins (TGF-β1/Smad) signaling pathway in quails (Lv et al. 2020). Therefore, the present study was performed to evaluate the median lethal dose (LD 50 ) of IMI to the domestic Japanese quail, Coturnix coturnix japonica, as a bioindicator for environmental contaminants (Zeid et al. 2019) and recommended by the Organization for Economic Co-operation and Development as a model organism for avian toxicity studies (OECD 2010). Also, the present work was carried out to evaluate the effects of a sub-lethal dose (3.014 mg/kg = 1/25 LD 50 ) of IMI for 1, 7, 14, and 28 days every other day on certain biochemical parameters such as cholinesterases (acetylcholinesterase, AChE, and butyrylcholinesterase, BuChE), aminotransferases (alanine transaminase, ALT, and aspartate transaminase, AST), phosphatases (acid phosphatase, ACP, and alkaline phosphatase, ALP), lactate dehydrogenase (LDH), adenosine tri-phosphatase (ATPase), superoxide dismutase (SOD) glutathione s-transferase (GST), glutathione-reduced (GSH), lipid peroxidation (LPO), and genetic levels (deoxyribonucleic acid, DNA, and ribonucleic acid, RNA) in Japanese quail.

Animals' husbandry
Male domestic Japanese quail, Coturnix coturnix japonica (6 weeks old with body weight of 150-200 g), was locally bred at the Poultry Research Farm, Faculty of Agriculture, Alexandria University, Egypt. Birds were housed indoors in stainless steel cages and kept under laboratory conditions (25±2 °C, 50-70% relative humidity, and 12-h light/dark cycle) for 2 weeks before starting the experiment with unrestricted amounts of standard poultry soft started feed and water ad libitum. The experiments were conducted with the guidelines of the Institutional Animal Care and Use Committee (IACUC), Alexandria University (AU0818123105).

Toxicity studies
The medium lethal dose (LD 50 value) and the confidence limits of IMI on quail were calculated according to Weill (1952) by administering different doses of IMI suspended in corn oil, vigorously mixed, and administered directly into the stomach of quail via needle oral gavage (5 quails in each group). Group I was treated with corn oil and kept as control, while groups II, III, IV, and V were treated with doses of 60, 72, 86.40, and 103.68 mg kg −1 body weight of IMI, respectively. The number of dead animals and symptoms of acute toxicity in the treated groups was recorded (Table 1).

Biochemical studies
Thirty-two male quails were used and randomly grouped into 5 groups. Group I consisted of 12 birds and was given only corn oil and kept as a control. Groups II, III, IV, and IIV (5 birds per each group) were treated with 3.014 mg kg −1 of IMI for 1, 4, 7, and 14 days for 28 days every other day, respectively. Twenty-four hours after the experiment periods, a blood sample was collected from the basilic wing vein into non-heparinized clean tubes, left at room temperature for about 30 min, and then centrifuged at 5000 rpm for 5 min using HS-21 centrifuge (MSE). The obtained serum was kept frozen until biochemical studies. At the end of the experimental period of each dose, birds were sacrificed and dissected, and then the brains, hearts, livers, and wing muscles were rapidly removed, washed with 0.9% cold NaCl, weighed, and kept frozen until biochemical analysis. Tissue aliquots from each organ were weighed and homogenized using a homogenizer (Homogen, MSA) for 20-25 s in 10 volumes (w/v) either of ice-cold 0.1 M phosphate buffer, pH 8.0 for the determination of cholinesterases (AChE and BuChE), phosphatases (ACP and ALP), and aminotransferases (ALT and AST), ice-cold physiological sodium saline solution (0.9%) for the determination of LDH, GST, SOD, and LPO levels, liver DNA, and RNA (at a ratio of 1:50 w/v), or 10 mM tris-HCl phosphate buffer pH 7.4, 10 mM EDTA containing 0.32 M sucrose in the case of ATPase. For the determination of GSH, tissues were weighed and pulverized in 3% ice-cold of metaphosphoric acid (HPO 4 ), while the serum was diluted with 3% of cold HPO 4 . The homogenates were centrifuged at 5000 rpm for 20 min at 4 °C using HS-21 centrifuge (MSE).

