AMELIORATIVE POTENTIAL OF Moringa oleifera LEAF EXTRACT AGAINST ARSENIC TOXICITY IN Labeo rohita

F. Khalid¹, H. Azmat¹, N. Khan² and Saima³

¹Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore, Pakistan.

²Institute of Zoology, University of the Punjab, Lahore, Pakistan.

³Department of Animal nutrition, University of Veterinary and Animal Sciences, Lahore, Pakistan.

Corresponding author’s email address Hamda.azmat@uvas.edu.pk .

ABSTRACT

Arsenic (As) is one of the most harmful pollutants in water bodies which accumulate in animals and bio magnify from lower trophic level to higher trophic level causing imbalance in physiological phenomenon, leading to retarded growth and mortality. Fish is an important resource of healthy protein and poly-unsaturated fatty acids for human diet, it must be free from contaminants and metal toxicants. However, the presence of various metalloids like arsenic in the aquatic environment significantly impart change in the fish meat quality making it unfit for human consumption and overall quantity of fish meat production is affected due to the presence of sufficient amount of arsenic in the water bodies. Therefore, its elimination becomes a global challenge. Moringa oleifera (M. oleifera), a medicinal plant containing several pharmacological properties, was evaluated for ameliorating adverse effects of sub-lethal concentration of arsenic (1/ 3^rd of 96 h LC₅₀ = 6.75 mgL^-1) in Labeo rohita. For this purpose, acclimatized individuals of Labeo rohita were randomly allocated to six experimental glass aquaria in triplicates. The experimental fish were exposed to arsenic alone and in a combination with 2 and 4 % M. oleifera leaves extract for 28 days. Results of current study revealed that immune biomarkers such as total protein, albumin and globulin contents remarkably (p ≤ 0.05) lowered on arsenic exposure. Moreover, upon arsenic exposure red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly (p ≤ 0.05) decreased, whereas white blood cells (WBC) mean corpuscular volume (MCV) and platelets significantly increased. Conversely, fish treated with 2% or 4% M. oleifera leaf extract showed significant improvement and normalized the immune and hematological alteration in Labeo rohita with respect to time and dose dependent manner. The results of present study thus concluded that arsenic induced immunological and hematological alterations were ameliorated by the M. oleifera leaves extract supplementation. Moreover, 2% or 4% M. oleifera leaf extract supplementation both ameliorate the arsenic induced toxicity but 4% M. oleifera leaf extract supplementation more significantly ameliorate arsenic induced toxic effect.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Arsenic, a toxic metalloid element, present in many ecosystems across the world, including China, Pakistan, Chile, India, Mexico, Argentina, Taiwan, Bangladesh, Mongolia, Japan, Poland, Vietnam, Nepal, and the United States (Ali, 2018; Basheer, 2018). Globally, arsenic contamination in water resources ranged from 0-360mgL^-1 (Shaji et al., 2021). Arsenic contamination in Pakistan groundwater resources has been documented in many areas including the Kalalanwala area near Lahore, Punjab (Farooqi et al., 2007), Muzaffargar (10-906 µg L^-1) (Nickson et al., 2005), Mailsi (11-828 µgL^-1) (Rasoola et al., 2016) and Tharparkar (12-812 µgL^-1) (Brahman et al., 2013). Jabeen and Javed 2011 reported arsenic contamination in water and bed sediments of Ravi River ranged from 19.20 to 21.47 mgL^-1, 24.92µg/g to 34.70µg/g, respectively. Arsenic levels that are too high are extremely dangerous for both the environment and human health. Because of the severe toxicity of As even at low concentration the USEPA set a limit of 150 µg L^-1 for chronic exposure to aquatic organisms (USEPA 2002).

Fish exposed to environmental toxins often show various kinds of immune, growth, histological and blood chemistry disturbances (Ramesh et al., 2014). Fish blood exhibits the early effects of arsenic poisoning as it is mostly absorbed through the large gill surface area, where there is a very weak barrier between the metal salt and blood as well as through the buccal cavity (Rabbane et al., 2022). Acute toxicity on fish blood is also caused by many other metals like mercury, nickel and chromium, and synthetic pyrethroids such azodrin, mancozeb, cypermethrin and fenvalerate (Kumar and Banerjee, 2016).

Hematological indices like red blood corpuscles (RBCs), hemoglobin (Hb), white blood cells (WBC), packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH) have frequently been used to determine the blood ability to carry oxygen (Shah and Altindag, 2004). They also play a significant role in environmental monitoring of pollutants in aquatic ecosystems, serve as biomarkers of disease and stress (Ololade, et al., 2009). Beside this, serum immune biomarkers analysis such as total protein, albumin and globulin can be used to determine the general health status and targeted toxicity in animals. These biomarkers also recommended to provide early warning signals of dangerous alterations in stressed organisms (Cataldi et al., 1998).

