Open Veterinary Journal, (2026), Vol. 16(4): 2260-2270

Research Article

10.5455/OVJ.2026.v16.i4.26

Laboratory hazard characterization of acute and sub-lethal urea exposure in Nile Tilapia (Oreochromis niloticus)

Nagi Mousa¹, Abdulrahman Aljali^2* and Hamzah Othman³

¹General Trend Section, College of Natural Resources and Environmental Sciences, Al-Quba Branch - University of Derna, Derna, Libya

²Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Omar Al-Mukhtar University, Al-Bayda, Libya

³Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, University of Omer Al-Mukhtar, Al-Bayda, Libya

*Corresponding Author: Abdulrahman Aljali. Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Omar Al-Mukhtar University, Al-Bayda, Libya.. Email: nge7396 [at] gmail.com

Submitted: 02/01/2026 Revised: 24/03/2026 Accepted: 24/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Urea is one of the most widely used nitrogen-based fertilizers worldwide. Although environmental concerns frequently focus on secondary ammonia formation, the intrinsic toxic potential of direct urea exposure under controlled laboratory conditions in freshwater.

Aim: This study aimed to determine the acute and sub-lethal toxicity profile of urea in juvenile Nile tilapia (Oreochromis niloticus) and characterize concentration-dependent physiological, oxidative, and histopathological responses.

Methods: Acute toxicity was evaluated using a 96-hour semi-static bioassay, in accordance with the OECD Test Guideline 203. The median lethal concentration (96-hour LC₅₀) was calculated using Probit regression analysis. Sub-lethal exposures corresponding to 1/10 (1.18 g/l) and 1/2 (5.90 g/l) of the LC₅₀ were applied for 14 days. We assessed behavioral responses, hematological parameters, antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)], lipid peroxidation Malondialdehyde (MDA), and histopathological alterations in gill, liver, and kidney tissues. Data were analyzed using analysis of variance (ANOVA) or Kruskal–Wallis tests as appropriate.

Results: The 96-hour LC₅₀ of urea was 11.8 g/l (95% CI: 11.1–12.6 g/l), indicating moderate acute intrinsic toxicity. Sub-lethal exposure produced significant concentration-dependent reductions in erythrocyte count (F(2,12)=36.42, p < 0.001, η²=0.86) and hemoglobin (F(2,12)=22.41, p < 0.001, η²=0.79), with increased leukocyte counts (F(2,12)=29.68, p < 0.001, η²=0.83). SOD, CAT, and GPx activities were elevated at 1/10 LC₅₀ but significantly suppressed at 1/2 LC₅₀ (p < 0.001), while MDA levels increased markedly (F(2,12)=58.36, p < 0.001, η²=0.91). The histopathological lesion scores differed significantly among groups (p < 0.01; ε² range: 0.66–0.75).

Conclusion: In juvenile O. niloticus, direct laboratory exposure to urea induces measurable acute toxicity and robust concentration-dependent hematological, oxidative, and tissue-level alterations. These findings define the urea intrinsic hazard profile under controlled conditions and provide foundational toxicodynamic data without constituting an environmental risk assessment.

Keywords: Acute toxicity, LC_50, Oreochromis niloticus, Sub-lethal effects, Urea fertilizer.