Determination of biomarkers
The assessment of AChE and BuChE activities (Ellman et al. 1961), GST activity (Vessey and Boyer 1984), ATPase activity (Koch 1969), SOD activity (Marklund and Marklund 1974;Roth Jr and Gilbert 1984), GSH levels (Owens and Belcher 1965), and LPO levels (Nair and Turner 1984) were determined in serum, brain, liver, heart, and wing muscles. LDH (Bais and Philcox 1994) in serum, brain, heart, and wing muscles and liver function parameters as AST and ALT activities in serum and liver (IFCC 1986) were determined using Spectrum Diagnostic Kits, Egypt. ACP and ALP activities (Bessey et al. 1946) were carried out in serum and liver, while fresh blood glucose levels were measured immediately in time of blood collection before sacrificing quails using SD Check Gold Biosensor kit.
The extraction and determination of nucleic acids (deoxyribonucleic acid, DNA, and ribonucleic acid, RNA) were carried out according to the method of Schneider (1945) which depends on heating the tissue with 5 % trichloroacetic acid (TCA) at 90 °C after removal of phospholipids and acid-soluble phosphorus compounds. In this method, the liver was weighed, homogenized in 0.9 % cold saline solution at ratio of 1:50 (w/v), mixed gently with 5 ml of cold 10% TCA, kept in ice for 30 min, and then centrifuged for 2 min at 3500 rpm. Acid-soluble fraction was discarded, while the other fraction was treated with 10 ml of 95% ethanol for 2 min to remove lipids and then centrifuged for 2 min at 3500 rpm. The extract was treated with 5 ml of 5% TCA at 90°C for 15 min and then was centrifuged for 2 min at 3500 rpm. Pellets were separated suspended, washed by 5 ml of cold 5% TCA as previously described, and the washing was added to the hot extract for the determination of nucleic acids. The extracts were used for DNA and ribonucleic acid RNA estimations using diphenylamine and orcinol reactions at wavelengths of 595 and 665 nm, respectively. Protein contents were estimated by the method of Lowry et al. (1951).

Statistical analysis
Statistical analysis of the obtained data was carried out using two independent sample t-test and one-way analysis of variance (ANOVA) test using SPSS version 24.0 (IBM-SPSS Inc., Chicago, IL) to compare between control and the treated groups, followed by post hoc multiple comparisons test (LSD test) at the probability of 0.05. Analyses were carried out at least in triplicates. All values were calculated as the mean ± standard deviation (SD).

Toxicity of IMI against Japanese quail
Birds are a suitable pattern of hazard detection from xenobiotic and can be used as bioindicators (Adout et al. 2007). One of these popular birds is the Japanese quail, Coturnix japonica (Huss et al. 2008;Krieger 2010;OECD 2010). The reason for choosing Japanese quails as bioindicators is because they have a short sexual maturation period, short lifespan, and have high sensitivity to environmental variables (Guthery 2006). One of the objectives of the present study was to establish the acute oral toxicity (LD 50 ) of IMI for Japanese quail. The recorded dead animals were 0, 1, 2, 3, and 4/5 in I, II, III, IV, and V groups, respectively ( Table 1). The percentage of died animals at each dose was then transformed to probit using Finney's method (Finney 1952). The percentage dead for 0 and 100 were corrected before the determination of probits, where for 0% dead 100 (0.25/n), for 100% dead 100 (n − 0.25/n), and n = 5 quails (Ghosh 1984).
The value of LD 50 was found to be 75.35 mg kg −1 body weight after 24 h under laboratory conditions with confidence limits and molar lethal dose of 56.28-100.90 and 0.295, respectively (Table 2) indicating that IMI has a highly toxic effect on Japanese quails. There are many factors that influence the LD 50 values such as the experimental conditions as well as the variance between animals of the same basic strain obtained from different suppliers (Russell and Overstreet 1987). Different LD 50 values of IMI in Japanese quail were obtained by different researchers, where the values ranged from 17.02 mg kg −1 body weight (Rawi et al. 2019) to 31 mg kg −1 body weight (Tomlin 2002;EFSA 2008). The slope of the probit can be used to predict the dose at which certain proportions of birds are expected to die (Mineau and Palmer 2013). Data in Table 2 showed that the slope of the probit was > 9 which reflect that Japanese quail is at high risk.
Regarding the observed signs of toxicity in the present study, different symptoms of toxicity following the oral administration of IMI such as closed eyes, sitting on backhock, apathy, and reduced voting at low levels of doses (60-86.40 mg kg −1 ) were observed. Also, fatigue, sitting on one side, forced movement, head dropping, tremors, and finally rapid death, especially at high doses (≥104 mg kg −1 ). Ataxia, wing drop, diarrhea, opisthotonos, fluffed feather coat, wing drop, and immobility were observed in bobwhite quail after ingestion of IM (Toll 1990a(Toll , 1990b. Moreover, house sparrows, Passer domesticus, showed inability to fly and morbidity (Stafford 1991), while Mallard ducks showed moderate toxicity such as ataxia, hyperactivity, diarrhea, and immobility after oral dosing with IMI. It can be concluded that neonicotinoid insecticides such IMI, clothianidin, and thiamethoxam cause behavioral and systemic impairments (Tonietto et al. 2022) and represent a high risk for granivorous birds as found by Hancock (1996) and Gibbons et al. (2015).