Plant-based medicines have long been used widely and played a significant part in healthcare in all civilizations and communities. In most developing countries, traditional herbal remedies are a part of societies and the main therapeutic approach. These alternative therapies are widely accepted by societies, cost-effective, readily available, and frequently efficient (Aziz et al., 2018). The moringa plant provides both nutritional and therapeutic benefits (Pareek et al., 2023). It has economic significance due to its medicinal applications and nutritional advantages (Milla et al., 2021). It is a member of the sub-Himalayan Moringaceae family, which is native to Afghanistan, Bangladesh, Pakistan, and India (Khan et al., 2023). The active phytoconstituents alkaloids, saponins, tannins, sterols, anthraquinones, terpenoids, flavonoids and vitamins are abundant in moringa leaves, in addition to the different minerals found in them. These elements are necessary for the antioxidant properties of this plant as well as its capacity to protect against free radicals (Pareek et al., 2023). The three main polyphenols found in moringa extracts are quercetin, kaempferol and chlorogenic acid, which have antiproliferative, antihypertensive, and anti-inflammatory activities (Vergara-Jimenez et al., 2017). Additionally, M. oleifera leaf extract has been demonstrated to successfully guard against growth, hematological and immune suppression brought on by cadmium (Mallya et al., 2017) and lead (Melebary and Elnaggar 2022). The aim of current research was to evaluate protective role of M. oleifera leaf extract against arsenic induced hematological alteration and immune suppression in Labeo rohita.

MATERIALS AND METHODS

Experimental Animal: Labeo rohita individuals (30±2g) were collected from fish pond of University of Veterinary and Animal Sciences Lahore, Pakistan and placed in cemented rectangular tanks containing flow through aerated water for acclimatization according to Hunn et al., (1968). The fish were fed with commercial pelleted diet (30 % CP) up to satiation twice a day.

Moringa oleifera leaves extract and diet preparation: Moringa oleifera leaves were collected and identified from Department of Biological Sciences, University of Veterinary and Animal Science, Lahore. The leaves were shade dried, powdered in grinding mill and then macerated with methanol for 72hr. After maceration the extract filtered and evaporated to dryness by using a rotatory evaporator (DAI HAN SCIENTIFIC WEV-100-1L) at 45 ̊C (Handa et al., 2008). The residues were collected and stored at 4 ̊C until use. The chemical composition of extract was determined by GC-MS analysis according to Hameed and Sayed (2019).

Feed preparation: Fish feed ingredients were purchased from local market Pattoki, Pakistan, and analyzed for chemical composition following AOAC (2016). Following ingredients as shown in Table 1 mixed and combined in ratio to make 30% CP (NRC, 2011). Ingredients were divided into three parts. Two parts supplemented with 2% and 4% (w/w) Ahmed et al. (2020) M. oleifera leaf extract respectively, named as D₁ and D₂. third part was without M. oleifera supplement (D₀). All ingredients were mixed and combined to form pallets. The prepared feed was air dried and stored in air tight container at 4°C.

Table 1 showing feed ingredients composition and percentage.

Vitamin and mineral mixture premixes cover the minerals &vitamins levels for Labeo rohita as recommended by (NRC, 2011). ­­­¹ Vitamins premix each 1 kg contains vit. A 580000 IU, vit. E. 720 mg, vit. D₃ 8600 IU, vit. K₃ 142 mg, folic acid 86 mg, B₁₂ 58 mg, vit B₁ 58 mg, vit B₂ 34 mg, vit. B₆ 34 mg, vit C 0.1 mg, vit. pantothenic acid 8 mg. ² Mineral premix each 1 kg contains copper sulfate 3400 mg, zinc methionine 3000 mg, iron sulfate 2000 mg, manganese sulfate 65 mg, cobalt sulfate 572 mg, calcium iodide 25 mg, sodium selenite 25 mg, and calcium carbonate (as carrier substance) 1000mg.

