{"paper_id":"33ca5a3d-be57-43e2-b29b-d99ae971d8b2","body_text":"Toxicological Effects of Waterborne Boric Acid and the Protective Role of Dietary Black Cumin (Nigella sativa L.) Oil in Nile Tilapia (Oreochromis niloticus): Hematological, Biochemical, Oxidative Stress and Histopathological Responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Toxicological Effects of Waterborne Boric Acid and the Protective Role of Dietary Black Cumin (Nigella sativa L.) Oil in Nile Tilapia (Oreochromis niloticus): Hematological, Biochemical, Oxidative Stress and Histopathological Responses Mustafa ÖZ, Enes ÜSTÜNER, Suat DİKEL This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6515353/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to evaluate the protective effects of dietary black cumin ( Nigella sativa L.) oil on hematological, biochemical, oxidative stress, and histopathological responses in Nile tilapia ( Oreochromis niloticus ) exposed to waterborne boric acid. After determining the 96-hour LC 50 value of boric acid, the experimental design included exposure to 1/20th of this concentration. Fish were fed for 21 days with diets either containing or lacking 1% black cumin oil. At the end of the feeding trial, blood parameters, oxidative stress biomarkers, and tissue histopathology were analyzed. The group receiving black cumin oil without boric acid showed the most favorable physiological and biochemical profiles. In contrast, the group exposed to boric acid alone exhibited significant negative alterations. Importantly, fish fed black cumin oil while exposed to boric acid showed improvements across all measured parameters compared to the toxicant-only group. The findings indicate that dietary black cumin oil effectively alleviates the toxic effects of waterborne boric acid on Nile tilapia, supporting its potential use as a functional dietary additive in aquaculture. Nile tilapia Boric acid Black cumin oil Oxidative stress Sustainable fish farming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Aquatic ecosystems worldwide face increasing risks from a variety of chemical contaminants emanating from industrial, agricultural, and domestic sources. Among these contaminants, boric acid (H₃BO₃) has emerged as a pollutant of concern due to its widespread use in glass production, ceramics, agriculture, and even in certain antiseptic formulations (Kan & Kucukkurt, 2023 ). Natural sources of boric acid typically include seawater and geological formations, where it is found in water layers at concentrations exceeding 4 mg/L (Baransi-Karkaby, Bass, & Freger, 2019 ). In freshwater environments, boric acid can bioaccumulate and may lead to toxicity in aquatic organisms, particularly when concentrations exceed threshold values of 0.5 to 1 ppm (Gaikwad, Gaikwad, & Kamble, 2021 ). The application of boric acid as a pesticide in urban areas increases the likelihood of surface runoff during rainfall, enhancing its presence in nearby water bodies (See et al., 2010 ).Boric acid reaching aquatic ecosystems has been shown to cause histopathological damage in fish tissues and negatively affect blood parameters (Öz, Yavuz, & Bolukbas, 2020 ; Topal, Oruç, Altun, Ceyhun, & Atamanalp, 2016 ). In recent years, environmental toxicology has witnessed increasing interest in identifying natural protective substances that may reduce the toxic effects of environmental pollutants. Black seed ( Nigella sativa L .) oil has attracted considerable attention due to its rich composition of bioactive compounds such as thymoquinone, which exhibits strong antioxidant, anti-inflammatory, and immunomodulatory properties (Bordoni et al., 2019 ). Thymoquinone enhances antioxidant enzyme activities which supports its protective effects against oxidative damage according to multiple animal model studies. (Hosseinzadeh, Monaghash, Ahmadi, Ghiasvand, & Shokoohinia, 2017 ). The toxic effects can be counteracted by using herbal supplements that contain natural antioxidants in aquaculture operations. The bioactive compounds thymoquinone and others in Nigella sativa (black cumin) oil provide antioxidant and anti-inflammatory and hepatoprotective effects. Research shows that adding black cumin seeds and oil to fish feed enhances growth rates while decreasing oxidative stress. (Latif, Faheem, Asmatullah, Hoseinifar, & Van Doan, 2020 ; Öz, Dikel, & Durmus, 2018 ; Öz, Üstüner, & Bölükbaş, 2024 ). The Nile tilapia ( Oreochromis niloticus ) serves as an appropriate model species for toxicological research in aquatic ecosystems because of several key factors. The species stands as a valuable organism for studying environmental stressors because it adapts to different conditions and shows strong physiological responses to pollutants and plays important ecological roles in freshwater habitats. The Nile tilapia stands out because of its ability to adapt to changing water conditions which makes it essential for toxicology research. The species demonstrates wide tolerance to environmental factors including salinity and temperature fluctuations and pollution while thriving in both high and low-quality aquatic environments. (Abdel-Mohsen, 2014 ). The researchers can study pollution effects under different water conditions because of this natural ability to adapt. This ability enables researchers to study pollution effects under realistic ecological conditions. Research indicates Nile tilapia maintains survival capabilities in polluted environments containing heavy metals which makes it an ideal species for detecting heavy metal exposure in aquatic ecosystems. (Shaalan, 2024 ). The research aims to assess the hematological, biochemical, oxidative stress and histopathological effects of boric acid exposure in aquatic environment on Nile tilapia ( Oreochromis niloticus ) and to investigate the protective role of dietary black cumin oil against these toxic effects. 2. MATERIALS AND METHODS 2.1. Research Design Ethical approval was obtained from Çukurova University ethics committee (Ref No: 7/2020). Feeding activities were carried out at Çukurova University, Faculty of Fisheries, Dr. Nazmi Tekelioğlu Freshwater Fisheries Production and Research Station. In order to determine the appropriate boron dosage for our study, we initially estimated the LC50 (96 h) value by probit analysis (Finney, 1971). A total of 120 fish were used to calculate the LC50 value and boron was applied at six different concentrations (0.00, 10.00, 50.00, 100.00, 150.00 and 200.00 mg/L) (Çelik, Dikel, & Öz, 2024). During the evaluation of the LC50 value, the fish were monitored three times a day for 96 hours and the dead fish were immediately removed from the environment. During the LC50 experiment, oxygen was continuously supplied to 50 liter capacity aquariums and water was changed every 24 hours using prepared stocks. In addition, the fish were not fed during the experiment. To determine the LC50 value, 120 fish were used and the experiment was designed in two replicates. In the feeding trial, 108 fish were used. A total of 228 male Nile tilapia with a starting weight of 36.38±0.83 g were used in the study. The research was carried out in 80 liter aquariums with 9 fish in 3 replications and 27 fish were used in each group. Boric acid was added to the water of the research groups at the rate of 1/20 of the determined Lc50 value. Eheim brand 100 Watt thermostat heater was used to keep the water temperature constant and the water temperature was kept constant at 25 0C in all groups during the research. The research groups and fish numbers are shown in detail in Table 1. Table 1. Research groups, black cumin seed oil ratios, boric acid amount and fish numbers Groups Black Cumin oil in the feed (%) The amount of Boric Acid in the water (mg/L) Numbers of fish BA1 0.00 0.00 27 (3*9) BA2 1.00 0.00 27 (3*9) BA3 0.00 LC 50 /20 27 (3*9) BA4 1.00 LC 50 /20 27 (3*9) Total 108 2.2. Preparation of Experimental Diets and Feeding Protocol The dietary regime used in the trial was based on commercially available tilapia feed (Hem Yem, Gaziantep, Turkey) containing 39% crude protein, 6.7% crude fat, 4.30% crude fiber and 6.79% crude ash. The cold-pressed black seed oil utilised in the study was procured from a commercial supplier. According to the protocol adapted from Öz et al. (2018), 1% black cumin oil was added to the diets of experimental groups BA3 and BA4. The feeds were prepared in 100 g portions to ensure accuracy and uniformity in the supplementation process. To enhance coating quality, black cumin oil was systematically blended with 2 ml of sunflower oil and subsequently uniformly sprayed onto the feed. In the control oil groups, 3 ml of sunflower oil was incorporated to standardise the total lipid volume across all treatments and to maintain dietary consistency among the groups. The feeding trial started one day after the first biometric assessments and continued for 21 days. Throughout the study, fish were fed twice daily - once at 08:30 am and once at 16:30 pm - at a rate equivalent to 2% of their total body weight. 2.3. Blood Sampling and Hematological Procedures At the end of the feeding trial, fish were anesthetized using 2-phenoxyethanol at a concentration of 300 ppm to ensure minimal stress during sampling. Following anesthesia, each sample was carefully rinsed with 70% ethanol to minimize external contamination. Blood samples were then collected from the caudal vein using heparinized syringes. For hematological analyses, a portion of each blood sample was transferred into lavender-colored tubes containing ethylenediaminetetetraacetic acid (EDTA) as anticoagulant. Another portion was placed in serum separator tubes (SST™ II Advance, red cap) for biochemical analysis. These samples were centrifuged at 13,000 × g for 10 min at 4 °C to obtain clear serum. Hematologic parameters were assessed immediately after collection, while serum samples were stored at -80 °C until biochemical assessments were performed. Hematological parameters such as red blood cell count (RBC), hematocrit (Hct), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were analyzed using an MS4-S hematology autoanalyzer (Melet Schloesing Laboratories, Osny, France). The precision of automated results was verified through manual counts of K₃EDTA tube samples that followed the protocol established by (Blaxhall & Daisley, 1973). 2.4. Determination of Serum Biochemical Parameters The biochemical parameters assessed in blood serum comprised alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), triglycerides (TRG), cholesterol (CHO), and glucose (GLU). These indicators were chosen for their appropriateness in evaluating the metabolic and physiological condition of fish. All biochemical assays were conducted via an automated clinical chemistry analyser (MINDRAY BS-400) following the manufacturer's guidelines. 2.5. Oxidative Stress Parameters Sera extracted from fish blood samples were analysed for Total Antioxidant Status (TAS), Total Oxidant Status (SOD), Superoxide Dismutase (CAT), Catase, GPx, Glutathione Peroxidase, and MDA (malondialdehyde). Relassay (Cat no: RL0017) commercial assays (Erel, 2004) were employed to quantify TAS levels. Relassay (Cat no: RL0024) commercial assays were employed to assess Total Oxidant Status (TOS) levels. (Erel, 2004). Total Oxidant Status (TOS) levels were evaluated using Relassay (Cat no: RL0024) commercial kits (Erel, 2005). The ratio of TOS to TAS is considered as oxidative stress index (OSI). The TAS unit obtained for calculation was converted to μmol/L and the OSI value was calculated according to the following formula. OSI = TOS (μmol H2O2 equivalent/L) / TAS (μmol Trolox equivalent/L) (Harma & Erel, 2003; Kosecik, Erel, Sevinc, & Selek, 2005; Yumru et al., 2009). Malondialdehyde (MDA) level, a product of lipid peroxidation, was determined according to Alak et al. Superoxide dismutase (SOD) enzyme activity was determined by spectrophotometer (560 nm) according to NBT (nitro blue tetrazolium chloride) reduction method with O-2 under light (Sun, Oberley, & Li, 1988). Measurement of catalase (CAT) activity; samples were mixed with 1 mL H2O2 (50 mM) and reacted at 37 °C for 1 min. Then 1 mL of ammonium molybdate was added to terminate the reaction, resulting in the formation of a yellowish complex containing residual H2O2. Finally, the UV-vis absorption of this complex was measured at 405 nm by a microplate reader (Aebi, 1984). Measurement of glutathione peroxidase (GPx) activity; Glutathione Peroxidase (GPx) catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione (GSSG), it is immediately converted to the reduced form by simultaneous oxidation of NADPH to NADP. GPx activity was measured by a change in absorbance (decrease in readings over 3 min) at 340 nm (Kraus & Ganther, 1980; Paglia & Valentine, 1967; Prohaska, Oh, Hoekstra, & Ganther, 1977). 