Effect on cholinesterase enzymes
AChE is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system to terminate synaptic transmission, preventing continuous nerve firings at nerve endings (Blotnick-Rubin and Anglister 2018). AChE activity and its inhibition have been early recognized to be a biological marker of pesticide poisoning (Lionetto et al. 2013). The effect of 3.014 mg kg −1 of IMI on the AChE activity in male Japanese quail treated orally with different doses is depicted in Fig. 1a. The activity of the enzyme was significantly induced after all the tested doses of IMI in all the tested tissues except in hearts of quails treated with 3.014 mg kg −1 of IMI for 28 days every other day; it was inhibited by 13% of control. The higher percentages of the enzyme activity were 417, 588, 271, 200, and 253% in serum, brain, liver, heart, and wing muscles of quail after treatment with IMI for 14, 28, 14, 7, and 28 days every other day, respectively. The present results are in parallel with the results of Abou-Donia et al. (2008) who illustrated that IMI significantly increased AChE activity in plasma and brain of rats (Abou-Donia et al. 2008). From these findings, IMI acts as an agonist by binding to specific nAChRs which reflects these effects on AChE activity (Buckingham et al. 1997;Matsuda et al. 2001), causing interfering with acetylcholine (ACh) signaling and prolonged activation of the receptor. By blocking these receptors, an excess of ACh accumulates causing symptoms of neurotoxicity such as paralysis and eventual death (Zaror et al. 2008;Anderson et al. 2015). However, the inhibition of the AChE after oral exposure to IMI was recorded in chickens (Kammon et al. 2010) and rats Kumar et al. 2014;Lonare et al. 2014;Vohra and Khera 2015;Moeen et al. 2018).
BuChE is known as pseudo-cholinesterase and nonspecific cholinesterase, found in the plasma, liver, and other tissues (Wilson 2010;Singh and Dixit 2014). It catalyzes the hydrolysis of esters of choline, including butyrylcholine, succinylcholine, and ACh (Darvesh et al. 2003), and serves transmission as a backup for ACh in nerve impulse (Duysen et al. 2007). Figure 1b displays the enhancement of serum BuChE activity in Japanese quail after oral administration of 3.014 mg kg −1 for 1, 4, 7, 14, and 28 days every other day, where the activities ranged from 2.00-to 3.14-, 1.75-to 6.75-, 1.50-to 3.33-, 1.07-to 1.86-, and 1.00-to 2.60-fold the control, in serum, brain, liver, heart, and wing muscles, respectively, and the activities increased in a dosedependent manner in the case of the brain, liver, and wing muscles. However, Lopez-Antia et al. (2012) noted a significant decrease in BuChE of workers exposed to IMI, while non-inhibition by of BuChE activity was recorded in patients with IMI poisoning (Karatas 2009).