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Introduction

Increasing agricultural intensification has substantially increased the global use of nitrogen-based fertilizers. Among these, urea is one of the most extensively applied compounds due to its high nitrogen content and cost-effectiveness. However, excessive application and subsequent runoff have raised concerns regarding potential adverse effects on aquatic organisms, particularly economically important freshwater fish species such as Nile tilapia (Oreochromis niloticus) (George and Ibok, 2024). Nitrogen fertilizers introduced into aquatic environments may undergo transformation processes, including hydrolysis and nitrification, potentially contributing to elevated ammonia (NH₃) levels—a well-recognized toxicant in aquatic systems (Edwards et al., 2024). Although numerous studies have investigated the ecological consequences of nitrogen enrichment under environmental exposure scenarios, few investigations have specifically characterized the intrinsic toxicodynamic effects of urea under controlled laboratory conditions. Distinguishing between environmental risk assessment and hazard characterization in aquatic toxicology is important. To estimate the likelihood of adverse outcomes under real-world conditions, environmental risk assessment integrates exposure probability, environmental persistence, and ecological effects. In contrast, hazard characterization focuses exclusively on the inherent toxic potential of a substance under defined experimental settings, independent of environmental exposure modeling (Rand, 2020; Food and Agriculture Organization of the United Nations, 2023). Therefore, laboratory-based investigations provide fundamental insight into the biological responses induced by direct chemical exposure. Biomarkers of hematological and oxidative stress are widely recognized as sensitive indicators of sub-lethal toxic stress in fish. Alterations in erythrocyte count, hemoglobin concentration, and leukocyte profiles often represent early physiological responses to chemical insult (Bojarski et al., 2025; Sultana et al., 2020). Similarly, the modulation of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), reflects adaptive or pathological responses to oxidative imbalance (Menon et al., 2023). The histopathological evaluation of vital organs—including gills, liver, and kidney—provides complementary structural evidence of tissue-level damage (Liebel et al., 2013; Deb and Das, 2019). Nile tilapia (O. niloticus) is widely cultured and frequently employed as a model species in aquatic toxicology because of its physiological resilience, well-characterized hematological baseline parameters, and suitability for controlled exposure studies (George and Ibok, 2024). Despite its ecological and economic importance, the comprehensive laboratory-based hazard characterization of urea in this species remains limited. Therefore, the present study was designed to characterize the intrinsic toxic effects of urea fertilizer on juvenile O. niloticus under controlled laboratory conditions. Acute toxicity was determined by calculating the 96-hour median lethal concentration (LC₅₀). Subsequently, sub-lethal exposure at 1/10 and 1/2 of the LC₅₀ was conducted to evaluate concentration-dependent behavioral, hematological, biochemical, and histopathological responses. This investigation was structured as a laboratory hazard characterization study and does not aim to extrapolate the findings to the probability of environmental exposure.

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Materials and Methods

Test organisms and acclimation

In this experiment, 120 juvenile Nile tilapia (O. niloticus), a common freshwater fish used in urea toxicity assessments, were collected from a fish farm in Al-Tamimi village. The fish used had a uniform average body weight (approximately 45.6 ± 0.3 g) and a uniform length to minimize size-related variation. The specimens were transported to the laboratory and acclimated for 14 days in glass tanks (55 × 40 × 40 cm) containing dechlorinated tap water. During the acclimation period, the fish were fed commercial pelleted feed containing approximately 45% crude protein twice daily. The water was changed daily to remove waste. Only clinically healthy fish exhibiting normal behavior were selected for the experiment (Maitra and Nath, 2014; Jena et al., 2025).

Determination of the median LC50

The 96-hour median LC₅₀ of a commonly used commercial urea fertilizer was used in this study to better simulate real-world environmental exposure conditions. The 96-hour LC₅₀ was determined using a semi-stationary bioassay (static regeneration) in accordance with OECD Directive No. 203. A preliminary range test was performed to identify concentrations that induce a 24-hour mortality rate between 0% and 100% mg/dl. Based on this test, five geometrically spaced concentrations within the effective toxic range (approximately 8.75–16.25 g/l) were selected, along with a control sample for the final test. 10 fish were exposed to each duplicate concentration. Mortality rates were recorded at 24, 48, 72, and 96 hours. LC₅₀ values and their corresponding 95% confidence intervals were calculated using the appropriate statistical software (Organization for Economic Co-operation and Development, 2019).