Experimental design: In current study 180 acclimatized individuals of Labeo rohita randomly selected from cemented rectangular tanks and divided into six glass aquaria (having 300L water) with 15 fish in each aquarium. These glass aquaria designated treatment name as T_1, T₂, T₃, T₄, T₅ and T₆. Treatment 1 (T₁) and 2 (T₂) were fed with (D₀) diet along with zero and 1/3^rd of 96h LC₅₀ of arsenic (6.75 mgL^-1) exposure. Treatment 3 (T₃) and 4 (T₄) were fed with D₁ and D₂ diet, respectively with zero arsenic exposure. Treatment 5 (T₅) and 6 (T₆) were fed on D₁ and D₂ diet, along with 1/3^rd of 96h LC₅₀ of arsenic (6.75 mgL^-1) exposure. Fish were fed with their respective diets up to satiation twice a day for 28 days. Whole experiment was conducted in triplicate. Aquaria water was replenished with clean and well aerated water twice a week containing the same arsenic concentration (6.75 mgL^-1) containing the same arsenic concentration (6.75 mgL^-1) to avoid from ammonia stress and maintain water quality. In each aquarium desired arsenic concentration was confirmed through absorption spectrophotometer analysis and was found significantly similar with corresponding concentration. Current experiment was conducted under constant water temperature, pH, dissolved oxygen, and hardness of 30°C, 7.5, 5.5 and 150 mgL­^-1 respectively. These water quality parameters like pH, dissolved oxygen, temperature and hardness were checked by pH (ST 300), DO (ST 300D Ohaus, Corporation, USA) and multimeter (AD-3000, Adwa, Romania) respectively.

During experimental trial, two fish were randomly selected from each aquarium (six fish per treatment) at 7^th, 14^th, 21^st and 28^th. To overcome handling stress Fish were anaesthetized with MS-222 (30 mgL^-1). Using 3ml sterile hypodermic micro syringe blood was collected from the caudal vein of fish. Collected blood apportioned into two groups one group poured into K₃EDTA tube for hematology and another group into gel & clot activator tubes for serum collection. Collected sera was stored at -40°C for biochemical analysis.

Immunological parameters: Serum total protein and albumin contents were measured by using their respective Bioactiva Diagnostic (Bad Homburg, Germany) kits. 1000µl of reagent was taken into the cuvette and 20µl of serum sample was also added. Obtained mixture was mixed properly and inserted into serum chemistry analyzer for results at 546nm and 37°C. Serum globulin was measured by subtracting the albumin contents from total protein of the same sample described by (Khalil and Korni, 2017).

Hematological analysis: Hematological analysis was performed by using Celltac α hematology analyzer (MEK-6500K) of Nihon Kohdens company). It is an automatic hematology analyzer and was calibrated for hematological analysis in fish according to manufacturer’s recommendation before analysis. New calibration coefficient was determined by using both manual and analyzer assessed values and entered into the analyzer for calibration. After this blood from K₃EDTA tubes was aspirated through capillary in open mode and results were obtained on screen after 60 seconds

Statistical analysis: Obtained data of biochemical parameters were checked for normality and homogeneity of variance via Kolmogorov-Smirnov and Levene’s tests, respectively. Data subjected to two-way repeated measure ANOVA using the GraphPad prism software (version 9.4.1 for Windows). Results were shown as mean ± SE.  Differences between groups were assessed by Tukey’s honest-significant difference test and effects with a probability of p ≤ 0.05 were considered significant.

RESULTS

GC-MS profile of M. oleifera leaf extract: The GC-MS analysis of identified M. oleifera leaf extract identified the main components as well as their proportionate contribution to the overall peak area and retention time (Fig. 1 and Table 2). The data showed that there were 22 constituents in the MLE, with the majority being quercetin (42.66%), acetic acid (17.77%), Ethyl iso-allocholate (12.66%), 1-Methyle cyclopentanole (4.4%), p-Xylene (2.8%), Octadecanoic acid (2.8%), Linoleic acid (2.66%).

Table 2 Peak area (%) and retention time of different compounds identified in M. oleifera leaf extract determined by GC-MS.

Effect of arsenic and/or M. oleifera on immunity biomarkers: Serum innates immune biomarkers, such as total protein, albumin and globulin and were significantly (p ≤ 0.05) lower in arsenic exposed group (T₄) compared to other groups as represented in (Fig. 2-4). 2 and 4 % M. oleifera leaf extract supplementation to arsenic exposed group T₅ and T₆ significantly restored serum immunological biomarker level compared to arsenic exposed group (T₄). But 4 % M. oleifera leaf extract supplementation significantly (p ≤ 0.0001) restored serum immunological biomarker level. Moreover, albumin, globulin and total protein level were higher in Moringa oleifera leaf extract supplementation treatment (T₂ and T₃) than control (T₁) group.

^a p value: T₁ versus T₂, T₃, T₄, T₅ and T₆.

^b p value: T₂ versus T₃, T₄, T₅ and T₆.

^c p value: T₃ versus T₄, T₅ and T₆.

^d p value: T₄ versus T₅ and T₆.

^e p value: T₅ versus and T₆.

^a p value: T₁ versus T₂, T₃, T₄, T₅ and T₆.

^b p value: T₂ versus T₃, T₄, T₅ and T₆.

^c p value: T₃ versus T₄, T₅ and T₆.

^d p value: T₄ versus T₅ and T₆.