Effect of on blood glucose levels
In the toxicity studies, a variety of biochemical parameters are measured to evaluate a broad range of biomarkers affecting target organ identification and tissue injury assessment. The blood glucose levels in blood of Japanese quail treated with 3.014 mg/kg for different days for 28 days every other day are shown in Fig. 1c. Non-significant increases in the blood glucose levels were recorded when the quails were treated for 1 or 7 days every other day, while significant elevations were noticed when the quails were treated orally every other day with 3.014 mg kg −1 for 14 and 28 days, where the percentages of elevation were 25 and 42, respectively, indicating that the more exposure to IMI, the higher levels of glucose in blood were recorded. From these findings, IMI is considered a hyperglycemic inductor. It is established that IMI induced stress and disrupt glucose homeostasis (Khalil et al. 2017). The present results are in parallel with many investigators, where IMI was found to elevate the glucose levels in male Japanese quails (Kamel and Cherif 2017), chickens (Kammon et al. 2010;Balani et al. 2011;Ivanova et al. 2013), rats Khalil et al. 2017), and water fish, Labeo rohita (Qadir et al. 2014). The increase of blood glucose levels might be related to glycogen metabolism and gluconeogenesis alteration depending on the hepatic condition denoted hepatotoxicity and disturbance of the carbohydrate's catabolism (Ivanova et al. 2013).
However, Lopez-Antia et al. (2015) found that when redlegged partridges were fed on wheat seed treated with a low dose of IMI, the levels of plasma glucose reduced.

Effect on ATPase
ATPase is a key transmembrane enzyme found in all kinds of tissues and is responsible for the conversion of ATP to ADP and releasing energy (Stewart et al. 2014). It plays a vital role to control the cell volume by preventing the swelling of the cell, keeping neurons in activity or resting state, and acts role in the active transport of some metabolites such as glucose and amino acids (Barrett et al. 1989;Chakraborti and Dhalla 2016). Therefore, the measuring of ATPase activity reflects the consumed energy into the cell (Silver and Stull 1982;Hai and Murphy 1988). The present data exhibited inter-tissue differences in the ATPase activities and the reaction of each tissue towards IMI treatment, where the brain was found to contain the highest activity of ATPase followed by the heart, serum, and then the wing muscles (Fig. 1d). However, other studies revealed a reduction in the activity of ATPase in the rat brains following IMI treatment  (Lonare et al. 2014;Moeen et al. 2018). The obtained results illustrated that ATPase activities were significantly increased in serum and liver either following all the tested doses, while the brain, heart, and wing muscles showed significant increases following treatment for >7, 1-14, and 28 days every other day, respectively. The recoded inductions in the activities of ATPase in serum, brain, liver, heart, and wing muscles were higher than the control by 1.47-2.48-, 1.85-2.87-, 1.43-2.04-, 1.32-2.97-, and 3.19-fold following treatment with IMI for 1-28, 14-28, 1-28, 1-14, and 28 days every other day, respectively. The present results are in parallel with the results of Abdel-Haleem et al. (2018) who found that IMI and thiamethoxam increased the activities of ATPase in adult house flies, while the activity was inhibited in rat brain and erythrocyte following treatment with 45 and 90 mg kg −1 of IMI (Lonare et al. 2014;Moeen et al. 2018). Moreover, ATPase enzyme was found to be altered after IMI exposure at different levels depending on each tissue demand. In the present study, the levels of brain ATPase were higher than other tissues indicating that brain has massive demand for Na + /K + ATPase, since neurons rely on the pump to reverse postsynaptic Na + flux reestablishing gradient and action potential, exhausting about 75% of energy for ATPase only (Attwell and Laughlin 2001). In serum, Na + and K + gradient is related to the secondary transport facilitation for sugar, neurotransmitters, and metabolites, therefore, ATPase being indirectly responsible for the demand of glycogen to glucose conversion, explaining the arising of glucose levels in the blood of the quail. The obtained reduction in the ATPase activity in the brain and wing muscles of quails treated with IMI may be associated with neuronal damage caused by generation of excessive reactive oxygen species, ROS (Uttara et al. 2009), and defect in mitochondrial bioenergetics, which is commonly observed in neurodegenerative diseases (Kinoshita et al. 2016).

