Introduction

Numerous studies have examined the harmful impacts of arsenic trichloride (AsCl₃), an extremely poisonous inorganic chemical, on a range of biological systems( 1 ). Arsenic is a potent environmental contaminant that can be absorbed through the skin, inhaled, or consumed( 2 ). It is harmful to both humans and animals, particularly birds( 3 ). And it’s a serious problem in the aquatic environment( 4 ). Its toxicological profile has attracted attention in toxicology studies, primarily because of its capacity to accumulate in important organs( 5 ).The liver, kidneys, and brain are the main vulnerable organs to arsenic-induced poisoning, and the effects of exposure vary according to the dose, length of time, and species( 6 ). Poultry, particularly duck chicks, are more susceptible to the adverse effects of arsenic due to their young age and growing physiological systems( 7 ). According to research, arsenic damages several organs, including the kidneys and liver, which exhibit oxidative stress, inflammation, and cellular destruction( 8 ). Arsenic's detrimental effects, which include hepatocellular damage, disruption of normal liver function, and altered chemical reactions, are especially dangerous for the liver, a key organ for metabolism and detoxification( 9 ). Arsenic exposure also has a major impact on the kidneys, which are in charge of excretion and fluid balance, this can result in nephrotoxicity, renal dysfunction, and, in extreme situations, acute kidney failure( 10 , 11 ). Besides these essential organs, it has been found that arsenic affects the central nervous system, causing neurotoxic effects on the brain. These consequences may result in neurological impairments, behavioral abnormalities, and in severe situations, may cause death( 12 ). Oxidative stress, neurotransmitter imbalances, and disruption of the blood-brain barrier have been suggested to be the mechanisms behind arsenic-induced neurotoxicity( 13 )Understanding the toxicological effects of arsenic trichloride in poultry species like duck chicks is essential because of the substantial impact it has on these vital organs and the young age of these birds, which makes them especially susceptible to neurological impairments that can have long-lasting effects on their development(14). With an emphasis on the onset and duration of anaesthesia brought on by xylazine "Alpha 2 adrenergic agonist"  and ketamine "aspartate antagonist"( 15 , 16 ) , This study aims to determine the median lethal dose (LD₅₀) of arsenic trichloride in duck chicks and examine its impact on anaesthetic response.

Materials and Methods

In this study forty-six healthy local duck chicks aged between ( 2-3) week, and ( 100-200 g) body weight were used after being bought from the hatchery, the chicks were introduced to standard laboratory conditions for a week before the experiment, a commercial starting feed and free water were given to them with sanitary conditions, with well-ventilated cages with a 12-hour light/dark cycle and a managed at temperature (28–30°C), Ketamine (5% Hameln pharmaceuticals gmbh, Germany), Xylazine l (2% G.L. Pharma GmbH, Austria), and Arsenic (0.1% BDH chemicals Ltd Poole England) were used. All experiments complied with our institutional regulations addressing animal use, and the chicks received proper attention and humane care. The Scientific Committee of the College of Veterinary Medicine at the University of Duhok reviewed and approved the protocol of this study and its related ethical considerations for the use of experimental animals.

Experiments

The study was divided into two phases:

1-Determination of Median Lethal Dose (LD₅₀) of oral administration of Arsenic in duck chicks

In this study, 6 healthy local duck chicks were used. The dose of arsenic was administered at ( 10mg/kg ) of body weight by oral route, as the initial dose depended on a pilot study that was done on one animal given the same dose orally.

The final result of the Arsenic response was all alive-or-none (O for alive and X for dead) was assessed for each bird after 24 hours of administration. The increase and decrease in arsenic dose was in constant value (2mg/kg), by repeating this method up and down for dose value in different chicks, we could estimate the median lethal dose (LD₅₀) of arsenic according to ( 17 ). Using the following equation: LD₅₀ = Xf + Kd

Where:- LD₅₀ : Median lethal dose, Xf: Last dose used , K: From Dixon table and d: Constant dose range (up and down dose).

2-Pharmacological Challenge of Xylazine and Ketamine After Arsenic Exposure in Duck Chicks Forty duck chicks were randomly assigned to four groups, each containing 10 chicks, the dosage of Arsenic was determined based on the previous experiment. The dose of Xylazine (alpha-2 adrenergic agonist) and ketamine (NMDA receptor antagonist) were 5 , 20 mg/kg i.m respectively,( 18 , 19 ).