^e p value: T₅ versus and T₆.

^a p value: T₁ versus T₂, T₃, T₄, T₅ and T₆.

^b p value: T₂ versus T₃, T₄, T₅ and T₆.

^c p value: T₃ versus T₄, T₅ and T₆.

^d p value: T₄ versus T₅ and T₆.

^e p value: T₅ versus and T₆.

Effects of arsenic and/or M. oleifera on hematological profile: The effects of arsenic and/ or M. oleifera leaf extract supplementation on hematological profile represented in Table. 3. The fish fed with 2 and 4 % M. oleifera leaf extract supplemented diet (T₂ and T₃) exhibits no significant variations in hematological indices but exhibited higher RBC, Hb, HCT as compared to control group (T₁). In contrast, fish exposed to arsenic (1/3^rd of the 96h LC₅₀) had significantly reduced RBC, Hb concentrations, HCT, MCH, MCHC values and total leukocyte counts than other groups. However, most of these components were significantly improved in arsenic exposed group fed with 2 and 4% M. oleifera leaf extract supplemented diet

Table 3. Hematological profile of Labeo rohita treated with arsenic and/or M. oleifera leaf extract supplemented diet.

Changes in hematological components in Labeo rohita exposed to arsenic (1/3^rd of 96hr LC₅₀) and / or fed with Moringa oleifera leaf extract supplemented diet. The values are presented as the mean ± SE. Values with different alphabets are statistically different at p ≤ 0.05.

^a p value: T₁ versus T₂, T₃, T₄, T₅ and T₆.

^b p value: T₂ versus T₃, T₄, T₅ and T₆.

^c p value: T₃ versus T₄, T₅ and T₆.

^d p value: T₄ versus T₅ and T₆

^e p value: T₅ versus and T₆.

 DISCUSSION

Because arsenic possess negative effect on non-target creatures like fish, there are now a greater number of concerns raised about its potential ecotoxicity to human beings and the environment (Briffa et al., 2020). In addition to its toxicity, arsenic exposure of even sub-lethal concentrations causes its bioaccumulation and biomagnification into aquatic food chains, which poses a severe danger to the sustainability of the environment (Shahjahan et al., 2022). Thus, in addition to the increasing focus on determining the potential hazards that arsenic poses to aquatic systems, there is also an urgent need to identify new therapeutic agents against arsenic toxicity, which is still a challenging issue to solve. In this context, the current study was designed to evaluate the toxic effects of arsenic in the Labeo rohita in terms of the hematological profile and immune biomarkers of fish, mainly in order to clarify the potential therapeutic benefits of Moringa oleifera leaf extracts on the general health status of fish.

Proteins play an important role in cell metabolism and are involved in cell physiology and architecture. In the current study, feeding fish diets supplemented with M. oleifera leaf extract significantly increased total protein, albumin, and globulin levels over the control diet. Previous research has shown that including plant materials or extracts in fish diets produces significantly higher levels of total protein, albumin, and globulin, suggesting increases in defensive molecules (Abdel-Wahab and El-Bahr, 2012; Abdel-Tawwab and Hamed, 2020). In addition, Nile tilapia fed diets based on pomegranate peel and moringa showed substantial increases in total protein, albumin, and globulin, according to Monir et al., (2020). In the current investigation, we found that arsenic-exposed fish had low levels of total protein, albumin, and globulin, which may have been caused by proteolysis, a failure in protein synthesis brought on by hepatic and renal dysfunction (Ellis et al., 1981). In arsenic-exposed fish, low level of albumin may represent low blood viscosity, whereas low levels of globulin may be due to the liver inability to synthesized sufficient globulin, an indication of weakened immunity (Zhang et al., 2021). These results are in agreement with those that have been observed after toxicant exposure in several fish species (Kannan et al., 2014; Singh et al., 2015; Ghaffar et al., 2017; Majumder and Kaviraj, 2017; Kumar et al., 2019). The restored total protein level in the blood of arsenic exposed fish fed M. oleifera leaf extract supplemented diet demonstrates the immune stimulatory effects of M. oleifera. In similar research, Hamed and El-Sayed, (2018) found that Nile tilapia fed M. oleifera leaves extract had hepatoprotective effect, as indicated by higher serum levels of total protein and albumin than those exposed to pendimethalin without moringa supplementation. Similar findings were reported by Abdel-Tawwab et al., (2020), who stated that European sea bass fed diets enriched with garlic and chitosan showed increased levels of blood total protein, albumin, and globulin.