Sub-lethal exposure design

Each experimental group in the lethal dose exposure experiment consisted of 10 fish. At the end of the 14-day exposure period, five fish were randomly selected from each group for hematological, biochemical, and histopathological analyses. Each fish was considered the experimental unit for all statistical analyses. Sub-lethal toxicity experiments were designed based on the determined 96-hour LC₅₀ values. Sub-lethal exposure concentrations were selected based on the 96-hour LC₅₀ value to ensure biological relevance and experimental reliability. 1/10 of the LC₅₀ was chosen to represent a low, environmentally realistic concentration capable of inducing subtle physiological and biochemical alterations without causing mortality. In contrast, 1/2 of the LC₅₀ was selected as a higher sub- LC₅₀ to impose significant physiological stress while maintaining fish survival throughout prolonged exposure. A control group with no urea was included (Essien-ibok et al., 2014; Jena et al., 2025).

Sub-lethal exposure and evaluation

Fish were exposed to the selected sub-LC₅₀ in a semi-static system for 14 days. The water quality parameters (temperature, pH, and dissolved oxygen) were monitored daily. Physiological behavior, including feeding, swimming activity, and gill ventilation, was observed.

Water quality monitoring

The physicochemical parameters of the sub-LC₅₀ test water were monitored daily throughout the exposure period. Water temperature was measured using a calibrated thermometer, dissolved oxygen was measured using a digital DO meter, and pH was measured using a digital pH meter. To ensure stable experimental conditions, total alkalinity and hardness were determined following standard APHA procedures (Deb and Das, 2019).

Hematological analysis

Blood samples were collected from anesthetized fish via caudal vein puncture or tail ablation using heparinized syringes at the end of the exposure period. Red blood cell (RBC) counts were determined using an improved Neubauer hemocytometer with 0.85% saline as a diluent. The hemoglobin concentration was measured using the cyanmethemoglobin method. Differential leukocyte counts were performed on Giemsa-stained blood smears and examined under a light microscope (Maitra and Nath, 2014).

Antioxidant enzyme activity and lipid peroxidation assays

At the end of the exposure period, the liver tissue was excised, washed with cold saline, and homogenized (10% w/v) in phosphate-buffered saline (0.1 M, pH 7.4). The homogenates were centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was analyzed. SOD activity was determined by inhibiting pyrogallol auto-oxidation, CAT activity by monitoring hydrogen peroxide decomposition at 240 nm, and GPx activity by measuring NADPH oxidation at 340 nm. Lipid peroxidation was assessed by measuring Malondialdehyde (MDA) using the TBARS method. Enzyme activities were titrated according to total protein content, determined by the Lowry method. Antioxidant enzymes (SOD, CAT, and GPx) and lipid peroxidation (MDA) were analyzed following standard oxidative stress assessment protocols described by Menon et al. (2023).

Histopathological analysis

Following hematological sampling, the fish were humanely euthanized, and the target organs, particularly the gills, kidney, and liver, were excised for histopathological examination. Tissues were fixed in 10% neutral buffered formalin, dehydrated through a graded ethanol series, cleared in xylene, embedded in paraffin, and sectioned at approximately 5-µm thickness. Sections were stained with hematoxylin and eosin and examined under a light microscope at up to 400× magnification to identify pathological alterations (Deb and Das, 2019; Jena et al., 2025) .

Statistical analysis

Data were analyzed using IBM SPSS Statistics (Version XX, IBM Corp., Armonk, NY). Continuous variables are presented as mean ± standard deviation (SD), whereas ordinal data (behavioral and histopathological lesion scores) are expressed as median and interquartile range (IQR). The normality of continuous variables was assessed using the Shapiro–Wilk test due to the small sample size (n=5 per group), and the homogeneity of variances was evaluated using Levene’s test. When normality and homoscedasticity assumptions were satisfied, differences among groups were analyzed using one-way analysis of variance (ANOVA). The effect size for ANOVA was estimated using eta-squared (η²). When significant differences were detected, Tukey’s honestly significant difference test was applied for post hoc multiple comparisons. The Kruskal–Wallis test was used when parametric assumptions were not met. Ordinal behavioral and histopathological lesion scores (graded 0–3) were treated as non-parametric variables and analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction for pairwise comparisons. The effect size for nonparametric tests was calculated using epsilon-squared (ε²).