2.6. Histopathologic Investigation Gill, liver, and kidney tissues of Nile tilapia were fixed in 0.1 M phosphate-buffered formaldehyde (pH 7.4). After trimming, tissues were washed under slow running water for 24 hours, dehydrated through an ethanol series, and treated with xylene and xylene-paraffin. Samples were incubated in soft paraffin (46–48°C) for 15 minutes and hard paraffin (56–58°C) for 30 minutes before embedding with a Leica EG 1150 H. Sections of 4 µm thickness were cut using a Leica RM2125 Rotary Microtome and stained with Haematoxylin-Eosin. Sections were mounted on glass slides using Entellan Merck, examined by light microscopy (Leica DM-750), and photographed in lesion areas (Culling, Allison, & Barr, 2014). Hematoxylin-Eosin (H-E) staining of the gill, liver and kidney tissues was used to score the pathological findings on a scale of “(-) to 3” (-: none, 1: mildly severe, 2: moderately severe and 3: severe). 2.7. Statistical Analyses Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL. USA) to determine the differences between the groups in growth parameters and changes in blood parameters of fish fed with feed supplemented with ALE at different rates. 3. RESULTS 3.1. Determination of LC 50 Value Based on the 96-hour acute toxicity test, the LC₅₀ value of boric acid for Nile tilapia (Oreochromis niloticus) was calculated to be 176.446 mg/L. Regression analysis using the Probit method gave the equation y = 1.24x + 2.2103 with a coefficient of determination of R² = 0.8846, indicating a strong correlation between boric acid concentration and mortality (Fig. 1 ). 3.2. Blood Parameters The results of hematological analysis of Nile tilapia subjected to different treatments are presented in Table 2 . Significant differences in red blood cell indices were observed between the experimental groups, indicating that both boric acid exposure and black cumin oil supplementation affected hematological profiles. Similarly, serum biochemical parameters including liver enzymes, glucose, lipid profile and total protein levels are summarized in Table 3 . The data reflect the physiological responses of fish to boric acid toxicity and the potential ameliorative effects of dietary black cumin oil. Table 2 Protective effect of black cumin oil on blood parameters parameters against boric acid toxicity in Nile tilapia BA 1 BA 2 BA 3 BA 4 RBC (m/mm3) 1.692 ± 0.029 c 2.230 ± 0.026 a 1.357 ± 0.019 d 1.913 ± 0.014 b Hb (g/dl) 13.517 ± 0.306 c 16.100 ± 0.167 a 11.550 ± 0.176 d 14.050 ± 0.259 b Hct (%) 37.550 ± 0.243 c 46.733 ± 1.097 a 31.733 ± 1.442 d 40.750 ± 0.666 b MCV (fL) 222.019 ± 3.635 b 209.565 ± 4.178 c 233.923 ± 10.689 a 212.971 ± 2.459 c MCH (pg) 79.918 ± 2.116 b 72.199 ± 0.414 c 85.146 ± 1.572 a 73.441 ± 1.759 c MCHC (g/dL) 35.998 ± 0.849 ab 34.463 ± 0.708 b 36.469 ± 1.960 a 34.491 ± 1.047 b Table 3 Protective effect of black cumin oil on blood biochemistry parameters against boric acid toxicity in Nile tilapia BA 1 BA 2 BA 3 BA 4 Cho (mg/dl) 151.370 ± 1.834 b 132.530 ± 2.777 d 182.027 ± 2.558 a 140.270 ± 0.846 c Trg (mg/dl) 154.780 ± 0.551 b 128.337 ± 1.382 d 166.930 ± 1.048 a 142.023 ± 3.835 c Glu (mg/dl) 46.312 ± 0.896 b 37.828 ± 0.485 d 69.010 ± 0.624 a 42.760 ± 0.685 c ALP (U/L) 40.586 ± 0.576 c 32.663 ± 0.568 d 56.693 ± 2.458 a 44.545 ± 0.625 b AST (U/L) 252.640 ± 3.515 b 190.717 ± 3.033 d 339.439 ± 4.566 a 215.582 ± 4.296 c ALT (U/L) 44.090 ± 0.246 b 34.327 ± 1.032 d 66.393 ± 0.637 a 38.448 ± 1.103 c TP (g/dl) 6.070 ± 0.056 b 7.707 ± 0.176 a 4.167 ± 0.040 c 5.997 ± 0.208 b 3.3. Oxidative Stress Parameters In the BA3 group (boric acid exposure), a significant increase was observed in MDA levels, indicating heightened lipid peroxidation and oxidative damage, whereas the activities of antioxidant defense parameters (TAS, CAT, SOD, and GPx) were significantly reduced compared to the control group (Table 4 ). However, contrary to expectations, TOS and OSI levels were not increased but were actually lower than those in the control group. These findings suggest that, while boric acid exposure predominantly triggers oxidative stress through increased lipid peroxidation (as indicated by elevated MDA levels) and suppression of antioxidant defenses, it does not lead to an increase in total oxidant status or oxidative stress index when compared directly to the control. Table 4 Protective effect of black cumin oil on oxidative stress parameters against boric acid toxicity in Nile tilapia Oxidative Stress Parameters BA 1-Control BA 2 BA 3 BA 4 TAS (mmol/L) 2.142 ± 0.008 c 3.259 ± 0.091 a 1.999 ± 0.018 d 2.475 ± 0.015 b TOS (µmol/L) 26.793 ± 0.628 c 34.629 ± 0.198 a 21.9991 ± 0.557 d 30.228 ± 1.013 b OSI 1.251 ± 0.029 a 1.063 ± 0.034 b 1.100 ± 0.031 b 1.221 ± 0.039 a CAT (U/ml) 216.990 ± 2.062 c 291.940 ± 1.901 a 180.920 ± 3.304 d 263.150 ± 3.847 b SOD (U/ml) 380.877 ± 2.641 c 415.010 ± 2.464 a 338.500 ± 3.153 d 398.430 ± 1.271 b MDA (mmol/L) 79.220 ± 0.886 b 63.400 ± 0.704 d 94.423 ± 0.666 a 74.650 ± 1.759 c GPx (U/ml) 194.063 ± 3.095 c 281.317 ± 1.880 a 131.903 ± 3.161 d 217.283 ± 2.466 b 3.4. Histopathologic Findings Histopathological evaluations of the kidney, liver, and gill tissues were performed using light microscopy (Table 5 ). In Group 1 (control group), no lesions were observed in the gills, and both primary and secondary lamellae exhibited normal histological features. In Group 3, separation of the epithelial cells in the secondary lamellae, fusion of secondary lamellae, and edema in the secondary lamellae were observed (Fig. 4 ). In Group 2, only minimal lesions were detected in the primary and secondary lamellae, and the overall histological appearance of the gills was close to normal. Group 4 gills exhibited minimal lesions compared to Group 3 and appeared largely similar to the normal histological structure. Examination of the liver tissue revealed that, in Group 1, the liver tissue, exocrine pancreas acinar cells, and sinusoidal spaces displayed normal histological architecture. In Group 3, severe hydropic and vacuolar degeneration, pronounced hemorrhages in the liver sinusoidal spaces, hyperactivated melanomacrophages in the liver (black arrow), vacuolization in pancreatic cells, and edema and erythrocytes in the acinar cells of the exocrine pancreas were observed (Fig. 3 ). In Group 2, the histological appearance was generally normal, but mild hemorrhage in the liver sinusoids was noted. In Group 4, minimal lesions were found, and the tissue structure resembled normal histology. Renal tissue showed a normal histological appearance in Group 1. In Group 3, hyperactivated melanomacrophages, vacuolization of the epithelial lining of proximal and distal tubules (T), congestion in the glomerulus (red arrow), and interstitial hemorrhage were recorded (Fig. 1 ). In Group 2, the kidney exhibited a nearly normal histology with only interstitial hemorrhage observed. In Group 4, minimal lesions were detected compared to Group 3, and the general renal tissue architecture was maintained. Table 5 Semiquantitative histopathological scoring of fish tissues in experimental groups exposed to boric acid and dietary black cumin seed oil Tissue Histopathological Alteration BA1 BA2 BA3 BA4 Gill Separation of epithelial cells of the secondary lamellae 0 0–1 2–3 2 Fusion of secondary lamellae 0 0–1 3 1–2 Edema in secondary lamellae 0–1 1–2 2–3 1 Liver Hyperactivated melanomacrophages 0 1–2 2 1 Vacuolization in pancreatic cells 0 0–1 2–3 1–2 Edema and erythrocytes in the acinar cells of the exocrine pancreas 0–1 1–2 2 1–2 Congestion in the liver 0–1 1–2 3 2 Vacuolar degeneration and hydropic degeneration of hepatocytes 0 1–2 3 1–2 Kidney Hyperactivated melanomacrophages 0 1–2 2–3 1 Vacuolization of epithelial lining of proximal and distal tubules 0–1 1–2 2–3 1–2 Congestion in the glomerulus 0 0–1 3 1–2 Interstitial hemorrhage 0 0–1 2 0–1 0: absent; 1: mild; 2: moderate; 3: severe. 4. DISCUSSION In this study, the 96-h LC₅₀ value of boric acid for Nile tilapia ( Oreochromis niloticus ) was 176.446 mg/L, indicating moderate acute toxicity. This value provides an important insight into the species-specific sensitivity of Nile tilapia to boron compounds and is consistent with findings from similar studies on Nile tilapia. For example, Acar, İnanan, Zemheri, Kesbiç, and Yılmaz ( 2018 ) reported a 96-h LC₅₀ value of 141.42 mg/L for Nile tilapia, while Abdel Aliem, Soliman, Khaled, Mourad, and Dighiesh ( 2022 ) found a value of 290 mg/L under different experimental conditions. The observed differences between the experiments demonstrate how water quality and fish physiological state and size together with experimental design affect toxicity results. The evaluation of boron-based compound ecological risks requires site-specific and species-specific assessments for both aquaculture systems and natural water bodies.The observed mortality pattern indicates boric acid toxicity increases with dose and may disrupt cellular and physiological processes including membrane permeability and ion regulation and enzyme activity. Research indicates boron toxicity in aquatic organisms results in oxidative stress and impaired osmoregulation and vital organ histopathological changes in liver gills and kidneys. (Topal et al., 2016 ). LC₅₀ values help determine environmental exposure limits for safety purposes and enable the creation of non-lethal toxicity experiments. The researchers chose 1/20th of the LC₅₀ value for chronic exposure experiments to establish an ecologically relevant concentration which would evaluate long-term physiological and histopathological effects. The study enabled researchers to test dietary black cumin oil as a potential mitigation strategy.The health status of fish depends on hematological and biochemical parameters which serve as indicators of environmental pollutant-induced physiological stress. The exposure to boric acid (BA3 group) resulted in substantial decreases of red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Hct) levels which indicated anemia and reduced oxygen delivery capacity. The current study supports earlier research which demonstrated that boron compounds produce adverse effects on fish hematological parameters. (Acar et al., 2018 ; Çelik et al., 2024 ).The liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were elevated which indicates liver damage. Fish exposed to boric acid have shown similar liver problems. (Topal et al., 2016 ).By contrast, fish in the BA4 group getting nutritional supplements with 1% black cumin oil (Nigella sativa) showed better haematological and biochemical profiles than the BA3 group. Studies on N. sativa's antioxidant and hepatoprotective effects clearly indicate how effective it is in raising haematological markers and liver function in fish. (Mohammed & Arias, 2016 ; Öz et al., 2024 ).Environmental toxins create their harmful effects on aquatic organisms through oxidative stress as a widespread mechanism. The study revealed that boric acid exposure led to elevated malondialdehyde (MDA) levels which indicate lipid peroxidation and reduced activities of catalase (CAT) and superoxide dismutase (SOD) and glutathione peroxidase (GPx) antioxidant enzymes. The observed results match previous research which demonstrated that boric acid induces oxidative stress in fish. (Ertürk Gürkan, Gürkan, Sarıtunç, İbiş, & Güneş, 2025 ; Öz, 2024 ). The N. sativa oil supplement in the BA4 group reduced oxidative stress through lower MDA levels and higher CAT, SOD and GPx activities. The antioxidant effects of N. sativa stem from its bioactive compounds including thymoquinone which neutralizes free radicals while enhancing antioxidant defense pathways. The Nile tilapia kidney tissue (Oreochromis niloticus) functions as a vital organ for both immune system response and detoxification operations. The head kidney of this organ serves as the main hematopoietic center while providing substantial immune defense through its resident lymphocytes and macrophages. The head kidney serves as a vital location for both innate and adaptive immune system development which underscores its essential role in maintaining tilapia health and immune system function. (Gan et al., 2016 ; Mo et al., 2024 ). For example, significant increases in chemokines are associated with immune responses against such infections and facilitate the recruitment of immune cells to effectively combat pathogens (Nakharuthai & Srisapoome, 2020 ). Heavy metal exposure in Nile tilapia kidneys leads to histopathological changes which include glomerular degeneration together with tubular necrosis and vacuolization. The kidneys of fish exposed to di-n-butyl phthalate (DBP) show major histopathological damage through glomerular structural disruption and tubular functional impairment. The observed changes indicate reduced kidney perfusion and filtration efficiency which results in elevated blood levels of urea and creatinine. (Zeid & Khalil, 2014 ). This deterioration shows a clear relationship between environmental pollutants and impaired kidney function; where heavy metals accumulate in kidney tissues, causing cellular damage and functional deficiencies. (Abdel Hakim, Helal, Salem, Zaghloul, & Hanbal, 2016 ). The exposure of fish to dimethoate pesticides results in major histopathological changes that affect the structural integrity of Garra mullya kidneys. The kidneys of control group fish remain healthy with their glomeruli clearly visible and intact. The toxic substance