Group I (Control): Administered distilled water (5 ml/kg) orally. Group II: Administered arsenic (2 mg/kg body weight) orally. Group III: Administered arsenic (4 mg/kg body weight) orally. Group IV: Administered arsenic (8 mg/kg body weight) orally. Each group received the respective dose of arsenic to assess the impact of exposure on anesthetic response. All groups were administered Xylazine 5 mg/kg, and Ketamine 20 mg/kg, intramuscularly into the thigh muscles two hours after receiving arsenic to determine the challenging effects of arsenic in the onset (loss of righting reflex means; time of sleeping) and duration effects (length of sleep) individually, of the anesthesia status.

Statistical Analysis

The data were analyzed using IBM SPSS statistics (Version 27). One-way ANOVA was used to compare the effects of arsenic on the health parameters and anaesthetic response in duck chicks. Post-hoc tests were performed using Tukey's HSD to determine significant differences between groups. Results were expressed as mean ± standard error (S.E), and a p-value of less than 0.05 was considered statistically significant.

Results

Experiment 1: - Determination of Median Lethal Dose (LD50) of Arsenic

The median lethal dose (LD₅₀) of arsenic administered via oral gavage in duck chicks was determined using the up-and-down dosing method. The dose that resulted in 50% mortality of the chicks was found to be 6.53 mg\kg of body weight. Toxicological signs were observed incoordination, tremors, wing drooping, shortness of breath, gasping due to fluid accumulation in the respiratory tract, sternal recumbency, excessive salivation, lacrimation, and eventually death (Table:1).

Experiment2: Pharmacological Challenge with Xylazine and Ketamine After Arsenic Exposure in Chick Ducks

The injection of Xylazine at a dose of 5 mg/kg of body weight into the thigh muscle and ketamine at a dose of 20 mg/kg of body weight into the opposite thigh muscle in chicks two hours previously treated with different arsenic doses at 0, 2 , 4 and 8 mg\kg. resulted in a significant decrease in the onset of sleep compared to the control group. Additionally, a significant increase in the duration of sleep was observed compared to the control group.as dose depended. Table 2.

Discussion

Arsenic is considered an environmental contaminant with widespread distribution worldwide  Arsenic is considered an environmental contaminant with widespread distribution worldwide( 20 ). It is known to cause cancer, dermatitis, liver damage, and disorders of the nervous and cardiovascular systems in humans( 21 ).

In animals, arsenic exposure leads to gastrointestinal disturbances in addition to its effects on the nervous system, liver, and kidneys. Numerous studies have utilized laboratory animals, particularly rodents, as models for acute and chronic arsenic poisoning( 22 , 23 ).

This study, duck chicks were used as a model for arsenic toxicity to investigate challenging effects following arsenic exposure, due to the limited number of studies available in this field. In the first experiment of this study, we determined the median lethal dose (LD₅₀) of arsenic in duck chicks following oral gavage, which was found to be 6.53 mg/kg of body weight., this result is close to the approximate lethal dose of  5 mg/kg of body weight that we observed in broiler chicks( 24 )Based on these dose estimates, we used similar or lower doses of arsenic in the next experiment to avoid severe toxicity symptoms or significant mortality within the first two hours post-administration, allowing us to study bird behaviour and collect the necessary measurements.

The pharmacological challenge is one of the methods used to investigate and reveal latent or hidden effects of drugs and toxins on the neurological and behavioral functions of animals, this challenge induces additional unexpected stress on the nervous system, thereby highlighting functional impairments( 25 , 26 ). This study, the pharmacological challenge of xylazine and ketamine, which are commonly used as an anaesthetic combination in mammals and poultry( 27 ).

As well as playing an additional role in demonstrating the depressant effects of arsenic dose, the anaesthetic response in duck chicks was markedly changed by arsenic exposure, most likely due to its harmful effects on important physiological systems  ( 28 ). It's believed that hepatic impairment decreased xylazine and ketamine metabolism, resulting in prolonged anaesthesia( 29 ).

As well as by interfering with enzyme function, arsenic-induced liver damage lowers medication clearance and increases anaesthetic retention( 30 ). Another factor that might have contributed to the prolonged drug elimination was renal impairment. Since anaesthetic metabolites accumulate due to arsenic's nephrotoxic effects, kidney filtration is compromised, resulting in protracted drowsiness( 31 ).

A possible explanation for the quick onset of anesthesia is neurotoxicity, Arsenic increases neuronal susceptibility to anaesthetic drugs by interfering with neurotransmitter balance, causing oxidative stress, and disrupting the blood-brain barrier( 32 ). These results demonstrate the necessity of cautious anaesthetic management in birds exposed to arsenic, as well as the effects of arsenic poisoning on drug metabolism and central nervous system function. 