For acute toxicity assessment, LC₅₀ values and their 95% confidence intervals were calculated using Probit regression analysis. The goodness-of-fit of the model was evaluated using chi-square statistics. Fish were randomly distributed into duplicate tanks per treatment under strictly standardized environmental conditions (temperature, dissolved oxygen, pH, and regular water renewal). No systematic differences were observed between replicate tanks during the exposure period. Because tanks were maintained under identical physicochemical conditions and functioned as technical replicates rather than independent experimental treatments, the individual fish were considered the experimental unit for statistical analysis. Statistical significance was set at p < 0.05.

Ethical approval

The study protocol adhered to international ethical standards for animal experimentation, including OECD guidelines for fish toxicity testing and ARRIVE recommendations. No procedures causing unnecessary pain or suffering were applied.

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Results

Acute toxicity of urea and determination of 96-hour LC50

The mortality of fish increased with increasing urea concentration over the 96-hour exposure period. At the lowest concentration (8.75 g/l), only 5% of fish died, whereas the highest concentration (16.25 g/l) caused 95% mortality. The intermediate concentrations showed a gradual increase in mortality, indicating a clear concentration-dependent response (Table 1, Fig. 1). Probit analysis of the mortality data yielded a 96-hour LC₅₀ value of 11.8 g/l, with a 95% confidence interval ranging from 11.1 to 12.6 g/l. At this concentration, 50% of the exposed fish population is expected to die under the same experimental conditions. Based on the determined 96-hour LC₅₀, sub-LC₅₀ for subsequent sub-acute and chronic studies were calculated as 1.18 g/l and 5.90 g/l of the LC₅₀, respectively. These concentrations were used to examine physiological and biochemical responses without causing significant mortality.

Table 1. Mortality of fish at different urea concentrations after 96-hour exposure.

Fig. 1. Concentration–mortality relationship for urea in fish over 96 hours, with LC₅₀ (red), 1/2 LC₅₀ (orange), and 1/10 LC₅₀ (green) indicated.

The concentration-mortality relationship is illustrated in Fig. 1, where mortality increased sharply above 11 g/l, highlighting the acute toxicity threshold for urea in this fish species.

Behavioral and clinical observations of 96-hour LC50 test

During the 96-hour acute toxicity test, urea-exposed fish exhibited clear concentration-dependent behavioral and clinical alterations compared with the control group. The control fish maintained normal swimming activity, feeding behavior, and opercular movements throughout the exposure period. In contrast, exposed fish showed progressive behavioral disturbances, including erratic swimming, increased opercular movement, surface gasping, loss of equilibrium, and excessive mucus secretion, with symptom severity increasing at higher urea concentrations and prior to mortality.

Sub-lethal exposure result

Exposure to sub-LC₅₀ of urea, calculated from the 96-hour LC₅₀ value (11.8 g/l), resulted in concentration-dependent biochemical responses in fish (Table 2).

Table 2. The experimental concentrations derived from the 96-hour LC₅₀.

Clinical signs of sub-lethal exposure

Fish exposed to a low sub-LC₅₀ (1/10 of the LC₅₀) exhibited mild behavioral alterations after the first few days of exposure. These changes included reduced feeding activity, occasional erratic swimming, and a slight increase in opercular movement (Table 3), suggesting early respiratory stress. Most fish maintained equilibrium, and no mortality was recorded during the exposure period. At the higher sub-LC₅₀ (1/2 of the LC₅₀), pronounced behavioral and clinical signs were observed. The fish initially displayed marked hyperactivity, followed by lethargy and impaired equilibrium. Signs of hypoxia were evidenced by frequent surfacing behavior and accelerated opercular movements. Body discoloration was occasionally observed, accompanied by reduced appetite and bottom-dwelling behavior (Table 3). The severity of these symptoms increased over time, indicating progressive urea exposure-associated physiological distress. The behavioral and clinical alterations observed are consistent with NH₃-mediated toxicity resulting from urea hydrolysis in aquatic systems and represent early sensitive indicators of sub-lethal stress in fish. These responses preceded hematological, biochemical, and histopathological changes, thereby confirming a concentration-dependent intrinsic toxic effect of urea in O. niloticus under controlled laboratory conditions. Behavioral scores differed significantly among groups for all assessed parameters (Kruskal–Wallis test, p < 0.01). Post hoc analysis revealed significantly higher scores in the 1/2 LC₅₀ group compared with both the control and 1/10 LC₅₀ groups (p < 0.05), indicating a concentration-dependent increase in behavioral disturbance. The effect size estimates (ε² range: 0.65–0.73) demonstrated large treatment effects.