Blood is a pathophysiological reflector of the whole organism body, and therefore, blood measurements are necessary for evaluating the structural and functional condition of fish exposed to pollutants. In current study the significant reductions in RBC count, Hb concentration, and Hct in the arsenic-exposed group may be caused by either a faster rate of erythrocyte destruction or a slower rate of RBC formation and hemoglobin synthesis (Ahmed and Fazio, 2022). The capacity of arsenic to lower blood iron concentrations, which inhibits the production of hemoglobin, or the rapid oxidation of hemoglobin to methemoglobin, among other factors, may have contributed to the decline in Hb content (Muttappa, 2015). In addition, Ambali et al., (2010), Deeba et al., (2017), and Quintana et al., (2018) stated that, as a result of oxidative stress and rapid lipoperoxidation of erythrocyte membranes, arsenic exposure also increases RBC fragility. Furthermore, the kidney impairment brought on by arsenic may have an impact on hematopoiesis (Xing et al., 2012; Zheng et al., 2014). Several fish species exposed to arsenic have been documented to exhibit similar anemic circumstances in the past, including Channa punctatus (Jha et al., 2017), Labeo rohita (Ghaffar et al., 2016), and Heteropneustes fossilis (Singh and Srivastava, 2015).

oleifera leaf extract supplementation significantly restored arsenic-induced anemia with the help of erythrocytes formation in the current investigation. Numerous studies have shown that M. oleifera includes a range of amino acids, vitamins, trace elements, minerals (including iron, copper, selenium, and zinc), and phytochemical components (like saponins and flavonoids) (Meireles et al., 2020). Vitamins are crucial for DNA synthesis and erythrocyte maturation, especially vitamins B6, B12, C, E, folic acid, and riboflavin (Bender and Mayes, 2006). The amino acid composition of M. oleifera is also necessary for the synthesis of globulin, which leads to the production of hemoglobin. Iron, one of the components used in the formation of hemoglobin, is also found in M. oleifera (Saini et al., 2014). Additionally, M. oleifera has been shown to strengthen and preserve the RBC membrane (Sarkar et al., 2017). The results of present study thus confirmed that arsenic induced immunological and hematological alterations were ameliorated by the Moringa oleiferaleaves extract supplementation.