causes kidney function impairment which results in the development of tubular degeneration and necrosis and glomerular alterations after exposure. (Borane, 2016 ). Heavy metals have been shown to produce harmful histological changes in fish kidneys. The toxic effects of metals such as lead, copper, and zinc have been demonstrated through histopathological analyses that show glomerular atrophy, tubular necrosis, and interstitial inflammation. (Al-Balawi et al., 2013 ; Al-Kshab, 2023 ). The importance of pesticides at sub-lethal concentrations, especially malathion and cypermethrin, is of critical importance. These chemicals are known to cause various histological changes in kidney tissues of species like Channa gachua, including hyperplasia, vacuolar degeneration, and cellular apoptosis. (Preeti & Garg, 2023 ; Singh, 2021 ). The study demonstrates that melanomacrophage hyperactivation in kidney tissue results from the inflammatory response which occurs because of elevated ROS levels produced by boric acid-induced oxidative stress. The vacuolization of proximal and distal tubules develops because boric acid disrupts membrane integrity and reduces Na⁺/K⁺-ATPase activity which causes ionic imbalance and increased osmotic pressure. The damage to capillary endothelial cells by boric acid leads to increased vascular permeability which results in blood accumulation and leakage. The toxicological effects of heavy metals and pesticides on fish gill tissues constitute a major concern in aquatic biology because these substances severely affect fish health and ecosystem integrity. The toxic substances cause consistent histopathological changes which appear as hyperplasia together with necrosis and edema. The use of chlorpyrifos pesticides leads to major changes in gill structure and operational capabilities. The exposure to chlorpyrifos results in long-term damage to fish Cyprinus carpio through necrosis and filament fusion of their gills. (Edwin, Ihsan, Rahmatika, & Darlis, 2019 ). Similar observations were documented in research on the Indian Flying Barb, showing increased mucus secretion and filament degeneration following exposure to Endosulfan (Das & Gupta, 2013 ). Studies have shown that lead exposure causes severe gill damage in Tilapia species, particularly leading to edema and necrosis (Batista, Triastuti, & Pursetyo, 2021 ). The research indicates epithelial cell separation in secondary lamellae of gill tissue occurs because boric acid weakens intercellular junction complexes. The fusion of secondary lamellae occurs through epithelial hyperplasia together with increased mucus secretion and inflammatory cell accumulation. The swelling of secondary lamellae occurs because boric acid makes capillary endothelial cells more permeable and disrupts the osmotic balance. The toxicological effects of heavy metals and pesticides on fish liver tissue result in numerous histopathological changes. The liver remains most vulnerable to chemical changes and structural damage because of its detoxification and metabolism roles after toxic substance exposure. Histopathological studies show that heavy metal exposure causes vacuolization of hepatocytes, degeneration of hepatic structure, and infiltration of immune cells which indicates significant tissue damage and stress responses. (Akter et al., 2020 ; Bibi, Naz, Saeed, & Chatha, 2021 ). Exposure to paraquat has been shown to cause liver necrosis and hemorrhage in African catfish ( Clarias gariepinus ) (Ladipo, Doherty, & Oyebadejo, 2011 ). The combined exposure to pesticides and heavy metals produces synergistic toxic effects which intensifies histopathological damage. Fish exposed to both types of pollutants show higher levels of hepatocyte degeneration and necrosis. (Ghafarifarsani et al., 2024 ; Ogbeide & Okoduwa, 2024 ). The research indicates that boric acid leads to liver tissue degeneration through membrane peroxidation in hepatocytes which disrupts mitochondrial function and reduces ATP production. Hemorrhages in sinusoidal spaces are believed to be associated with structural damage to endothelial cells. Oxidative stress, mitochondrial dysfunction, ion imbalance, and protein denaturation caused by boric acid are thought to play roles as common molecular mechanisms in the formation of all these histopathological lesions. CONCLUSION The present study revealed that even non-lethal exposure to waterborne boric acid can significantly impair the physiological, biochemical and histopathological integrity of Nile tilapia (Oreochromis niloticus). In particular, fish exposed to 1/20 of the 96-hour LC₅₀ (176,446 mg/L) exhibited hematological suppression, elevated hepatic enzyme levels, increased oxidative stress biomarkers, and marked tissue damage in gill, liver, kidney and muscle tissues. Dietary supplementation with 1% black cumin (Nigella sativa) oil showed a remarkable protective effect against boric acid-induced toxicity. The black cumin supplemented group showed improved hematological profiles, recovered antioxidant enzyme activity and markedly reduced histopathological lesions compared to the toxicant-only group. These results support the functional role of black cumin oil as a natural antioxidant and tissue protective agent in aquaculture nutrition (Fig. 5 ). Taken together, the findings highlight the dual importance of this research: first, contributing to the toxicological understanding of boric acid as an emerging aquatic contaminant; second, highlighting the efficacy of a phytogenic feed additive in reducing such toxicity. Future research should investigate the dose-dependent effects of Nigella sativa, examine its long-term physiological consequences and assess the interaction between environmental stressors and dietary antioxidants in aquaculture systems. Declarations Author Contributions Mustafa OZ: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Validation; Visualization; review & editing Writing- Original draft preparation. Enes ÜSTÜNER: Taking tissue samples, taking histological sections, pathological evaluation, writing histopathology findings, Data curation , review & editing Writing- Original draft preparation Suat DIKEL: Methodology; Project administration, Data curation Funding While preparing this manuscript, the authors affirm that they did not receive any financial assistance, grants, or other forms of support. The availability of data and material The data supporting the findings of this study can be obtained from the corresponding author upon a reasonable request. Competing interests The authors explicitly affirm that they have no identifiable financial conflicts of interest or personal ties that could have potentially influenced the findings presented in this research. References Abdel-Mohsen, H. A. (2014). Monitoring of Oreochromis responses to metal pollution in Lake Mariut, Alexandria, Egypt. Journal of King Abdulaziz University, 25 (1), 21. Abdel Aliem, R., Soliman, B., Khaled, H., Mourad, M., & Dighiesh, H. (2022). Toxicological effect of boric acid and cadmium chloride on the Nile tilapia, Oreochromis niloticus. Egyptian J Aquatic Biol Fish, 26 (5), 667-680. Abdel Hakim, N. F., Helal, A. F., Salem, M. F., Zaghloul, A. M., & Hanbal, M. M. (2016). Effect of some heavy metals on physiological and chemical parameters in Nile tilapia (Oreochromis niloticus L.). Journal of Egyptian Academic Society for Environmental Development. D, Environmental Studies, 17 (1), 81-95. Acar, Ü., İnanan, B. E., Zemheri, F., Kesbiç, O. S., & Yılmaz, S. (2018). Acute exposure to boron in Nile tilapia (Oreochromis niloticus): Median-lethal concentration (LC50), blood parameters, DNA fragmentation of blood and sperm cells. Chemosphere, 213 , 345-350. Aebi, H. (1984). [13] Catalase in vitro. In Methods in enzymology (Vol. 105, pp. 121-126): Elsevier. Akter, R., Pervin, M. A., Jahan, H., Rakhi, S. F., Reza, A. M., & Hossain, Z. (2020). Toxic effects of an organophosphate pesticide, envoy 50 SC on the histopathological, hematological, and brain acetylcholinesterase activities in stinging catfish (Heteropneustes fossilis). The Journal of Basic and Applied Zoology, 81 , 1-14. Al-Balawi, H. F. A., Al-Akel, A. S., Al-Misned, F., Suliman, E. A. M., Al-Ghanim, K. A., Mahboob, S., & Ahmad, Z. (2013). Effects of sub-lethal exposure of lead acetate on histopathology of gills, liver, kidney and muscle and its accumulation in these organs of Clarias gariepinus. Brazilian archives of biology and technology, 56 , 293-302. Al-Kshab, A. A. (2023). Physiological and histopathological effect of lead chloride on kidney of Gambusia affinis. Iraqi Journal of Agricultural Sciences, 54 (6), 1574-1582. Baransi-Karkaby, K., Bass, M., & Freger, V. (2019). In situ modification of reverse osmosis membrane elements for enhanced removal of multiple micropollutants. Membranes, 9 (2), 28. Batista, F. R., Triastuti, J., & Pursetyo, K. T. (2021). The Role of Salinity in Histopathology Description of Jatim Bulan Tilapia Juvenile (Oreochromis niloticus) Exposed by Lead (PB). World's Veterinary Journal, 11 (3), 448-455. Bibi, S., Naz, S., Saeed, S., & Chatha, A. M. M. (2021). A review on histopathological alterations induced by heavy metals (Cd, Ni, Cr, Hg) in different fish species. Punjab Univ J Zool, 36 (1), 81-89. Blaxhall, P., & Daisley, K. (1973). Routine haematological methods for use with fish blood. Journal of fish biology, 5 (6), 771-781. Borane, V. (2016). Histopathological Impact of Dimethoate on the Liver of Freshwater Fish, Garra mullya (Sykes). Int. J. Life. Sci. Scienti. Res, 2 (6), 708-711. Bordoni, L., Fedeli, D., Nasuti, C., Maggi, F., Papa, F., Wabitsch, M., . . . Gabbianelli, R. (2019). Antioxidant and anti-inflammatory properties of Nigella sativa oil in human pre-adipocytes. Antioxidants, 8 (2), 51. Culling, C. F. A., Allison, R., & Barr, W. (2014). Cellular pathology technique : Elsevier. Çelik, M., Dikel, S., & Öz, M. (2024). Investigation of the effect of water and feed sourced boron on the growth performance and blood parameters of Nile tilapia, Oreochromis niloticus. Journal of the World Aquaculture Society, 55 (6), e13104. Das, S., & Gupta, A. (2013). Effect of endosulfan (EC 35) on oxygen consumption patterns and gill morphology of the Indian flying Barb, Esomus danricus. Ceylon Journal of Science (Biological Sciences), 41 (2). Edwin, T., Ihsan, T., Rahmatika, A., & Darlis, N. (2019). Impact of chlorpyrifos toxicity on gill damage of two species of freshwater fish in Lake Diatas. Environmental Health Engineering and Management Journal, 6 (4), 241-246. Erel, O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical biochemistry, 37 (4), 277-285. Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical biochemistry, 38 (12), 1103-1111. Ertürk Gürkan, S., Gürkan, M., Sarıtunç, V., İbiş, E. C., & Güneş, B. (2025). Evaluation of Possible Toxic Effects of Boric Acid in Palourde Clam (Ruditapes decussatus) Through Histological Changes and Oxidative Responses. Biological Trace Element Research, 203 (2), 1151-1161. Finney, D. (1971). Probit analysis, Cambridge University Press. Cambridge, UK . Gaikwad, J., Gaikwad, S., & Kamble, N. (2021). Boric acid toxicity in some selected molluscan species. Int J Biol Environ Investig, 1 , 49-61. Gan, Z., Wang, B., Zhou, W., Lu, Y., Zhang, Y., Jian, J., . . . Nie, P. (2016). Molecular characterization and expression of ZAP-70 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus. Genes & Genomics, 38 , 321-331. Ghafarifarsani, H., Rohani, M. F., Raeeszadeh, M., Ahani, S., Yousefi, M., Talebi, M., & Hossain, M. S. (2024). Pesticides and heavy metal toxicity in fish and possible remediation–a review. Annals of Animal Science, 24 (4), 1007-1024. Harma, M., & Erel, O. (2003). Increased oxidative stress in patients with hydatidiform mole. Swiss medical weekly, 133 (4142), 563-566. Hosseinzadeh, L., Monaghash, H., Ahmadi, F., Ghiasvand, N., & Shokoohinia, Y. (2017). Bioassay-guided isolation of neuroprotective fatty acids from nigella sativa against 1-methyl-4-phenylpyridinium-induced neurotoxicity. Pharmacognosy magazine, 13 (52), 627. Kan, F., & Kucukkurt, I. (2023). The effects of boron on some biochemical parameters: a review. Journal of Trace Elements in Medicine and Biology, 79 , 127249. Kosecik, M., Erel, O., Sevinc, E., & Selek, S. (2005). Increased oxidative stress in children exposed to passive smoking. International journal of cardiology, 100 (1), 61-64. Kraus, R. J., & Ganther, H. E. (1980). Reaction of cyanide with glutathione peroxidase. Biochemical and biophysical research communications, 96 (3), 1116-1122. Ladipo, M., Doherty, V., & Oyebadejo, S. (2011). Acute toxicity, behavioural changes and histopathological effect of paraquat dichloride on tissues of catfish (Clarias gariepinus). International Journal of Biology, 3 (2), 67. Latif, M., Faheem, M., Asmatullah, Hoseinifar, S. H., & Van Doan, H. (2020). Dietary black seed effects on growth performance, proximate composition, antioxidant and histo-biochemical parameters of a culturable fish, rohu (Labeo rohita). Animals, 11 (1), 48. Mo, J., Li, J., Qiu, L., Wang, Y., Mu, L., & Ye, J. (2024). Collectin-K1 Plays a Role in the Clearance of Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus). International Journal of Molecular Sciences, 25 (5), 2508. Mohammed, H. H., & Arias, C. R. (2016). Protective efficacy of N igella sativa seeds and oil against columnaris disease in fishes. Journal of Fish Diseases, 39 (6), 693-703. Nakharuthai, C., & Srisapoome, P. (2020). Molecular identification and dual functions of two different CXC chemokines in Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare. Microorganisms, 8 (7), 1058. Ogbeide, O., & Okoduwa, K. (2024). Impact of Urban Runoff on Benthic and Pelagic Fish Fauna in Ikpoba River: Heavy Metals and Pathology of Liver Tissues. NIPES-Journal of Science and Technology Research, 6 (2). Öz, M. (2024). Effects of boric acid on oxidative stress parameters, growth performance and blood parameters of rainbow trout (Oncorhynchus Mykiss). Biological Trace Element Research , 1-9. Öz, M., Dikel, S., & Durmus, M. (2018). Effect of black cumin oil (Nigella sativa) on the growth performance, body composition and fatty acid profile of rainbow trout (Oncorhynchus mykiss). Iranian Journal of Fisheries Sciences, 17 (4), 713-724. Öz, M., Üstüner, E., & Bölükbaş, F. (2024). Effects of dietary black cumin (Nigella sativa L.) oil on growth performance, hemato‐biochemical and histopathology of cypermethrin‐intoxicated Nile tilapia (Oreochromis niloticus). Journal of the World Aquaculture Society, 55 (1), 273-288. Öz, M., Yavuz, O., & Bolukbas, F. (2020). Histopathology changes in the rainbow trout (Onchorhyncus mykiss) consuming boric acid supplemented fish fodder. Journal of Trace Elements in Medicine and Biology, 62 , 126581. Paglia, D. E., & Valentine, W. N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. The Journal of laboratory and clinical medicine, 70 (1), 158-169. Preeti, A., & Garg, V. (2023). Histopathological Alteration in Tissues of Channa Punctatus (Bloch) Induced by Lambda-Cyhalothrin. International Journal of Zoological Investigations, 9 (2), 92-99. doi:10.33745/ijzi.2023.v09i02.010 Prohaska, J., Oh, S.-H., Hoekstra, W., & Ganther, H. (1977). Glutathione peroxidase: inhibition by cyanide and release of selenium. Biochemical and biophysical research communications, 74 (1), 64-71. See, A. S., Salleh, A. B., Bakar, F. A., Yusof, N. A., Abdulamir, A. S., & Heng, L. Y. (2010). Risk and Health Effect of Boric Acid. American Journal of Applied Sciences, 7 (5). doi:10.3844/ajassp.2010.620.627 Shaalan, W. M. (2024). Hazardous Effects of Heavy Metal Pollution on Histological and Gene Expression Profiles of Nile Tilapia in the Eastern Delta, Egypt Aquatic Ecosystems. Singh, B. M. (2021). Histopathological Study of Channa Gachua Exposed to Sublethal Concentration of Malathion. International Journal of Fisheries and Aquatic Studies, 9 (3), 174-179. doi:10.22271/fish.2021.v9.i3c.2472 Sun, Y., Oberley, L. W., & Li, Y. (1988). A simple method for clinical assay of superoxide dismutase. Clinical chemistry, 34 (3), 497-500. Topal, A., Oruç, E., Altun, S., Ceyhun, S. B., & Atamanalp, M. (2016). The effects of acute boric acid treatment on gill, kidney and muscle tissues in juvenile rainbow trout. Journal of Applied Animal Research, 44 (1), 297-302. Yumru, M., Savas, H. A., Kalenderoglu, A., Bulut, M., Celik, H., & Erel, O. (2009). Oxidative imbalance in bipolar disorder subtypes: a comparative study. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 33 (6), 1070-1074. Zeid, E. A., & Khalil, A. (2014). Toxicological consequences of Di-n-Butyl-Phthalate (DBP) on health of Nile Tilapia fingerlings. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6515353\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":447625397,\"identity\":\"d12f7e44-58ec-4ce9-8346-285c64597bed\",\"order_by\":0,\"name\":\"Mustafa ÖZ\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBADfgb2BmY4T4KwhgQGyQaeAyhaDIjQIpFApBZ+/tNpkj9/2Enwz3z82PBHzT15gwPMB2/zMPzJx6VFsuHsNmmehGQJidtpxsk8x4oNNxxgS7bmYTCwbMChxeBg7zZphgTmOobbCcaHGdgSGDcc4DGTBmrB6TKDw7zbJH8k1EvI3zz++eCPfwn2Gw7wf8Ov5RjvNgmehMMSBjd4jBN42xISgbaw4dUi2cO72Zon7biE4ZmcYmPevoTkmYfZjC3nGBjj1MLPf3bjzR821RJyx49vlvzxLcG273jzwxtvKuQIRQwSUDgMdjDxGhgY5BtIUT0KRsEoGAUjAQAAA6JRPX2WWJ8AAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0001-5264-7103\",\"institution\":\"Aksaray University: Aksaray Universitesi\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Mustafa\",\"middleName\":\"\",\"lastName\":\"ÖZ\",\"suffix\":\"\"},{\"id\":447625398,\"identity\":\"71aeed51-3796-43f9-aec5-8b5035c29288\",\"order_by\":1,\"name\":\"Enes ÜSTÜNER\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Aksaray University: Aksaray Universitesi\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Enes\",\"middleName\":\"\",\"lastName\":\"ÜSTÜNER\",\"suffix\":\"\"},{\"id\":447625399,\"identity\":\"38fc046d-3fc4-4991-92a3-4b553e994175\",\"order_by\":2,\"name\":\"Suat DİKEL\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Cukurova University: Cukurova Universitesi\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Suat\",\"middleName\":\"\",\"lastName\":\"DİKEL\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-04-23 20:16:04\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6515353/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6515353/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81536409,\"identity\":\"d489eb4c-5048-4973-85ba-7d49021cd17c\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 10:22:18\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":16820,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProbit regression curve used to determine the 96-hour LC₅₀ of boric acid in Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/5f5566729e1b9910eae5856a.jpg\"},{\"id\":81536412,\"identity\":\"90fc96f3-ae73-4436-9ece-dc5000115201\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 10:22:18\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":278829,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Normal histological appearance of the kidney of Nile tilapia (\\u003cem\\u003eO. niloticus\\u003c/em\\u003e); Group 1, hematoxylin and eosin stain (H\\u0026amp;E). \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Hyperactivated melanomacrophages (black arrow), vacuolization of the epithelial lining of proximal and distal tubules (T) (black lines), congestion in the glomerulus (red arrow), and interstitial hemorrhage (blue arrow) in the kidney; Group 3, hematoxylin and eosin stain (H\\u0026amp;E). \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Interstitial hemorrhage (black arrow) in the kidney; Group 2, hematoxylin and eosin stain (H\\u0026amp;E). \\u003cstrong\\u003e(D)\\u003c/strong\\u003eHyperactivated melanomacrophages (black arrow) and interstitial hemorrhage (red arrow) in the kidney; Group 4, hematoxylin and eosin stain (H\\u0026amp;E).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/828be4f9c97cf650cdbf8a9a.jpg\"},{\"id\":81537388,\"identity\":\"06356c95-75f8-4bf6-aa9d-f87b7c4fcb1e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 10:30:18\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":324048,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Histological appearance of normal liver in Nile tilapia (\\u003cem\\u003eO. niloticus\\u003c/em\\u003e), showing acini of the exocrine pancreas (PA) and liver sinusoids (S), Group 1, stained with hematoxylin and eosin (H\\u0026amp;E).\\u003cstrong\\u003e(B) \\u003c/strong\\u003eHyperactivated melanomacrophages observed in the liver (black arrow), vacuolization in pancreatic cells (red arrow), and edema and erythrocytes in the acinar cells of the exocrine pancreas (asterisk); Group 3, stained with hematoxylin and eosin (H\\u0026amp;E).\\u003cstrong\\u003e(C) \\u003c/strong\\u003eCongestion in the liver (black arrow); Group 2, stained with hematoxylin and eosin (H\\u0026amp;E).\\u003cstrong\\u003e(D) \\u003c/strong\\u003eVacuolar degeneration and hydropic degeneration of hepatocytes in the liver (black arrow); Group 4, stained with hematoxylin and eosin (H\\u0026amp;E).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/103889b05d5c69235537829a.jpg\"},{\"id\":81536418,\"identity\":\"6435a1c5-845b-4dc7-86d3-9623ad23ee69\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 10:22:18\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":290588,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGill tissue of Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e). \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Normal histological image of Nile tilapia (O. niloticus) gills; Group 1, hematoxylin-eosin staining (H\\u0026amp;E). \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Separation of epithelial cells of the secondary lamellae (black arrow), fusion of secondary lamellae (red arrow); Group 3, hematoxylin-eosin staining (H\\u0026amp;E). \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Fusion of secondary lamellae (star); Group 2, hematoxylin-eosin staining (H\\u0026amp;E). \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Separation of epithelial cells of the secondary lamellae (black arrow), edema in secondary lamellae (red arrow); Group 4, hematoxylin-eosin staining (H\\u0026amp;E).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/184540de1af67fd904cc2e1d.jpg\"},{\"id\":81536416,\"identity\":\"87ddb332-2d3c-4bcd-b5fb-b6581884a9e2\",\"added_by\":\"auto\",\"created_at\":\"2025-04-28 10:22:18\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":78936,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic representation of the toxic mechanisms induced by waterborne boric acid exposure and the protective effects of dietary black cumin (\\u003cem\\u003eNigella sativa L\\u003c/em\\u003e.) oil in Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/fc1ec1513a9fdf087990cc12.jpg\"},{\"id\":81784970,\"identity\":\"8ab26aee-64bf-4cd1-9097-0aaaaaf9a5bb\",\"added_by\":\"auto\",\"created_at\":\"2025-05-01 20:08:40\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1962227,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6515353/v1/48df55ef-5216-415e-b438-210507435e2b.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Toxicological Effects of Waterborne Boric Acid and the Protective Role of Dietary Black Cumin (Nigella sativa L.) Oil in Nile Tilapia (Oreochromis niloticus): Hematological, Biochemical, Oxidative Stress and Histopathological Responses\",\"fulltext\":[{\"header\":\"1. INTRODUCTION\",\"content\":\"\\u003cp\\u003eAquatic ecosystems worldwide face increasing risks from a variety of chemical contaminants emanating from industrial, agricultural, and domestic sources. Among these contaminants, boric acid (H₃BO₃) has emerged as a pollutant of concern due to its widespread use in glass production, ceramics, agriculture, and even in certain antiseptic formulations (Kan \\u0026amp; Kucukkurt, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Natural sources of boric acid typically include seawater and geological formations, where it is found in water layers at concentrations exceeding 4 mg/L (Baransi-Karkaby, Bass, \\u0026amp; Freger, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). In freshwater environments, boric acid can bioaccumulate and may lead to toxicity in aquatic organisms, particularly when concentrations exceed threshold values of 0.5 to 1 ppm (Gaikwad, Gaikwad, \\u0026amp; Kamble, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The application of boric acid as a pesticide in urban areas increases the likelihood of surface runoff during rainfall, enhancing its presence in nearby water bodies (See et al., \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e).Boric acid reaching aquatic ecosystems has been shown to cause histopathological damage in fish tissues and negatively affect blood parameters (\\u0026Ouml;z, Yavuz, \\u0026amp; Bolukbas, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Topal, Oru\\u0026ccedil;, Altun, Ceyhun, \\u0026amp; Atamanalp, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). In recent years, environmental toxicology has witnessed increasing interest in identifying natural protective substances that may reduce the toxic effects of environmental pollutants. Black seed (\\u003cem\\u003eNigella sativa L\\u003c/em\\u003e.) oil has attracted considerable attention due to its rich composition of bioactive compounds such as thymoquinone, which exhibits strong antioxidant, anti-inflammatory, and immunomodulatory properties (Bordoni et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Thymoquinone enhances antioxidant enzyme activities which supports its protective effects against oxidative damage according to multiple animal model studies. (Hosseinzadeh, Monaghash, Ahmadi, Ghiasvand, \\u0026amp; Shokoohinia, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). The toxic effects can be counteracted by using herbal supplements that contain natural antioxidants in aquaculture operations. The bioactive compounds thymoquinone and others in \\u003cem\\u003eNigella sativa\\u003c/em\\u003e (black cumin) oil provide antioxidant and anti-inflammatory and hepatoprotective effects. Research shows that adding black cumin seeds and oil to fish feed enhances growth rates while decreasing oxidative stress. (Latif, Faheem, Asmatullah, Hoseinifar, \\u0026amp; Van Doan, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; \\u0026Ouml;z, Dikel, \\u0026amp; Durmus, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; \\u0026Ouml;z, \\u0026Uuml;st\\u0026uuml;ner, \\u0026amp; B\\u0026ouml;l\\u0026uuml;kbaş, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e) serves as an appropriate model species for toxicological research in aquatic ecosystems because of several key factors. The species stands as a valuable organism for studying environmental stressors because it adapts to different conditions and shows strong physiological responses to pollutants and plays important ecological roles in freshwater habitats. The Nile tilapia stands out because of its ability to adapt to changing water conditions which makes it essential for toxicology research. The species demonstrates wide tolerance to environmental factors including salinity and temperature fluctuations and pollution while thriving in both high and low-quality aquatic environments. (Abdel-Mohsen, \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). The researchers can study pollution effects under different water conditions because of this natural ability to adapt. This ability enables researchers to study pollution effects under realistic ecological conditions. Research indicates Nile tilapia maintains survival capabilities in polluted environments containing heavy metals which makes it an ideal species for detecting heavy metal exposure in aquatic ecosystems. (Shaalan, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The research aims to assess the hematological, biochemical, oxidative stress and histopathological effects of boric acid exposure in aquatic environment on Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e) and to investigate the protective role of dietary black cumin oil against these toxic effects.