Table 3. Behavioral alteration scores [median (IQR), n=5 per group].

Physicochemical parameters of the test water

The physicochemical parameters of the test water were monitored daily throughout the exposure period to ensure stable experimental conditions. The water temperature ranged from 24.5°C to 25.5°C, dissolved oxygen was maintained at 6.8–7.2 mg/l, and the pH ranged from 7.2 to 7.6. The total alkalinity and hardness remained stable at 85–90 and 120–125 mg/l as CaCO₃, respectively (Table 4). These results indicate that the water quality parameters were within the optimal range for the tested fish species and that additional stress factors that could confound the observed effects of urea exposure were not introduced.

Table 4. Physicochemical parameters of the test water.

Hematological and histopathological responses to sub-lethal exposure

All parametric variables were assumed to have normality and homogeneity of variance (Shapiro–Wilk test, p > 0.05; Levene’s test, p > 0.05). RBC counts differed significantly among the experimental groups (one-way ANOVA, F(2,12)=36.42, p < 0.001, η²=0.86), indicating a significant treatment effect. Tukey’s post hoc test revealed significant reductions in both the 1/10 LC₅₀ and 1/2 LC₅₀ groups compared with the control (p < 0.05), with the lowest RBC values observed in the 1/2 LC₅₀ group. Hemoglobin concentration also showed significant group differences (F(2,12)=22.41, p < 0.001, η²=0.79), reflecting a large effect size. The fish exposed to 1/2 LC₅₀ exhibited significantly lower hemoglobin levels than the control and 1/10 LC₅₀ groups (p < 0.05). White blood cell (WBC) counts increased significantly across treatments (F(2,12)=29.68, p < 0.001, η²=0.83), demonstrating a large treatment effect. The 1/2 LC₅₀ group showed significantly higher WBC counts than the control and 1/10 LC₅₀ groups (p < 0.05) (Table 5). Collectively, these findings indicate a clear concentration-dependent hematological response to urea exposure.

Table 5. Hematological parameters in fish exposed to sub-lethal urea concentrations (n=5 per group).

Significant differences in antioxidant enzyme activities were detected among the experimental groups. SOD activity varied significantly (one-way ANOVA, F(2,12)=42.15, p < 0.001, η²=0.88), indicating a very large treatment effect. SOD activity increased significantly in the 1/10 LC₅₀ group but decreased markedly in the 1/2 LC₅₀ group relative to the control (p < 0.05). CAT activity also differed significantly among the groups (F(2,12)=31.77, p < 0.001, η²=0.84). Moderate exposure (1/10 LC₅₀) resulted in enhanced CAT activity, whereas higher exposure (1/2 LC₅₀) led to significant suppression compared with the control (p < 0.05). Similarly, GPx activity demonstrated significant group differences (F(2,12)=39.24, p < 0.001, η²=0.87), reflecting a large effect size. MDA levels differed markedly among treatments (F(2,12)=58.36, p < 0.001, η²=0.91). Lipid peroxidation increased progressively with concentration, with the highest values observed in the 1/2 LC₅₀ group. Overall, the effect size analysis revealed large to very large treatment effects across all oxidative stress biomarkers (η² range: 0.84–0.91), supporting a strong concentration-dependent oxidative response to urea exposure. Table 6 illustrates these results.

Table 6. Oxidative stress biomarkers in fish exposed to sub-lethal urea concentrations (n=5 per group).