REFERENCES

1. Abdel-Tawwab, M. and H.S. Hamed (2020). Antagonistic effects of dietary guava (Psidium guajava) leaves extract on growth, hemato-biochemical, and immunity response of cypermethrin-intoxicated Nile tilapia, Oreochromis niloticus, fingerlings. Aquaculture, 529: https://doi.org/10.1016/j.aquaculture.2020.735668
2. Abdel-Tawwab, M., R.H. Khalil, A. Ahmed, M. Medhat, S. Shakweer, A. Mohamed, K. Hany and M.R. Abdel-Latif (2020). Effects of black soldier fly (Hermetia illucens) larvae meal on growth performance, organs-somatic indices, body composition, and hemato-biochemical variables of European sea bass, Dicentrarchus labrax. Aquaculture. 522: 735136. https://doi.org/10.1016/j.aquaculture.2020.735136
3. Abdel-Wahab, A.M. and S.M. El-Bahr (2012). Influence of black cumin seeds (Nigella sativa) and turmeric (Curcuma longa Linn.) mixture on performance and serum biochemistry of Asian sea bass, Lates calcarifer. World. J. Fish Mar. Sci. 4(5):496-503. http://dx.doi.org/10.5829/idosi.wjfms.2012.04.05.6478
4. Ahmed, I., A. Zakiya and F. Fazio (2022). Effects of aquatic heavy metal intoxication on the level of hematocrit and hemoglobin in fishes: A review. Front. Environ. Sci. 10:919204. https://doi.org/10.3389/fenvs.2022.919204
5. Ali, I. (2018). Microwave assisted economic synthesis of multi walled carbon nanotubes for arsenic species removal in water: batch and column operations. J. Mol. Liq. 271: 677-685. https://doi.org/10.1016/j.molliq.2018.09.021
6. Ambali, S., A. Abubakar, M. Shittu, L. Yaqub, P. Kobo and A. Giwa (2010). Zinc ameliorates chlorpyrifos-induced increased erythrocyte fragility and lipoperoxidative changes in Wistar rats. New York Sci. J. 3: 117–122.
7. O.A.C. (2016). Natural Poisons, 20^th ed. Association of Official Analytical Chemists, Washington, DC., USA http://www.eoma.aoac.org/app_f.pdf
8. Aziz, M.A., M. Adnan and A.H. Khan (2018). Traditional uses of medicinal plants practiced by the indigenous communities at Mohmand Agency, FATA, Pakistan. J. Ethnobiol. Ethnomed.14: 120-129. https://doi.org/10.1186/s13002-017-0204-5
9. Basheer, A.A (2018). Chemical chiral pollution: impact on the society and science and need of the regulations in the 21^st Chirality. 30(4): 402-406. https://doi.org/10.1002/chir.22808
10. Bender, D. and P. Mayes (2006). Micronutrients: Vitamins and minerals. In: Murray, (Eds.), Harper's Illustrated Biochemistry. New York, McGraw-Hill Lange. 489-505. https://accessmedicine.mhmedical.com/content.aspx?bookid=2386&sectionid=187836268.
11. Brahman, K.D., T.G. Kazi and H.I. Afridi (2013). Simultaneously evaluate the toxic levels of fluoride and arsenic species in underground water of Tharparkar and possible contaminant sources: a multivariate study. Ecotoxicol. Environ. Saf. 89: 95-107. https://doi.org/10.1016/j.ecoenv.2012.11.023
12. Briffa, J., E. Sinagra, R. Blundell (2020). Heavy metal pollution in the environment and their toxicological effects on humans. Heliyon. 6(9): 13-18. https://doi.org/10.1016/j.heliyon.2020.e04691
13. Cataldi, , P. Di-Marco, A. Mandich and S. Cataudella (1998). Serum parameters of adriatic sturgeon Acipenser naccarii(Pisces:Acipenseriformes): effects of temperature and stress. Comp. Biochem. Physiol. 121: 351–354. http://203.250.122.194/blood/b_chem/b_chem_010.pdf
14. Deeba, F., I. Raza, N. Muhammad, H. Rahman, Z. Rehman, A. Azizullah, B. Khatta F. Ullah and M.K. Daud (2017). Chlorpyrifos and lambda cyhalothrin-induced oxidative stress in human erythrocytes. Toxicol. Ind. Health. 33: 297–307. https://doi.org/10.1177/0748233716635003
15. Ellis,, H. Yavuzcan, J.F.L. Olmeda and M.T. Spedicato (2011). Cortisol and finfish welfare. Fish Physiol. Biochem.38(1): 163-88. https://doi.org/10.1007/s10695-011-9568-y
16. Farooqi, A., H. Masuda and N. Firdous (2007). Toxic fluoride and arsenic contaminated groundwater in the Lahore and Kasur districts, Punjab, Pakistan and possible contaminant sources. Pollut. 145(3): 839-849. https://doi.org/10.1016/j.envpol.2006.05.007
17. Ghaffar, A., R. Hussain, M. Aslam, G. Abbas and A. Khan (2016). Arsenic and urea in combination alters the hematology, biochemistry and protoplasm in exposed rahu fish (Labeo rohita) (Hamilton, 1822). J. Fish. Aqu. Sci. 16: 289-296.http://dx.doi.org/10.4194/1303-2712-v16_2_09
18. Hamed, H.S. and Y.S.E. Sayed (2018). Antioxidant activities of Moringa oleifera leaf extract against pendimethalin-induced oxidative stress and genotoxicity in Nile tilapia, Oreochromis niloticus (L.). Fish Physiol. Biochem. 45(2): 71-82. https://doi.org/10.1007/s10695-018-0535-8