\\u003c/p\\u003e\"},{\"header\":\"2. MATERIALS AND METHODS\",\"content\":\"\\u003ch2\\u003e\\u003cstrong\\u003e2.1. Research Design\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eEthical approval was obtained from \\u0026Ccedil;ukurova University ethics committee (Ref No: 7/2020). Feeding activities were carried out at \\u0026Ccedil;ukurova University, Faculty of Fisheries, Dr. Nazmi Tekelioğlu Freshwater Fisheries Production and Research Station. In order to determine the appropriate boron dosage for our study, we initially estimated the LC50 (96 h) value by probit analysis (Finney, 1971). A total of 120 fish were used to calculate the LC50 value and boron was applied at six different concentrations (0.00, 10.00, 50.00, 100.00, 150.00 and 200.00 mg/L) (\\u0026Ccedil;elik, Dikel, \\u0026amp; \\u0026Ouml;z, 2024). During the evaluation of the LC50 value, the fish were monitored three times a day for 96 hours and the dead fish were immediately removed from the environment. During the LC50 experiment, oxygen was continuously supplied to 50 liter capacity aquariums and water was changed every 24 hours using prepared stocks. In addition, the fish were not fed during the experiment. To determine the LC50 value, 120 fish were used and the experiment was designed in two replicates. In the feeding trial, 108 fish were used. A total of 228 male Nile tilapia with a starting weight of 36.38\\u0026plusmn;0.83 g were used in the study. The research was carried out in 80 liter aquariums with 9 fish in 3 replications and 27 fish were used in each group. Boric acid was added to the water of the research groups at the rate of 1/20 of the determined Lc50 value. \\u0026nbsp;Eheim brand 100 Watt thermostat heater was used to keep the water temperature constant and the water temperature was kept constant at 25 0C in all groups during the research. The research groups and fish numbers are shown in detail in Table 1.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1.\\u0026nbsp;\\u003c/strong\\u003eResearch groups, black cumin seed oil ratios, boric acid amount and fish numbers\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGroups\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eBlack Cumin oil in the feed (%)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eThe amount of Boric Acid in the water (mg/L)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eNumbers of fish\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBA1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27 (3*9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBA2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27 (3*9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBA3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLC\\u003csub\\u003e50\\u003c/sub\\u003e/20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27 (3*9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBA4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLC\\u003csub\\u003e50\\u003c/sub\\u003e/20\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27 (3*9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"3\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTotal\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e108\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e2.2.\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ePreparation of Experimental Diets and Feeding Protocol\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThe dietary regime used in the trial was based on commercially available tilapia feed (Hem Yem, Gaziantep, Turkey) containing 39% crude protein, 6.7% crude fat, 4.30% crude fiber and 6.79% crude ash. The cold-pressed black seed oil utilised in the study was procured from a commercial supplier. According to the protocol adapted from \\u0026Ouml;z et al. (2018), 1% black cumin oil was added to the diets of experimental groups BA3 and BA4. The feeds were prepared in 100 g portions to ensure accuracy and uniformity in the supplementation \\u0026nbsp;process. \\u0026nbsp; \\u0026nbsp;To enhance coating quality, black cumin oil was systematically blended with 2 ml of \\u0026nbsp;sunflower oil and subsequently uniformly sprayed onto the feed. \\u0026nbsp; \\u0026nbsp;In the control oil groups, 3 ml \\u0026nbsp; of sunflower oil was incorporated to standardise the total lipid volume across all treatments and to maintain dietary \\u0026nbsp; consistency among the groups. The feeding trial started one day after the first biometric assessments and continued for 21 days. Throughout the study, fish were fed twice daily - once at 08:30 am and once at 16:30 pm - at a rate equivalent to 2% of their total body weight.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;2.3. Blood Sampling and Hematological Procedures\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAt the end of the feeding trial, fish were anesthetized using 2-phenoxyethanol at a concentration of 300 ppm to ensure minimal stress during sampling. Following anesthesia, each sample was carefully rinsed with 70% ethanol to minimize external contamination. Blood samples were then collected from the caudal vein using heparinized syringes. For hematological analyses, a portion of each blood sample was transferred into lavender-colored tubes containing ethylenediaminetetetraacetic acid (EDTA) as anticoagulant. Another portion was placed in serum separator tubes (SST\\u0026trade; II Advance, red cap) for biochemical analysis. These samples were centrifuged at 13,000 \\u0026times; g for 10 min at 4 \\u0026deg;C to obtain clear serum. Hematologic parameters were assessed immediately after collection, while serum samples were stored at -80 \\u0026deg;C until biochemical assessments were performed. Hematological parameters such as red blood cell count (RBC), hematocrit (Hct), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were analyzed using an MS4-S hematology autoanalyzer (Melet Schloesing Laboratories, Osny, France). The precision of automated results was verified through manual counts of K₃EDTA tube samples that followed \\u0026nbsp;the protocol established by (Blaxhall \\u0026amp; Daisley, 1973).\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e2.4. Determination of Serum Biochemical Parameters\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThe biochemical parameters assessed in blood serum comprised alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), triglycerides (TRG), cholesterol (CHO), and glucose (GLU). These indicators were chosen for their appropriateness in evaluating the metabolic and physiological condition of fish. All biochemical assays were conducted via an automated clinical chemistry analyser (MINDRAY BS-400) following the manufacturer\\u0026apos;s guidelines.\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e2.5. Oxidative Stress Parameters\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eSera extracted from fish blood samples were analysed for Total Antioxidant Status (TAS), Total Oxidant Status (SOD), Superoxide Dismutase (CAT), Catase, GPx, Glutathione Peroxidase, and MDA (malondialdehyde). \\u0026nbsp;Relassay (Cat no: RL0017) commercial assays (Erel, 2004) were employed to quantify TAS levels. \\u0026nbsp;Relassay (Cat no: RL0024) commercial assays were employed to assess Total Oxidant Status (TOS) levels. (Erel, 2004). Total Oxidant Status (TOS) levels were evaluated using Relassay (Cat no: RL0024) commercial kits (Erel, 2005). The ratio of TOS to TAS is considered as oxidative stress index (OSI). The TAS unit obtained for calculation was converted to \\u0026mu;mol/L and the OSI value was calculated according to the following formula.\\u003c/p\\u003e\\n\\u003cp\\u003eOSI = TOS (\\u0026mu;mol H2O2 equivalent/L) / TAS (\\u0026mu;mol Trolox equivalent/L) (Harma \\u0026amp; Erel, 2003; Kosecik, Erel, Sevinc, \\u0026amp; Selek, 2005; Yumru et al., 2009).\\u003c/p\\u003e\\n\\u003cp id=\\\"_Toc179459533\\\"\\u003eMalondialdehyde (MDA) level, a product of lipid peroxidation, was determined according to Alak et al. Superoxide dismutase (SOD) enzyme activity was determined by spectrophotometer (560 nm) according to NBT (nitro blue tetrazolium chloride) reduction method with O-2 under light\\u0026nbsp;(Sun, Oberley, \\u0026amp; Li, 1988). Measurement of catalase (CAT) activity; samples were mixed with 1 mL H2O2 (50 mM) and reacted at 37 \\u0026deg;C for 1 min. Then 1 mL of ammonium molybdate was added to terminate the reaction, resulting in the formation of a yellowish complex containing residual H2O2. Finally, the UV-vis absorption of this complex was measured at 405 nm by a microplate reader (Aebi, 1984). Measurement of glutathione peroxidase (GPx) activity; Glutathione Peroxidase (GPx) catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione (GSSG), it is immediately converted to the reduced form by simultaneous oxidation of NADPH to NADP. GPx activity was measured by a change in absorbance (decrease in readings over 3 min) at 340 nm (Kraus \\u0026amp; Ganther, 1980; Paglia \\u0026amp; Valentine, 1967; Prohaska, Oh, Hoekstra, \\u0026amp; Ganther, 1977).\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e2.6. Histopathologic Investigation\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eGill, liver, and kidney tissues of Nile tilapia were fixed in 0.1 M phosphate-buffered formaldehyde (pH 7.4). After trimming, tissues were washed under slow running water for 24 hours, dehydrated through an ethanol series, and treated with xylene and xylene-paraffin. Samples were incubated in soft paraffin (46\\u0026ndash;48\\u0026deg;C) for 15 minutes and hard paraffin (56\\u0026ndash;58\\u0026deg;C) for 30 minutes before embedding with a Leica EG 1150 H. Sections of 4 \\u0026micro;m thickness were cut using a Leica RM2125 Rotary Microtome and stained with Haematoxylin-Eosin. Sections were mounted on glass slides using Entellan Merck, examined by light microscopy (Leica DM-750), and photographed in lesion areas (Culling, Allison, \\u0026amp; Barr, 2014).\\u003c/p\\u003e\\n\\u003cp\\u003eHematoxylin-Eosin (H-E) staining of the gill, liver and kidney tissues was used to score the pathological findings on a scale of \\u0026ldquo;(-) to 3\\u0026rdquo; (-: none, 1: mildly severe, 2: moderately severe and 3: severe).\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e2.7. Statistical Analyses\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eStatistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL. USA) to determine the differences between the groups in growth parameters and changes in blood parameters of fish fed with feed supplemented with ALE at different rates.\\u003c/p\\u003e\"},{\"header\":\"3. RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Determination of LC\\u003csub\\u003e50\\u003c/sub\\u003e Value\\u003c/h2\\u003e \\u003cp\\u003eBased on the 96-hour acute toxicity test, the LC₅₀ value of boric acid for Nile tilapia (Oreochromis niloticus) was calculated to be 176.446 mg/L. Regression analysis using the Probit method gave the equation y\\u0026thinsp;=\\u0026thinsp;1.24x\\u0026thinsp;+\\u0026thinsp;2.2103 with a coefficient of determination of R\\u0026sup2; = 0.8846, indicating a strong correlation between boric acid concentration and mortality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Blood Parameters\\u003c/h2\\u003e \\u003cp\\u003eThe results of hematological analysis of Nile tilapia subjected to different treatments are presented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. Significant differences in red blood cell indices were observed between the experimental groups, indicating that both boric acid exposure and black cumin oil supplementation affected hematological profiles.