Histopathological descriptions were restricted to directly observable morphological alterations without functional or mechanistic interpretation. Histological examination of kidney sections demonstrated disruption of the normal renal architecture, characterized by interstitial inflammatory cell infiltration, tubular epithelial degeneration, and reduced tubular lumen diameter (Fig. 2A and B).

Fig. 2. (A-B): Photomicrograph of kidney tissue of Oreochromis niloticus exposed to sub-LC₅₀ of urea, illustrating concentration-dependent histopathological alterations. Renal sections show mild-to-moderate tubular epithelial degeneration (TD; score 1–2), characterized by cytoplasmic vacuolation and loss of cellular integrity, accompanied by narrowing of the tubular lumen (TLN; score 1). In the higher exposure groups, the lesions progressed to marked tubular necrosis (TN; score 2–3) with proteinaceous casts (PP; score 2) within the tubular lumen and interstitial inflammatory cell infiltration (IC; score 2). These changes indicate impaired renal function. Hematoxylin and eosin (H&E), 400×. Scale bar=50 µm. Abbreviations (Kidney): RT: Renal tubules. TD: Tubular epithelial degeneration. TN: Tubular necrosis. TLN: Tubular lumen narrowing. PP: proteinaceous casts. IC: interstitial inflammatory cells.

Histological examination of the Nile tilapia (O. niloticus) gills exposed to low concentrations of urea revealed epithelial hyperplasia (EPH; Grade 1), characterized by an increased number of epithelial cells and increased lamellar thickness (Fig. 3A). Partial fusion of secondary lamellae lamellar fusion (FL; Grade 1) and mild epithelial lifting (EL; Grade 1) were also observed (Fig. 3B).

Fig. 3. Histopathological alterations in Nile tilapia gill tissues exposed to elevated urea concentrations under laboratory conditions (A) Moderate EPH and EL. (B) Marked FL with vascular congestion. (C) Severe EPH with extensive FL and focal degeneration. Sections stained with hematoxylin and eosin (H&E, 400×. Scale bar=50 µm. Abbreviations (Gills): SL: Secondary lamellae. FL: lamellar fusion. EPH: Epithelial hyperplasia and hypertrophy. EL: Epithelial lifting. LT: Lamellar telangiectasia. AD: Aneurysmal dilation. ED: Epithelial desquamation. MCD: Marginal canal dilation. H: Hemorrhage.

More pronounced structural alterations were recorded at higher exposure concentrations. These included lamellar telangiectasis (Grade 2), aneurysmal dilation of secondary lamellae (AD; Grades 2–3), and extensive FL; Grade 2. Additional findings included epithelial desquamation (Grades 2–3), marginal channel dilation (Grade 2), and focal hemorrhage (Grade 2) (Fig. 3C). The observed lesions’ severity increased in a concentration-dependent manner.

In control fish, hepatocytes exhibited a typical polygonal morphology with centrally located nuclei arranged in cords separated by hepatic sinusoids. Melanomacrophage centers were observed within the hepatic parenchyma.

Marked histopathological alterations were recorded in fish exposed to low concentrations of urea compared to the control group. These included extensive hepatocellular necrosis, cellular debris accumulation, focal hemorrhagic areas, and bacterial aggregates within the hepatic tissue. Increased numbers of inflammatory cells were also observed. The severity of these alterations was greater in the exposed groups than in the control group (Fig. 4A and B).

Fig. 4. Histopathological sections of the liver of Nile tilapia fish exposed to urea (A) Liver tissue showing focal hepatic degeneration with inflammatory cell infiltration, hepatic cord distortion, and sinusoidal irregularity, indicating a moderate toxic effect. (B) Liver tissue exhibiting mild hepatic congestion and sinusoidal dilation with relatively preserved architecture, suggesting early-stage or sub-lethal toxic stress induced by urea exposure. Hematoxylin and eosin (H&E) stain, 400×. Scale bar=20 µm.