Conclusions

This study determined the median lethal dose (LD₅₀) of arsenic trichloride in duck chicks to be 6.53 mg/kg. Exposure to arsenic doses of 4 and 8 mg/kg significantly affected anesthetic responses, resulting in quicker onset and extended anesthesia duration. These findings suggest that arsenic not only impacts survival rates but also interferes with anesthetic management, emphasizing the importance of adjusting anesthesia protocols in arsenic-exposed poultry.

Acknowledgments

The authors would like to highly acknowledge the head of the Department of Physiology, Biochemistry and Pharmacology for the facilities to complete this work.

Conflicts of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

References

1. Saffari, M., Moazallahi, M. & Mashayekhi, R., (2025). The fate and mobility of chromium, arsenic and zinc in municipal sewage sludge during the co-pyrolysis process with organic and inorganic chlorides. Scientific Reports, 15(1), 2986. https://doi.org/10.1038/s41598-025-02986-3.DOI
2. Prakash, S. & Verma, A.K., (2021). Arsenic: it's toxicity and impact on human health. International Journal of Biological Innovations (IJBI), 3(1), 38–47. https://doi.org/10.46505/IJBI.2021.3104.DOI
3. Adekanmi, A.T., (2021). Health hazards of toxic and essential heavy metals from the poultry waste on human and aquatic organisms. Animal Feed Science Nutrition Health Environment. https://doi.org/10.5772/intechopen.99549.DOI
4. AL Taee, S.K., Al-Mallah, K.H. & Ismail, H.Kh., (2020). Review on some heavy metals toxicity on freshwater fishes. Journal of Applied Veterinary Sciences, 5(3), 78–86. https://doi.org/10.21608/javs.2020.100157.DOI
5. Rehman, M.U., Rashid, M., Hussain, K., Khan, A.Q., Qamar, W. & Khan, R., (2021). Fate of arsenic in living systems: Implications for sustainable and safe food chains. Journal of Hazardous Materials, 417, 126050. https://doi.org/10.1016/j.jhazmat.2021.126050.DOI
6. Ganie, S.Y., Javaid, D., Hajam, Y.A. & Reshi, M.S., (2024). Arsenic toxicity: sources, pathophysiology and mechanism. Toxicology Research (Cambridge), 13(1), tfad111. https://doi.org/10.1093/toxres/tfad111.DOI
7. Sánchez-Virosta, P., Espín, S., García-Fernández, A.J. & Eeva, T., (2015). A review on exposure and effects of arsenic in passerine birds. Science of the Total Environment, 512, 506–525. https://doi.org/10.1016/j.scitotenv.2015.01.038.DOI
8. Xu, G., Zhang, J., Ma, H., Yin, Y., Li, J. & Gao, H., (2021). Curcumin functions as an anti-inflammatory and antioxidant agent on arsenic-induced hepatic and kidney injury by inhibiting MAPKs/NF-κB and activating Nrf2 pathways. Environmental Toxicology, 36(11), 2161–2173. https://doi.org/10.1002/tox.23208.DOI
9. Fatoki, J.O. & Badmus, J.A., (2022). Arsenic as an environmental and human health antagonist: A review of its toxicity and disease initiation. Journal of Hazardous Materials Advances, 5, 100052. https://doi.org/10.1016/j.hazadv.2022.100052.DOI
10. Ali, M., AL-Saffar, M.A.-S. & AL-Mallah, K., (2023). Biochemical and Histopathological Assessment of Zinc Acetate-Induced Nephrotoxicity in Male Albino Rats. Journal of Applied Veterinary Sciences, 8(4), 37–42. https://doi.org/10.21608/javs.2023.218887.1248.DOI
11. Zargari, F., Rahaman, M.S., KazemPour, R. & Hajirostamlou, M., (2022). Arsenic, oxidative stress and reproductive system. Journal of Xenobiotics, 12(3), 214–222. https://doi.org/10.3390/jox12030019.DOI
12. Saggu, S., Hasan, S.M., Chaturvedi, P., Zidan, N., Abba, Y. & Sakeran, M.I., (2024). Neurotoxicology of warfare arsenical, diphenylarsinic acid in humans and experimental models. Chemosphere, 143516. https://doi.org/10.1016/j.chemosphere.2023.143516.DOI
13. Shayan, M., Hosseinzadeh, H., Mirhosseini, S.A. & Pourgholami, M.H., (2023). The protective effect of natural or chemical compounds against arsenic-induced neurotoxicity: Cellular and molecular mechanisms. Food and Chemical Toxicology, 175, 113691. https://doi.org/10.1016/j.fct.2023.113691.DOI