\\u003c/p\\u003e \\u003cp\\u003eSimilarly, serum biochemical parameters including liver enzymes, glucose, lipid profile and total protein levels are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e. The data reflect the physiological responses of fish to boric acid toxicity and the potential ameliorative effects of dietary black cumin oil.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eProtective effect of black cumin oil on blood parameters parameters against boric acid toxicity in Nile tilapia\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBA 1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBA 2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eBA 3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eBA 4\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eRBC (m/mm3)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.692\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.029\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.230\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.026\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.357\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.019\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.913\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.014\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHb (g/dl)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e13.517\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.306\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e16.100\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.167\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e11.550\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.176\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e14.050\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.259\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eHct (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e37.550\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.243\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e46.733\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.097\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e31.733\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.442\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e40.750\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.666\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMCV (fL)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e222.019\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.635\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e209.565\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.178\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e233.923\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10.689\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e212.971\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.459\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMCH (pg)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e79.918\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.116\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e72.199\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.414\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e85.146\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.572\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e73.441\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.759\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMCHC (g/dL)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e35.998\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.849\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e34.463\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.708\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e36.469\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.960\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e34.491\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.047\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eProtective effect of black cumin oil on blood biochemistry parameters against boric acid toxicity in Nile tilapia\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBA 1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBA 2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eBA 3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eBA 4\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCho (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e151.370\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.834\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e132.530\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.777\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e182.027\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.558\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e140.270\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.846\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTrg (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e154.780\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.551\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e128.337\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.382\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e166.930\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.048\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e142.023\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.835\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGlu (mg/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e46.312\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.896\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e37.828\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.485\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e69.010\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.624\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e42.760\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.685\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eALP (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e40.586\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.576\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e32.663\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.568\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e56.693\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.458\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e44.545\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.625\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAST (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e252.640\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.515\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e190.717\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.033\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e339.439\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.566\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e215.582\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.296\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eALT (U/L)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e44.090\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.246\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e34.327\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.032\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e66.393\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.637\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e38.448\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.103\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTP (g/dl)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.070\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.056\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.707\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.176\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e4.167\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.040\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5.997\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.208\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Oxidative Stress Parameters\\u003c/h2\\u003e \\u003cp\\u003eIn the BA3 group (boric acid exposure), a significant increase was observed in MDA levels, indicating heightened lipid peroxidation and oxidative damage, whereas the activities of antioxidant defense parameters (TAS, CAT, SOD, and GPx) were significantly reduced compared to the control group (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). However, contrary to expectations, TOS and OSI levels were not increased but were actually lower than those in the control group. These findings suggest that, while boric acid exposure predominantly triggers oxidative stress through increased lipid peroxidation (as indicated by elevated MDA levels) and suppression of antioxidant defenses, it does not lead to an increase in total oxidant status or oxidative stress index when compared directly to the control.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eProtective effect of black cumin oil on oxidative stress parameters against boric acid toxicity in Nile tilapia\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c5\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003eOxidative Stress Parameters\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBA 1-Control\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBA 2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eBA 3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eBA 4\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTAS (mmol/L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.142\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.008\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.259\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.091\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.999\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.018\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.475\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.015\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTOS (\\u0026micro;mol/L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e26.793\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.628\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e34.629\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.198\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e21.9991\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.557\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e30.228\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.013\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eOSI\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.251\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.029\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.063\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.034\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.100\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.031\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.221\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.039\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eCAT (U/ml)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e216.990\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.062\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e291.940\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.901\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e180.920\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.304\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e263.150\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.847\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eSOD (U/ml)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e380.877\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.641\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e415.010\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.464\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e338.500\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.153\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e398.430\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.271\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMDA (mmol/L)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e79.220\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.886\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e63.400\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.704\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e94.423\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.666\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e74.650\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.759\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eGPx (U/ml)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e194.063\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.095\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e281.317\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.880\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e131.903\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.161\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e217.283\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.466\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Histopathologic Findings\\u003c/h2\\u003e \\u003cp\\u003eHistopathological evaluations of the kidney, liver, and gill tissues were performed using light microscopy (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). In Group 1 (control group), no lesions were observed in the gills, and both primary and secondary lamellae exhibited normal histological features. In Group 3, separation of the epithelial cells in the secondary lamellae, fusion of secondary lamellae, and edema in the secondary lamellae were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). In Group 2, only minimal lesions were detected in the primary and secondary lamellae, and the overall histological appearance of the gills was close to normal. Group 4 gills exhibited minimal lesions compared to Group 3 and appeared largely similar to the normal histological structure. Examination of the liver tissue revealed that, in Group 1, the liver tissue, exocrine pancreas acinar cells, and sinusoidal spaces displayed normal histological architecture. In Group 3, severe hydropic and vacuolar degeneration, pronounced hemorrhages in the liver sinusoidal spaces, hyperactivated melanomacrophages in the liver (black arrow), vacuolization in pancreatic cells, and edema and erythrocytes in the acinar cells of the exocrine pancreas were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). In Group 2, the histological appearance was generally normal, but mild hemorrhage in the liver sinusoids was noted. In Group 4, minimal lesions were found, and the tissue structure resembled normal histology. Renal tissue showed a normal histological appearance in Group 1. In Group 3, hyperactivated melanomacrophages, vacuolization of the epithelial lining of proximal and distal tubules (T), congestion in the glomerulus (red arrow), and interstitial hemorrhage were recorded (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). In Group 2, the kidney exhibited a nearly normal histology with only interstitial hemorrhage observed. In Group 4, minimal lesions were detected compared to Group 3, and the general renal tissue architecture was maintained.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab5\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 5\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSemiquantitative histopathological scoring of fish tissues in experimental groups exposed to boric acid and dietary black cumin seed oil\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTissue\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eHistopathological Alteration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBA1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eBA2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eBA3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eBA4\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eGill\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSeparation of epithelial cells of the secondary lamellae\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u0026ndash;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eFusion of secondary lamellae\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eEdema in secondary lamellae\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u0026ndash;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eLiver\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eHyperactivated melanomacrophages\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eVacuolization in pancreatic cells\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u0026ndash;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eEdema and erythrocytes in the acinar cells of the exocrine pancreas\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCongestion in the liver\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eVacuolar degeneration and hydropic degeneration of hepatocytes\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eKidney\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eHyperactivated melanomacrophages\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u0026ndash;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eVacuolization of epithelial lining of proximal and distal tubules\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u0026ndash;3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCongestion in the glomerulus\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1\\u0026ndash;2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eInterstitial hemorrhage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u0026ndash;1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003e0: absent; 1: mild; 2: moderate; 3: severe.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. DISCUSSION\",\"content\":\"\\u003cp\\u003eIn this study, the 96-h LC₅₀ value of boric acid for Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e) was 176.446 mg/L, indicating moderate acute toxicity. This value provides an important insight into the species-specific sensitivity of Nile tilapia to boron compounds and is consistent with findings from similar studies on Nile tilapia. For example, Acar, İnanan, Zemheri, Kesbiç, and Yılmaz (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) reported a 96-h LC₅₀ value of 141.42 mg/L for Nile tilapia, while Abdel Aliem, Soliman, Khaled, Mourad, and Dighiesh (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) found a value of 290 mg/L under different experimental conditions. The observed differences between the experiments demonstrate how water quality and fish physiological state and size together with experimental design affect toxicity results. The evaluation of boron-based compound ecological risks requires site-specific and species-specific assessments for both aquaculture systems and natural water bodies.The observed mortality pattern indicates boric acid toxicity increases with dose and may disrupt cellular and physiological processes including membrane permeability and ion regulation and enzyme activity. Research indicates boron toxicity in aquatic organisms results in oxidative stress and impaired osmoregulation and vital organ histopathological changes in liver gills and kidneys. (Topal et al., \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). LC₅₀ values help determine environmental exposure limits for safety purposes and enable the creation of non-lethal toxicity experiments. The researchers chose 1/20th of the LC₅₀ value for chronic exposure experiments to establish an ecologically relevant concentration which would evaluate long-term physiological and histopathological effects. The study enabled researchers to test dietary black cumin oil as a potential mitigation strategy.The health status of fish depends on hematological and biochemical parameters which serve as indicators of environmental pollutant-induced physiological stress. The exposure to boric acid (BA3 group) resulted in substantial decreases of red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Hct) levels which indicated anemia and reduced oxygen delivery capacity. The current study supports earlier research which demonstrated that boron compounds produce adverse effects on fish hematological parameters. (Acar et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Çelik et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).The liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were elevated which indicates liver damage. Fish exposed to boric acid have shown similar liver problems. (Topal et al., \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e).By contrast, fish in the BA4 group getting nutritional supplements with 1% black cumin oil (Nigella sativa) showed better haematological and biochemical profiles than the BA3 group. Studies on N. sativa's antioxidant and hepatoprotective effects clearly indicate how effective it is in raising haematological markers and liver function in fish. (Mohammed \\u0026amp; Arias, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Öz et al., \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).Environmental toxins create their harmful effects on aquatic organisms through oxidative stress as a widespread mechanism. The study revealed that boric acid exposure led to elevated malondialdehyde (MDA) levels which indicate lipid peroxidation and reduced activities of catalase (CAT) and superoxide dismutase (SOD) and glutathione peroxidase (GPx) antioxidant enzymes. The observed results match previous research which demonstrated that boric acid induces oxidative stress in fish. (Ertürk Gürkan, Gürkan, Sarıtunç, İbiş, \\u0026amp; Güneş, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; Öz, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe N. sativa oil supplement in the BA4 group reduced oxidative stress through lower MDA levels and higher CAT, SOD and GPx activities. The antioxidant effects of \\u003cem\\u003eN. sativa\\u003c/em\\u003e stem from its bioactive compounds including thymoquinone which neutralizes free radicals while enhancing antioxidant defense pathways. The Nile tilapia kidney tissue (Oreochromis niloticus) functions as a vital organ for both immune system response and detoxification operations. The head kidney of this organ serves as the main hematopoietic center while providing substantial immune defense through its resident lymphocytes and macrophages. The head kidney serves as a vital location for both innate and adaptive immune system development which underscores its essential role in maintaining tilapia health and immune system function. (Gan et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Mo et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). For example, significant increases in chemokines are associated with immune responses against such infections and facilitate the recruitment of immune cells to effectively combat pathogens (Nakharuthai \\u0026amp; Srisapoome, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Heavy metal exposure in Nile tilapia kidneys leads to histopathological changes which include glomerular degeneration together with tubular necrosis and vacuolization. The kidneys of fish exposed to di-n-butyl phthalate (DBP) show major histopathological damage through glomerular structural disruption and tubular functional impairment. The observed changes indicate reduced kidney perfusion and filtration efficiency which results in elevated blood levels of urea and creatinine. (Zeid \\u0026amp; Khalil, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). This deterioration shows a clear relationship between environmental pollutants and impaired kidney function; where heavy metals accumulate in kidney tissues, causing cellular damage and functional deficiencies. (Abdel Hakim, Helal, Salem, Zaghloul, \\u0026amp; Hanbal, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). The exposure of fish to dimethoate pesticides results in major histopathological changes that affect the structural integrity of \\u003cem\\u003eGarra mullya\\u003c/em\\u003e kidneys. The kidneys of control group fish remain healthy with their glomeruli clearly visible and intact. The toxic substance causes kidney function impairment which results in the development of tubular degeneration and necrosis and glomerular alterations after exposure. (Borane, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). Heavy metals have been shown to produce harmful histological changes in fish kidneys. The toxic effects of metals such as lead, copper, and zinc have been demonstrated through histopathological analyses that show glomerular atrophy, tubular necrosis, and interstitial inflammation. (Al-Balawi et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Al-Kshab, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The importance of pesticides at sub-lethal concentrations, especially malathion and cypermethrin, is of critical importance. These chemicals are known to cause various histological changes in kidney tissues of species like Channa gachua, including hyperplasia, vacuolar degeneration, and cellular apoptosis. (Preeti \\u0026amp; Garg, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Singh, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The study demonstrates that melanomacrophage hyperactivation in kidney tissue results from the inflammatory response which occurs because of elevated ROS levels produced by boric acid-induced oxidative stress. The vacuolization of proximal and distal tubules develops because boric acid disrupts membrane integrity and reduces