Effect on phosphatases
The cellular membrane damage has been evaluated by measuring the activity of ACP and ALP, where ACP takes part in processes such as digestion (Cheung and Low 1975), ion transport ( Fig. 2a-b illustrate that livers were found to have higher activities either of ACP or ALP than serum and the activities were significantly induced compared to the control values in a cumulative dose-dependent manner. The activities increased in case of ACP by 2-6-and 1.59-5.75-fold the control, while in case of ALP, the activities increased by 1.41-2.67-and 1.87-5.46-fold the control in serum and liver, respectively. ACP is known as tartrate-resistant acid, being a hydrolytic enzyme, and found to be correlated to cell phagocytosis and autolysis (Weber and Niehus 1961) and considered as a good marker for the metabolism of phosphorus in many tissues (Kirstein et al. 2006). Many pesticides were found to increase the levels of these enzymes (Kumari and Sinha 2010) and cause a release of some hydrolytic enzymes from lysosomes into the bloodstream (Lozano-Paniagua et al. 2021). The obtained elevated activities seem to result from enhanced enzyme turnover under the pesticide stress. The present results are in parallel with many researchers, where IMI induced ACP in birds' serum (Kammon et al. 2010;Ivanova et al. 2013) or rat liver (Vohra and Khera 2015). However, Lopez-Antia et al. (2012) established an inhibition of ALP levels in the IMI-ingested red-legged partridge, while Toor et al. (2013) found a non-significant decrease in ALP activity of rats. It can be concluded that any changes in the ACP and ALP activities may reflect the damage to the cell membrane.

Effect of IMI on aminotransferases
Metabolic disorders are correlated to enzyme system failure caused by the nutritional, environmental, and some factors as feed intoxication (Senanayake et al. 2015). Therefore, one of the objectives of this study was to understand how either serum or liver enzyme levels of Japanese quails altered during the exposure to sublethal doses of IMI under different numbers of doses over 28 days of the study. ALT and AST belong to enzymes that catalyze the transferring of amino group from amino acids to oxo-acids and are common markers of liver injury. The determinations of serum enzyme activities were established as a routine tool in the diagnosis of avian diseases (Sakas 2002). A significant induction of ALT and AST enzymes either in serum or livers of Japanese quail after oral dosing of 3.014 mg kg −1 of IMI for all the tested doses was recorded and the induction was in a dose-dependent manner ( Fig. 3c-d). The recorded inductions in serum ALT activities were 3.84, 8.55, 13.97, and 15.32, while in case of serum AST, the recorded values were 1.76-, 2.61-, 3.79-, and 4.01-fold the control in quails treated for 1, 7, 14, and 28 days every other day, respectively. In case of liver ALT, the activities increased by 2.10-, 3.03-, 3.90-, and 5.78-fold, while the activities of liver AST increased by 1.63-, 2.87-, 3.87-, and 6.64-fold the control after treatment with IMI by the same regimen, respectively. The present results are in parallel with many studies, where ALT and AST liver levels were significantly raised in the liver of quail (Lv et al. 2020), chickens (Kammon et al. 2010), fish, Labeo rohita (Qadir et al. 2014), rats (Soujanya et al. 2013;Vohra and Khera 2015), and mice  when treated with IMI. The higher enzyme activity of ALT and AST refers to the acute liver failures (Orlewick and Vovchuk 2012). Therefore, the increases in the activities ALT and AST in treated quail might be responsible for the hepatic damage. Many studies indicated hepatotoxicity and alteration in the activity of aminotransferases in quails exposed to neonicotinoids (Eissa 2004;Ertl et al. 2018;Lv et al. 2020) because they were found to be accumulated in livers of quails (Eissa 2004;Ertl et al. 2018) resulting in the induction of liver fibrosis in quail (Lv et al. 2020). Also, it was reported that hepatic alteration was recorded in bobwhite quail (Toll 1990b), red-legged partridge (Lopez-Antia et al. 2015), and chickens (Salvaggio et al. 2018) exposed to neonicotinoids. It can be concluded that the elevation in the activities of AST, ALT, ALP, and ACP indicate the utilization of amino acids for the oxidation or glucogenesis and can be used to determine liver, heart, and skeletal muscle dysfunction (Kumari and Sinha 2010).