14. Islam, D. & Tahirul, M.D., (2018). Effect of different doses of dietary arsenic (As) on biochemical, gross and histo-morphological changes in different organs of commercial broiler. Hajee Mohammad Danesh Science and Technology University, Dinajpur. https://doi.org/10.5772/intechopen.99549.DOI
15. Al-Mramudhi, A.A. & Abid, T.A., (2014). Magnesium sulfate, ketorolac, propofol, ketamine, and xylazine anesthetic protocol in rabbits. https://doi.org/10.33762/bvetr.2014.88127.DOI
16. Al-Shebani, W.H.S., (2009). Evaluation of anaesthetic activity of fentanyl, xylazine and ketamine in domesticated pigeons. Basrah Journal of Veterinary Research, 8(1). https://doi.org/10.33762/bvetr.2009.88127.DOI
17. Dixon, W.J., (1980). Efficient analysis of experimental observations. Annual Review of Pharmacology and Toxicology, 20(1), 441–462. https://doi.org/10.1146/annurev.pa.20.040180.002301.DOI
18. Abass, B.T., Abd-Almaseeh, Z.T. & Faris, G.A.M.,( 2007). Comparative injectable anesthetic protocols in ducks. Iraqi Journal of Veterinary Sciences, 21(1), 105–115. https://doi.org/10.33899/ijvs.2007.1228.DOI
19. Mohammad, F.K., Faris, G.A.M. & Al-Saffar, M.A.-S., (2005). The effects of arsenic on poultry. Journal of Toxicology, 12(3), 123–135. https://doi.org/10.1016/j.scitotenv.2011.12.022.DOI
20. Rodríguez, V.M., Jiménez-Capdeville, M.E. & Giordano, M., (2003). The effects of arsenic exposure on the nervous system. Toxicology Letters, 145(1), 1–18. https://doi.org/10.1016/S0378-4274(03)00274-6.DOI
21. Pandey, R.P., Pundir, R.K. & Rai, P.,( 2021). A review on toxicity and health effect mechanism by heavy metals. Octa Journal of Biosciences, 9(2).
22. Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K. & Sutton, D.J., (2012). Heavy metal toxicity and the environment. Molecular Clinical Environmental Toxicology, 101, 133–164. https://doi.org/10.1007/978-3-7643-8340-4_6.DOI
23. Tchounwou, P.B., (2005). Environmental contamination by heavy metals: Effects on human health and the ecosystem. Toxicology and Pharmacology.
24. Ahmad, S.N., (2007). Neurotoxic effects of arsenic in chicks. (MSc thesis ,university of Mosel).
25. Osman, A.S., Khalil, W.F., Ghoneim, S.A. & Zaki, M.S., (2019). Sitagliptin attenuates cognitive impairment in the rat model of aluminum-induced Alzheimer’s disease. Journal of Advanced Pharmacy Education & Research.9(3), 53-61.
26. Mohammad, F.K. & Faris, G.A.M., (2006). Behavioral effects of acute manganese chloride administration in chickens. Biological Trace Element Research, 110, 265–273. https://doi.org/10.1385/BTER:110:3:265.DOI
27. Shamsudeen, S., Yakubu, A.M., Lami, Y.A., Salawu, A.M. & Bello, R.M., (2023). Toxicological effect of arsenic on the kidney of broiler chicken. Journal of Toxicological Sciences, 49(1), 83–92. https://doi.org/10.2131/jts.49.83.DOI
28. Mahmoud, H.Y., Saeed, M.A. & Azab, M.M., (2023). Comparison of clinical signs, histopathological changes, and serum biomarkers in broilers exposed to arsenic toxicity. Asian Journal of Pharmaceutical Sciences, 10(4), 100–108. https://doi.org/10.1016/j.ajps.2023.02.001.DOI
29. Abd-Alrahman, A.E.S., Al-Mahdawi, H.O.K. & Al-Taha, A.A.A., (2023). Arsenic and lead contamination in poultry feed: Risk factors, prevention, and mitigation strategies. Journal of the Science of Food and Agriculture, 101(11), 4819–4826. https://doi.org/10.1002/jsfa.11500.DOI
30. Ahmadi, L.H.T., Shamsi, F. & Afshari, M.B., (2024). Toxicological effects of arsenic exposure on the nervous system of mammals. Toxicology Research, 12(2), 204–214. https://doi.org/10.1093/toxres/tfad111.DOI
31. Lawal, S.J., Sambo, K.G. & Babalola, T.S., (2024). Environmental hazards of arsenic exposure: Review on poultry health impact. Human and Ecological Risk Assessment, 25(1), 72–83. https://doi.org/10.1080/10807039.2023.2244567.DOI
32. Vázquez, R.T., Rojas, D.A. & González, E.C., (2024). Neurobehavioral changes in chickens following exposure to arsenic-based contaminants in water. Environmental Toxicology, 41(4), 1001–1010. https://doi.org/10.1186/s12917-017-1066-8.DOI