19. Handa, S.S., S.P.S. Khanuja, G. Longo and D.D. Rakesh (2008). Extraction technologies for medicinal and aromatic plants. UNIDO. 3(8): 22-30. https://www.unido.org/sites/default/files/200910/Extraction technologies for medicinal and aromatic plants 0.pdf
20. Hunn, J.B., R.A. Schoettger and E.W. Whealdon (1968). Observations on the handling and maintenance of bioassay fish. Prog Fish Cult. 30(3): 164-167. https://pubs.usgs.gov/publication/1003266
21. Jabeen, G. and M. Javed (2011). Evaluation of arsenic toxicity to biota in river Ravi (Pakistan) aquatic ecosystem. Int. J. Agric. Biol. 13(6): 929-934. http://www.fspublishers.org
22. Jha, D.K., B.B. Mishra, K.R. Thakur, K. Pranay, K. Sayrav, V. Kumar, P. Verma and P.K. Khan (2017). Toxicological effects of arsenic exposure on haematology of fresh water fish Channa punctatus. Der. Pharma. Chemica. 9(16): 1-5. http://www.derpharmachemica.com/archive.html)
23. Kannan, M.M., V. Padmanaban and Uthayakuma (2014). Response of synthetic pyrethroid cypermethrin (10% EC) induced stress in biochemical and haematological parameters of Indian major carp Catla catla (Hamilton, 1822). World. J. Pharm. Res. 3. https://www.researchgate.net/publication/272510042_Response_of_synthetic_pyrethroid_cypermethrin_10_EC_induced_stress_in_biochemical_and_haematological_parameters_of_Indian_major_carp_Catla_catla_Hamilton_1822
24. Khalil, F. and F.M.M. Korni (2017). Evaluation of Moringa oleifera leaves and their aqueous extract in improving growth, immunity and mitigating effect of stress on common carp (Cyprinus Carpio) fingerlings. Turkish. J. Aqua. Sci. 32(3): 170-177. http://dx.doi.org/10.18864/TJAS201715
25. Khan, D.R., H. Uslu and O.G. Osman (2023). Moringa oleifera: A Sustainable Intervention to Address Malnutrition and Poverty in Khyber Pakhtunkhwa (KPK)- Pakistan. I. C. S. I. S. (Turkey) 1(1) 225-229 http://dx.doi.org/10.59287/icsis.605.
26. Kumar N, Gupta SK, Bhushan S and N.P. Singh (2019). Impacts of acute toxicity of arsenic (III) alone and with high temperature on stress biomarkers, immunological status and cellular metabolism in fish. Aquat Toxicol. 214:105233. https://doi.org/10.1016/j.aquatox.2019.105233
27. Kumar, R.R. and T.K. Banerjee (2012). Analysis of arsenic bioaccumulation in different organs of the nutritionally important catfish, Clarias batrachus (L.) exposed to the trivalent arsenic salt, sodium arsenite. Bull. Environ. Contam. Toxicol. 89: 445–449. https://doi.org/10.1007/s00128-012-0714-8
28. Majumder, R. and A. Kaviraj (2017). Cypermethrin induced stress and changes in growth of freshwater fish Oreochromis niloticus. Int. Aquat. Res.9: 117–128. https://doi.org/10.1007/s40071-017-0161-6
29. Mallya, R., P.K. Chatterjee, N.A. Vinodini, P. Chatterjee and P. Mithra (2017). Moringa oleiferaleaf extract: beneficial effects on cadmium induced toxicities - A Review. J. Clin. Diagn. Res. 11(4): 01-04. https://doi.org/10.7860/jcdr/2017/21796.9671
30. Meireles, D., J. Gomes and L. Lopes (2020). A review of properties, nutritional and pharmaceutical applications of Moringa oleifera: integrative approach on conventional and traditional Asian medicine. Adv. Tradit. Med. 20: 495-515. https://doi.org/10.1007%2Fs13596-020-00468-0
31. Melebary, S.J. and M.H.R., Elnaggar (2023). Impact of Moringa oleiferaleaf extract in reducing the effect of lead acetate toxicity in mice. Saudi. J. Biol. Sci. 30(1): 103507. https://doi.org/10.1016%2Fj.sjbs.2022.103507
32. Milla PG, R. Peñalver and G. Nieto (2021). Health benefits of uses and applications of moringa oleiferain bakery products. Plants (Basel). 10(2): 318. https://doi.org/10.3390%2Fplants10020318
33. Monir, W., M.A. Abdel-Rahman, E.D.S. Hassan, E.S. Mansour and S.M.M. Awad (2020) Pomegranate peel and moringa-based diets enhanced biochemical and immune parameters of Nile tilapia against bacterial infection by Aeromonas hydrophila. Microb. Pathog. 145:104202. https://doi.org/10.1016/j.micpath.2020.104202
34. Muttappa, K. (2015). Haematological and histological changes in tilapia (Oreochromis mossambicus) exposed to cadmium and chlorpyrifos. Karn. Veter. 23: 23-28. https://www.semanticscholar.org/paper/Haematological-and-Histological-Changes-in-Tilapia-Muttappa/66c1133e6db80bc5e09aa516d1d19a44cfc99fd5
35. Nickson, R.T., J.M. McArthur, B. Shrestha, T.O. Kyaw-Myint and D. Lowry (2005). Arsenic and other drinking water quality issues, Muzaffargarh District, Pakistan. Appl. Geochem. 20(1): 55-68. https://doi.org/10.1016/j.apgeochem.2004.06.004
36. National research council (NRC) 2011. Nutrient Requirements of Fish and Shrimp. National academies press. https://doi.org/10.1007/s10499-011-9480-6