Effect of IMI on glutathione S-transferase
Birds are usually exposed to a wide spectrum of environmental stressors; therefore, birds have an efficient antioxidant enzymatic system such as GST which belongs to phase II enzymes to detoxify xenobiotics (Dasari et al. 2018). Data in Fig. 3a illustrate that tissues of quails had different activities of GST and arranged as the following: liver > wing muscles >heart > brain > serum. The activities of GST in all the tested tissues were increased, and the increases were in a cumulative dose-dependent manner in all the tested tissues except serum. The recoded activities were higher than the control by 1.95, 5.68, 7.03, and 1.08 in serum; 1.78, 2.26, 5.25, and 9.75 in the brain; 1.52, 1.73, 2.03, and 2.65 in the liver; 1.34, 1.48, 1.73, and 2.92 in the wing muscles; and 1.36, 4.37, 3.33, and 1.99-fold in the heart following dosing with 3.014 mg kg −1 IMI for 1, 7, 14, and 28 days every other day, respectively. Pigeons were found to have different GST activities in different tissues and arranged as follows: kidney> liver> testes> brain> lung> heart (Kumar et al. 1980). The present results are in parallel with the results of many investigators, where IMI significantly elevated the GST activity in mice (El-Gendy et al. 2010) and rat (Moeen et al. 2018) suggesting that this pesticide exerts its toxic effect by the induction of oxidative stress and this enzyme is involved in the detoxification of imidacloprid (Yang et al. 2021). However, when male rats were orally administered with IMI, a non-significant decrease in the brain GST activity was recorded following 4 weeks of treatment (Lonare et al. 2014).

Effect of IMI on superoxide dismutase of Japanese quail
SOD is a vital antioxidant enzyme having a role in the mitigation of exhausting state by decreasing stress, motivating apoptosis, and protecting lipids in avian tissues (Surai 2016). The tissues of quail were found to have different activities of SOD and arranged as follows: brain> liver> wing muscle> serum> heart (Fig. 3b). Serum SOD activity showed significant induction after dosing with 3.014 mg kg −1 of IMI for 1, 7, and 14 days every other day, where the recorded percentages of induction were 130, 137, and 119 of control, respectively, while the activity showed significant inhibition by 36% after dosing with IMI for 28 days every other day. Brain SOD activity showed inhibition in the activity when dosing for 1 and 7 days every other day, while quails dosed either for 14 or 28 days every other day showed significant and nonsignificant increases with percentages of activities equaled to 155 and 106 of control, respectively (Fig. 3b). Also, the data showed that liver SOD activities were induced after a single dose by 32%, while the activities were inhibited by 16, 41, and 50% when quails were orally dosed with IMI for 7, 14, and 28 days every other day, respectively. In case of the heart, the SOD activity was increased by 20 and 50% after dosing with IMI either for 1 or 7 days every other day, while the activities were reduced by 13 and 85% after dosing for 14 and 28 days every other day, respectively. In case of wing muscles SOD, the activities were increased in a cumulative dose-dependent manner and the recoded increases ranged from 1.08-to 1.47-fold the control. The present results are in parallel with the study of Duzguner and Erdogan (2010), Lopez-Antia et al. (2015), and Ali et al. (2018) who recorded stimulation of SOD activities in birds following either IMI or acetamiprid treatments. However, SOD activities were inhibited in different tissues of quails (Lopez-Antia et al. 2012;Lv et al. 2020) and rats Lonare et al. 2014;Moeen et al. 2018). It can be concluded that IMI produces cytotoxicity by triggering the ROS and alters the balance of the antioxidant system (Banaee 2013) inducing apoptosis (Li et al. 2022).