The severity of histopathological lesions differed significantly among experimental groups across all examined tissues. Gill lesion scores showed significant variation (Kruskal–Wallis test, χ²(2)=10.80, p=0.004, ε²=0.72). Post hoc Dunn’s test with Bonferroni correction revealed significantly higher scores in the 1/2 LC₅₀ group compared with both the control and 1/10 LC₅₀ groups (p < 0.05). Liver tissue also demonstrated significant group differences (χ²(2)=9.95, p=0.007, ε²=0.66), whereas kidney lesions exhibited marked variation among treatments (χ²(2)=11.20, p=0.003, ε²=0.75) (p 0.05). Overall, the effect size estimates indicated large treatment effects across all examined tissues (ε² range: 0.66–0.75) (Table 7), supporting a clear concentration-dependent increase in histopathological damage following urea exposure.

Table 7. Histopathological lesion scores [median (IQR), n=5 per group].

Values are expressed as median IQR. Differences among groups were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Differences were considered significant at p < 0.05.

Collectively, the effect size estimates demonstrated large to very large treatment effects across hematological, oxidative, and histopathological biomarkers (η² range: 0.79–0.91; ε² range: 0.66–0.75), confirming the robust concentration-dependent biological impact of urea exposure.

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Discussion

Urea fertilizer induced measurable acute toxicity in Nile tilapia (O. niloticus), with a 96-hour LC₅₀ value of 11.8 g/l under semi-static laboratory conditions. This value represents a quantitative hazard threshold independent of environmental exposure modeling and is consistent with previously reported LC₅₀ values for freshwater fish exposed to urea and other nitrogenous compounds (George and Ibok, 2024). Mortality increased progressively with concentration, indicating the clear sensitivity of this species to elevated nitrogen levels under controlled conditions (Edwards et al., 2024). Prolonged exposure to sub-LC₅₀ produced behavioral and physiological alterations, including gill EPH and structural hepatic changes, suggesting cumulative tissue responses to sustained N2 stress (Deb and Das, 2019; Jena et al., 2025). Hematological assessment revealed significant reductions in erythrocyte count and Hb concentration, accompanied by elevated leukocyte counts. These changes were more pronounced at 1/2 LC₅₀ than at 1/10 LC₅₀, demonstrating a graded exposure-related response (Maitra and Nath, 2014; Bojarski et al., 2025). Oxidative stress biomarkers exhibited differential modulation across exposure levels. Moderate exposure enhanced the activities of SOD, CAT, and GPx, whereas higher sub-lethal exposure was associated with the suppression of antioxidant defenses and increased MDA levels, indicating intensified lipid peroxidation (Menon et al., 2023; Grădinariu et al., 2025). Histopathological evaluation revealed concentration-related structural alterations in the gill, liver, and kidney tissues. The observed changes included epithelial thickening and FL in the gills, cytoplasmic vacuolation and sinusoidal dilation in the liver sections, and degenerative tubular changes in the renal tissue. Lesion severity scores increased consistently with concentration, as confirmed by semi-quantitative grading (Liebel et al., 2013), providing objective support for exposure-related tissue damage (Deb and Das, 2019; Makaras et al., 2025). Importantly, these findings delineate intrinsic biological hazards under standardized laboratory conditions and do not extrapolate to the probability of environmental exposure. Overall, the tested concentrations represent a laboratory-based hazard scenario and were not intended to simulate realistic environmental exposure conditions. Accordingly, the results should be interpreted as toxicodynamic responses under controlled experimental conditions.

Osmotic stress as a potential confounding factor

The potential contribution of osmotic stress at exposure concentrations expressed in the g/l range warrants consideration. Urea may increase the osmolarity of the surrounding medium due to its high solubility, potentially disturbing water and ion homeostasis in exposed fish. Such an osmotic imbalance could influence gill permeability, renal function, and hematological parameters independently of direct toxicodynamic mechanisms. Therefore, some of the observed physiological alterations may reflect a combined response to intrinsic chemical toxicity and elevated osmotic load under controlled laboratory conditions. Future studies incorporating osmolarity measurements would help distinguish between specific toxic effects and nonspecific osmotic stress responses.