Na⁺/K⁺-ATPase activity which causes ionic imbalance and increased osmotic pressure. The damage to capillary endothelial cells by boric acid leads to increased vascular permeability which results in blood accumulation and leakage. The toxicological effects of heavy metals and pesticides on fish gill tissues constitute a major concern in aquatic biology because these substances severely affect fish health and ecosystem integrity. The toxic substances cause consistent histopathological changes which appear as hyperplasia together with necrosis and edema. The use of chlorpyrifos pesticides leads to major changes in gill structure and operational capabilities. The exposure to chlorpyrifos results in long-term damage to fish Cyprinus carpio through necrosis and filament fusion of their gills. (Edwin, Ihsan, Rahmatika, \\u0026amp; Darlis, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Similar observations were documented in research on the Indian Flying Barb, showing increased mucus secretion and filament degeneration following exposure to Endosulfan (Das \\u0026amp; Gupta, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Studies have shown that lead exposure causes severe gill damage in Tilapia species, particularly leading to edema and necrosis (Batista, Triastuti, \\u0026amp; Pursetyo, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The research indicates epithelial cell separation in secondary lamellae of gill tissue occurs because boric acid weakens intercellular junction complexes. The fusion of secondary lamellae occurs through epithelial hyperplasia together with increased mucus secretion and inflammatory cell accumulation. The swelling of secondary lamellae occurs because boric acid makes capillary endothelial cells more permeable and disrupts the osmotic balance. The toxicological effects of heavy metals and pesticides on fish liver tissue result in numerous histopathological changes. The liver remains most vulnerable to chemical changes and structural damage because of its detoxification and metabolism roles after toxic substance exposure. Histopathological studies show that heavy metal exposure causes vacuolization of hepatocytes, degeneration of hepatic structure, and infiltration of immune cells which indicates significant tissue damage and stress responses. (Akter et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Bibi, Naz, Saeed, \\u0026amp; Chatha, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Exposure to paraquat has been shown to cause liver necrosis and hemorrhage in African catfish (\\u003cem\\u003eClarias gariepinus\\u003c/em\\u003e) (Ladipo, Doherty, \\u0026amp; Oyebadejo, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The combined exposure to pesticides and heavy metals produces synergistic toxic effects which intensifies histopathological damage. Fish exposed to both types of pollutants show higher levels of hepatocyte degeneration and necrosis. (Ghafarifarsani et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Ogbeide \\u0026amp; Okoduwa, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The research indicates that boric acid leads to liver tissue degeneration through membrane peroxidation in hepatocytes which disrupts mitochondrial function and reduces ATP production. Hemorrhages in sinusoidal spaces are believed to be associated with structural damage to endothelial cells. Oxidative stress, mitochondrial dysfunction, ion imbalance, and protein denaturation caused by boric acid are thought to play roles as common molecular mechanisms in the formation of all these histopathological lesions.\\u003c/p\\u003e \"},{\"header\":\"CONCLUSION\",\"content\":\"\\u003cp\\u003eThe present study revealed that even non-lethal exposure to waterborne boric acid can significantly impair the physiological, biochemical and histopathological integrity of Nile tilapia (Oreochromis niloticus). In particular, fish exposed to 1/20 of the 96-hour LC₅₀ (176,446 mg/L) exhibited hematological suppression, elevated hepatic enzyme levels, increased oxidative stress biomarkers, and marked tissue damage in gill, liver, kidney and muscle tissues.\\u003c/p\\u003e\\u003cp\\u003eDietary supplementation with 1% black cumin (Nigella sativa) oil showed a remarkable protective effect against boric acid-induced toxicity. The black cumin supplemented group showed improved hematological profiles, recovered antioxidant enzyme activity and markedly reduced histopathological lesions compared to the toxicant-only group. These results support the functional role of black cumin oil as a natural antioxidant and tissue protective agent in aquaculture nutrition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTaken together, the findings highlight the dual importance of this research: first, contributing to the toxicological understanding of boric acid as an emerging aquatic contaminant; second, highlighting the efficacy of a phytogenic feed additive in reducing such toxicity. Future research should investigate the dose-dependent effects of Nigella sativa, examine its long-term physiological consequences and assess the interaction between environmental stressors and dietary antioxidants in aquaculture systems.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMustafa OZ: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Validation; Visualization; \\u0026nbsp;review \\u0026amp; editing Writing- Original draft preparation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEnes \\u0026Uuml;ST\\u0026Uuml;NER: Taking tissue samples, taking histological sections, pathological evaluation, writing histopathology findings, Data curation , review \\u0026amp; editing Writing- Original draft preparation\\u003c/p\\u003e\\n\\u003cp\\u003eSuat DIKEL: Methodology; Project administration, Data curation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWhile preparing this manuscript, the authors affirm that they did not receive any financial assistance, grants, or other forms of support.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe availability of data and material\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data supporting the findings of this study can be obtained from the corresponding author upon a reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors explicitly affirm that they have no identifiable financial conflicts of interest or personal ties that could have potentially influenced the findings presented in this research.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAbdel-Mohsen, H. A. (2014). Monitoring of Oreochromis responses to metal pollution in Lake Mariut, Alexandria, Egypt. \\u003cem\\u003eJournal of King Abdulaziz University, 25\\u003c/em\\u003e(1), 21.\\u003c/li\\u003e\\n\\u003cli\\u003eAbdel Aliem, R., Soliman, B., Khaled, H., Mourad, M., \\u0026amp; Dighiesh, H. (2022). Toxicological effect of boric acid and cadmium chloride on the Nile tilapia, Oreochromis niloticus. \\u003cem\\u003eEgyptian J Aquatic Biol Fish, 26\\u003c/em\\u003e(5), 667-680.\\u003c/li\\u003e\\n\\u003cli\\u003eAbdel Hakim, N. F., Helal, A. F., Salem, M. F., Zaghloul, A. M., \\u0026amp; Hanbal, M. M. (2016). Effect of some heavy metals on physiological and chemical parameters in Nile tilapia (Oreochromis niloticus L.). \\u003cem\\u003eJournal of Egyptian Academic Society for Environmental Development. D, Environmental Studies, 17\\u003c/em\\u003e(1), 81-95.\\u003c/li\\u003e\\n\\u003cli\\u003eAcar, \\u0026Uuml;., İnanan, B. E., Zemheri, F., Kesbi\\u0026ccedil;, O. S., \\u0026amp; Yılmaz, S. (2018). Acute exposure to boron in Nile tilapia (Oreochromis niloticus): Median-lethal concentration (LC50), blood parameters, DNA fragmentation of blood and sperm cells. \\u003cem\\u003eChemosphere, 213\\u003c/em\\u003e, 345-350.\\u003c/li\\u003e\\n\\u003cli\\u003eAebi, H. (1984). [13] Catalase in vitro. In \\u003cem\\u003eMethods in enzymology\\u003c/em\\u003e (Vol. 105, pp. 121-126): Elsevier.\\u003c/li\\u003e\\n\\u003cli\\u003eAkter, R., Pervin, M. A., Jahan, H., Rakhi, S. F., Reza, A. M., \\u0026amp; Hossain, Z. (2020). Toxic effects of an organophosphate pesticide, envoy 50 SC on the histopathological, hematological, and brain acetylcholinesterase activities in stinging catfish (Heteropneustes fossilis). \\u003cem\\u003eThe Journal of Basic and Applied Zoology, 81\\u003c/em\\u003e, 1-14.\\u003c/li\\u003e\\n\\u003cli\\u003eAl-Balawi, H. F. A., Al-Akel, A. S., Al-Misned, F., Suliman, E. A. M., Al-Ghanim, K. A., Mahboob, S., \\u0026amp; Ahmad, Z. (2013). 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Histopathology changes in the rainbow trout (Onchorhyncus mykiss) consuming boric acid supplemented fish fodder. \\u003cem\\u003eJournal of Trace Elements in Medicine and Biology, 62\\u003c/em\\u003e, 126581.\\u003c/li\\u003e\\n\\u003cli\\u003ePaglia, D. E., \\u0026amp; Valentine, W. N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. \\u003cem\\u003eThe Journal of laboratory and clinical medicine, 70\\u003c/em\\u003e(1), 158-169.\\u003c/li\\u003e\\n\\u003cli\\u003ePreeti, A., \\u0026amp; Garg, V. (2023). Histopathological Alteration in Tissues of Channa Punctatus (Bloch) Induced by Lambda-Cyhalothrin. \\u003cem\\u003eInternational Journal of Zoological Investigations, 9\\u003c/em\\u003e(2), 92-99. doi:10.33745/ijzi.2023.v09i02.010\\u003c/li\\u003e\\n\\u003cli\\u003eProhaska, J., Oh, S.-H., Hoekstra, W., \\u0026amp; Ganther, H. (1977). 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Histopathological Study of Channa Gachua Exposed to Sublethal Concentration of Malathion. \\u003cem\\u003eInternational Journal of Fisheries and Aquatic Studies, 9\\u003c/em\\u003e(3), 174-179. doi:10.22271/fish.2021.v9.i3c.2472\\u003c/li\\u003e\\n\\u003cli\\u003eSun, Y., Oberley, L. W., \\u0026amp; Li, Y. (1988). A simple method for clinical assay of superoxide dismutase. \\u003cem\\u003eClinical chemistry, 34\\u003c/em\\u003e(3), 497-500.\\u003c/li\\u003e\\n\\u003cli\\u003eTopal, A., Oru\\u0026ccedil;, E., Altun, S., Ceyhun, S. B., \\u0026amp; Atamanalp, M. (2016). The effects of acute boric acid treatment on gill, kidney and muscle tissues in juvenile rainbow trout. \\u003cem\\u003eJournal of Applied Animal Research, 44\\u003c/em\\u003e(1), 297-302.\\u003c/li\\u003e\\n\\u003cli\\u003eYumru, M., Savas, H. A., Kalenderoglu, A., Bulut, M., Celik, H., \\u0026amp; Erel, O. (2009). Oxidative imbalance in bipolar disorder subtypes: a comparative study. \\u003cem\\u003eProgress in Neuro-Psychopharmacology and Biological Psychiatry, 33\\u003c/em\\u003e(6), 1070-1074.\\u003c/li\\u003e\\n\\u003cli\\u003eZeid, E. A., \\u0026amp; Khalil, A. (2014). Toxicological consequences of Di-n-Butyl-Phthalate (DBP) on health of Nile Tilapia fingerlings. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Nile tilapia, Boric acid, Black cumin oil, Oxidative stress, Sustainable fish farming\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6515353/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6515353/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThis study aimed to evaluate the protective effects of dietary black cumin (\\u003cem\\u003eNigella sativa\\u003c/em\\u003e L.) oil on hematological, biochemical, oxidative stress, and histopathological responses in Nile tilapia (\\u003cem\\u003eOreochromis niloticus\\u003c/em\\u003e) exposed to waterborne boric acid. After determining the 96-hour LC\\u003csub\\u003e50\\u003c/sub\\u003e value of boric acid, the experimental design included exposure to 1/20th of this concentration. Fish were fed for 21 days with diets either containing or lacking 1% black cumin oil. At the end of the feeding trial, blood parameters, oxidative stress biomarkers, and tissue histopathology were analyzed. The group receiving black cumin oil without boric acid showed the most favorable physiological and biochemical profiles. In contrast, the group exposed to boric acid alone exhibited significant negative alterations. Importantly, fish fed black cumin oil while exposed to boric acid showed improvements across all measured parameters compared to the toxicant-only group.\\u003c/p\\u003e \\u003cp\\u003eThe findings indicate that dietary black cumin oil effectively alleviates the toxic effects of waterborne boric acid on Nile tilapia, supporting its potential use as a functional dietary additive in aquaculture.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Toxicological Effects of Waterborne Boric Acid and the Protective Role of Dietary Black Cumin (Nigella sativa L.) Oil in Nile Tilapia (Oreochromis niloticus): Hematological, Biochemical, Oxidative Stress and Histopathological Responses\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-28 10:22:13\",\"doi\":\"10.21203/rs.3.rs-6515353/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"8b0d6e5c-f75c-4520-8e1f-a5140b0615db\",\"owner\":[],\"postedDate\":\"April 28th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-05-01T20:00:32+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-28 10:22:13\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6515353\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6515353\",\"identity\":\"rs-6515353\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}