Effect of IMI on lactate dehydrogenase
LDH enzyme is a tetrameric protein and widely distributed in the heart, liver, muscle, and kidney catalyzing the conversion of lactate to pyruvate in the presence of NADH within the glycolysis from anaerobic to aerobic respiration (Schumann et al. 2002). The results showed that different tissues of quail had different activities of LDH and the activities were increased in a cumulative dose-dependent manner in case of serum, liver, and wing muscles (Fig. 3c). The obtained LDH activities were higher than the control by 1. 76, 2.82, 3.05, and 3.24 in serum; 1.66, 2.04, 2.44, and 4.01 in the liver; and 2.01, 2.22, 2.74, and 4.75-fold in the wing muscles, when quails were treated for 1, 7, 14, and 28 days every other day, respectively. In case of the heart, the LDH activity increased after oral IMI administration for 1, 7, and 14 days every other day and then slightly reduced to 95% of control after treatment for 28 days every other day. Many studies established the induction of LDH due to IMI exposure as obtained in the present study. It was reported that LDH was significantly raised in male rats (Lonare et al. 2014) and water fish, Labeo rohita (Qadir et al. 2014). The increased LDH activity in the quails exposed to IMI may be due to cellular hypoxia and damage to hepatopancreatic cells, where the energy demands are met by anaerobic respiration and related with the increase of LDH activity (Kumari and Sinha 2010). However, a significant depletion in plasma LDH was established in red-legged partridge fed on seeds treated with IMI (Lopez-Antia et al. 2015). Under the physiological and pathological conditions, the occurrence of an energetic metabolism dominated by the glycolytic generation of lactate can be associated with a decrease of intracellular pH which linked to hypoxic glycolysis and ATP consumption and could trigger a parallel decrease in the activity of LDH, depressing in turn the energetic potential of lactate generation (Iacovino et al. 2022). The changes in the LDH activity in quail treated with IMI may be due to severe cellular damage, leading to an increase in the release of LDH that impaired carbohydrate and protein metabolism (Sivakumari et al. 1997). LDH is considered one of the most important enzymes involved in the anaerobic metabolism of carbohydrates under stress conditions, and the increase in LDH activity is considered as a physiological mechanism for energy supply to cope with the toxic effects of pesticides . Therefore, LDH can be used as an indicator for cellular damage, clinical practice, cytotoxicity, and a marker of common injuries and diseases such as heart failure (Stambaugh and Post 1966).

Effect of IMI on lipid peroxidation levels
ROS are produced either endogenously within metabolism processes or by exogenous xenobiotics such as pesticides and can affect the cell components, especially biological molecules containing a lipid component of polyunsaturated fatty acids (Phaniendra et al. 2015) as well as affect the scavenging enzyme system (Etemadi-Aleagha et al. 2002). The production of the free radicals was found to occur permanently in cells as a result of both enzymatic and nonenzymatic reactions (Abdollahi et al. 2004). Because ROS cause LPO to the cellular membrane and then cell death as a result of polyunsaturated fatty acid oxidation (Aydemir et al. 2000); therefore, the malondialdehyde (MDA) estimation is considered as a vital biomarker of LPO under conditions of cellular stress (Zhang et al. 2004;Osman et al. 2021). Data in Fig. 3d show that different tissues of quail had different levels of LPO as follows: liver > heart > wing muscles> serum> brain, and these levels were increased in a cumulative dose-dependent manner in serum, brain, and wing muscles post-IMI treatment. The recorded levels were higher than the control by 1.90, 4.51, 6.3,1 and 16.13 in serum; 1.27, 2.20, 2.61, and 4.05 in the brain; 1.27, 1.70, 2.09, and 2.16 in the liver; 2.56, 3.52, 3.73, and 4.47 in wing muscles; and 1.12, 1.84, 2.10, and 1.98-fold the control in the heart when quails were treated with IMI for 1, 7, 14, and 28 days every other day, respectively. The results of the present study are matching with previous results, where neonicotinoids such as IMI and acetamiprid can induce the production of LPO levels in the liver of the treated birds (Sasidhar-Babu et al. 2014), female rats Erdogan 2010, 2012;El-Gendy et al. 2010;Kapoor et al. 2010;Bal et al. 2012;Moeen et al. 2018), and male mice . The increased levels of lipid peroxidation are related to glycolipid metabolism disorders, loss of integrity of the cell membrane, and increase energy demand (Dornelles and Oliveira 2014). However, IMI failed to change the LPO levels in the liver of red-legged partridge fed on either low (0.7 mg/g) or high (1.4 mg/g) doses treated wheat seeds for 10 days (Lopez-Antia et al. 2012). In the present study, it can be concluded that IMI may alter the antioxidant balance and induces the production of LPO and inflammation in the tissue of male Japanese quail.