37. Ololade,A. and O. Ogini (2009). Behavioural and hematological effects of zinc on African catfish, Clarias gariepinus. Internation. J. of Fisher. and Aquacul.1.2: 22-27. http://www.academicjournals.org/IJFA
38. Pareek, A., M. Pant, M.M. Gupta, P. Kashania, Y. Ratan, V. Jain, A. Pareek and A.A. Chuturgoon (2023). Moringa oleifera: An Updated Comprehensive Review of Its Pharmacological Activities, Ethnomedicinal, Phytopharmaceutical Formulation, Clinical, Phytochemical, and Toxicological Aspects. Int. J. Mol. Sci. 24(3): 2098. https://doi.org/10.3390/ijms24032098
39. Quintana, M.M., R.V. Osimani, G. Magnarelli, M.G. Rovedatti and N. Guiñazú (2018). The insecticides chlorpyrifos and acetamiprid induce redox imbalance in umbilical cord blood erythrocytes in vitro. Pest. Biochem. Physiol. 148: 87–92. https://doi.org/10.1016/j.pestbp.2018.04.001
40. Rabbane, M.G. M.A. Kabir, M. Habibullah-Al-Mamun and M.G. Mustafa, (2022). Toxic Effects of Arsenic in Commercially Important Fish Rohu Carp, Labeo rohitaof Bangladesh. Fishes. 7: https://doi.org/10.3390/fishes7050217
41. Ramesh, M.S., M. Veera-Gowtham and V.R.k. Poopal (2013). Hematological, biochemical and enzymological responses in an Indian major carp Labeo rohita induced by sublethal concentration of waterborne selenite exposure. Chemico-biolo. interac. 207. https://doi.org/10.1016/j.cbi.2013.10.018
42. Rasoola, A., A. Farooqia, S. Masoodb and K. Hussainc (2016). Arsenic in groundwater and its health risk assessment in drinking water of Mailsi, Punjab, Pakistan. Hum. Ecol. Risk Assess. 22(1) 187-202. http://dx.doi.org/10.1080/10807039.2015.1056295
43. Saini, R.K., P. Manoj, N.P. Shetty, K. Srinivasan and P. Giridhar (2014). Dietary iron supplements and Moringa oleifera leaves influence the liver hepcidin messenger RNA expression and biochemical indices of iron status in rats. Nutr Res.34(7):630-8. https://doi.org/10.1016/j.nutres.2014.07.003
44. Sarkar, S., S. Mukherjee, A. Chattopadhyay and S. Bhattacharya (2017). Differential modulation of cellular antioxidant status in zebrafish liver and kidney exposed to low dose arsenic trioxide. Ecotoxicol. Environ. Saf. 135: 173- https://doi.org/10.1016/j.ecoenv.2016.09.025
45. Shah, S.L. and A. Altindag (2004). Hematological parameters on tench (Tinca tinca) after acute and chronic exposure to lethal and sublthal mercury treatments. Bull. Environ. Contam. Toxicol. 73: 911–918. https://doi.org/10.1007/s00128-004-0513-y
46. Shahjahan, Md. K. Taslima, M.S. Rahman, Md. Al-Emran, S.I. Alam and C. Faggio (2022). Effects of heavy metals on fish physiology – A review. Chemosphere. 300: 134519. https://doi.org/10.1016/j.chemosphere.2022.134519
47. Shaji, E., M. Santosh, K.V. Sarath, P. Prakash, V. Deepchand and B.V. Divya (2021). Arsenic contamination of groundwater: A global synopsis with focus on the Indian Peninsula.Front. 12(3): 1-66. https://doi.org/10.1016/j.gsf.2020.08.015
48. Singh, S., and A.K. Srivastava (2015). Sub lethal effects of copper and arsenic on some biochemical and hematological parameters of the blood in an air breathing fish Heteropneustes fossilis. J. Ecophysiol. Occup. Hlth. 15 (3): 105-114. http://dx.doi.org/10.15512/joeoh/2015/v15i3-4/121592
49. (2002). United states Environmental Protection Agency. Short term methods for estimating the chronic toxicity of effluents and receiving water of freshwater organisms. Fourth edition. US Environmental Protection Agency, Office of water, Washington, DC. EPA-821-R-02-013. https://www.epa.gov/sites/default/files/2015-08/documents/short-term-chronic-marine-and-estuarine-wet-manual_2002.pdf
50. Vergara-Jimenez, , M.M. Almatrafi and M.L. Fernandez (2017). Bioactive Components in Moringa oleifera Leaves Protect against Chronic Disease. Antioxidants (Basel). 6(4): 91. https://doi.org/10.3390%2Fantiox6040091
51. Xing, H., S. Li, Z. Wang, X. Gao, S. Xu and X. Wang X. 2012. Histopathological changes and antioxidant response in brain and kidney of common carp exposed to atrazine and chlorpyrifos. Chemosphere. 88: 377-383. https://doi.org/10.1016/j.chemosphere.2012.02.049
52. Zhang, J., T. Wang and Y. Fang (2021). Clinical significance of serum albumin/globulin ratio in patients with pyogenic liver abscess. Front Surg. 677799. https://doi.org/10.3389/fsurg.2021.677799
53. Zheng, L., C.C. Kuo, J. Fadrowski, J. Agnew, V.M. Weaver and A. Navas-Acien (2021). Arsenic and chronic kidney disease: a systematic review. Curr. Environ. Health. Rep. 1(3): 192-207. https://doi.org/10.1007/s40572-014-0024-x