Effect of IMI on glutathione-reduced content of Japanese quail
GSH is a non-enzymatic antioxidant found in cells and acts as a substrate for several metabolic enzymes that mitigate or restrain the adverse effects of free radicals (Hayes and Mclellan 1999;Banerjee et al. 2001;Lushchak 2012). The potential effect of IMI on the levels of GSH in Japanese quails showed a significant reduction in the GSH contents in all the tested tissues with levels of 94-61, 84-31, 84-53, 89-61, and 74-52% of the control in serum, brain, liver, heart, and wing muscles of quails, after dosing with IMI for 1-28 days every other day, respectively (Fig. 4a-b). The obtained results are in parallel with those reported by many investigators, where IMI reduced the levels of GSH in layer chickens (Sasidhar-Babu et al. 2014), red-legged partridge (Lopez-Antia et al. 2015), and rat (Bal et al. 2012;Duzguner and Erdogan 2012;Soujanya et al. 2013). The reduction in GSH levels in the present study indicated that quails suffered from serious damage due to reduction in their antioxidant capacity and could be related to the use of GSH itself in the metabolic processes and to compensate the increased production of free radicals ) and scavenge ROS which recovered again due to the inhibition of glutathione reductase by IMI (Halliwell and Gutteridge 2015). Therefore, GSH can be used as an important indicator of oxidative stress (Osman et al. 2021).

Effect of IMI on liver nucleic acids (DNA and RNA)
The determination of nucleic acid concentration is considered a biomarker of xenobiotic exposure, where a frequency of apoptosis and fragmentations of DNA in the IMI-treated animals were recorded (Bal et al. 2012). The effect of 3.014 mg kg −1 of IMI on the levels of DNA and RNA in livers of quails after dosing for different days is depicted in Fig. 5. It was noticed that IMI significantly decreased the DNA and RNA levels in a dose-dependentmanner, where the recorded percentages of DNA were 92, 79, 75, and 71 of control, while the percentages of RNA were 81, 29, 20, and 11 of control after treating quails with IMI for 1, 7, 14, and 28 days every other day, respectively. IMI was found to decrease DNA and RNA in mice (Prasanna and Vardhani 2013;Kumar et al. 2014) and cause DNA damage in fish (Vieira et al. 2018). The obtained depletion in DNA and RNA levels in the present study may be due to the genotoxic action of IMI as reported by many studies (Feng et al. 2005;Karabay and Oguz 2005;Demsia et al. 2007;Costa et al. 2009). It is well known that genetic responses to oxidative stress are occurring in bacteria, yeast, and mammalian cell lines (Farr and Kogoma 1991;Hidalgo and Demple 1995). The damaged or impaired DNA was suggested as a reason for the reduction of DNA level (Blasiak et al. 1990) resulting in the apoptotic induction (Awasthi et al. 1984). The impairment in the synthesis of the protein which mediate DNA repair and known as Ku protein because of ROS or nitrogenous species could lead to DNA strand breaks, where the oxyradicals have been found to act as chemical nucleases on DNA, resulting in an ultimate DNA strand breakage (Sodhi et al. 2008;Celik et al. 2009). The DNA damage can be an important event able to influence the survival, breeding, and perpetuation of avian species, because it makes them more susceptible to environmental variations (Gomes de Faria et al. 2018). Therefore, DNA Fig. 4 In vivo effect of 3.014 mg kg −1 of IMI on GSH contents in serum (a) and brain, liver, heart, and wing muscles (b) of quail treated orally for different days for 28 days every other day. Means having the same letter(s) are not significantly different from each other p≤ 0.05, n= 5 in each group

Conclusion
Pesticides not only affect pests but also affect non-target organisms like wild birds. Because IMI was found to be highly toxic to avian; therefore, the present study aimed to assess the median lethal dose and the sublethal effect the most widely applied insecticide, IMI, to crops in the Egyptian fields on certain biomarkers of the Japanese quail as a bioindicator for xenobiotic detection. Based on the obtained results, IMI is highly toxic to quail and can cross the bloodbrain barrier and alters the levels of some chemical and genetic parameters of the quail leading to neurotoxicity, hepatotoxicity, oxidative stress, and genotoxicity in quail. The changes in these biomarkers can be used to detect the toxic effects of imidacloprid on quail as a wild avian.