Study limitations

A major limitation of the present study is the absence of speciation analysis of nitrogen forms, particularly total NH₃ nitrogen and unionized NH₃. Since urea may undergo hydrolysis and contribute to NH₃ formation under aqueous conditions, the lack of quantitative differentiation between urea-derived nitrogen species limits the ability to attribute the observed toxic effects exclusively to urea. Therefore, the present findings should be interpreted as reflecting the combined nitrogen-related toxicodynamic responses under controlled laboratory conditions.

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Conclusion

This investigation defines the laboratory hazard profile of urea in juvenile O. niloticus through the determination of acute toxicity and sub-lethal biomarker analysis. Urea demonstrated moderate acute intrinsic toxicity and induced concentration-dependent physiological, biochemical, and histological alterations under controlled exposure conditions. These findings contribute to the toxicodynamic understanding of nitrogenous compounds and provide foundational data for subsequent environmental risk modeling. However, they do not constitute an environmental risk assessment. Further research is warranted to investigate the long-term and cumulative effects of urea exposure and its interactions with other environmental stressors to better inform sustainable aquaculture and water quality management practices.

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Acknowledgments

None.

Conflict of interest

The authors declare no known financial interests or personal relationships that could influence the work mentioned in this paper.

Funding

None.

Authors’ contributions

Nagi Mousa contributed to the conceptualization, experimental design, data collection, and manuscript drafting. Abdulrahman Aljali provided expertise in toxicology, supervised laboratory analyses, and critically revised the manuscript. Hamzah Othman performed histopathological examinations and data interpretation. All authors have reviewed and approved the final version of the manuscript.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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References

American Public Health Association, American Water Works Association, & Water Environment Federation. 2017. Standard Methods for the Examination of Water and Wastewater 24th ed. Washington, DC: American Public Health Association.

Bojarski, B., Witeska, M. and Kondera, E. 2025. Blood biochemical biomarkers in fish toxicology—a review. Animals 15(7), 965; doi:10.3390/ani15070965

Deb, N. and Das, S. 2019. Impact of fertilizer urea on liver histopathology of freshwater snakehead (Channa punctata). Int. J. Scientific. Res. Biol. Sci. 6(1), 216–219. Available via https://www.academia.edu/download/

Edwards, T.M., Puglis, H.J., Kent, D.B., Durán, J.L., Bradshaw, L.M. and Farag, A.M. 2024. Ammonia and aquatic ecosystems: a review of global sources, biogeochemical cycling, and effects on fish. Sci. Total. Environ. 907, 167911; doi:10.1016/j.scitotenv.2023.167911

Essien-Ibok, M.A., Asuquo, I.E. and Ekpo, I.E. 2014. The assessment of acute toxicity of urea fertilizer against Heterobranchus bidorsalis fingerlings. Global J. Fish. Aquaculture, 2(5), 169–176. Available via https://www.globalscienceresearchjournals.org/articles/the-assessment-of-acute-toxicity-of-urea-fertilizer-against-heterobranchus-bidorsalis-fingerlings.pd

Food and Agriculture Organization of the United Nations. 2023. The state of food and agriculture 2023: revealing the true cost of food to transform agrifood systems. Rome, Italy: FAO; doi10.4060/cc7724en

George, U.U., Akpan, E.R. and Akpan, M.M. 2020. Assessing the impacts of coastal activities on the water quality of Qua Iboe River Estuary, South-South, Nigeria. New. York. Sci. J. 13(3), 1–15. Available via http://www.sciencepub.net/newyork/nys130320/01_36012nys130320_1_15.pdf

George, U.U. and Ibok, E. 2024. Investigating the acute toxic effects of urea fertilizer on juvenile Nile tilapia (Oreochromis niloticus). Asian J. Fisheries Aquatic Res. 26(6), 68–75; doi:10.9734/ajfar/2024/v26i6776

Grădinariu, L., Crețu, M., Vizireanu, C. and Dediu, L. 2025. Oxidative stress biomarkers in fish exposed to environmental concentrations of pharmaceutical pollutants: a review. Biology 14(5), 472; doi:10.3390/biology14050472

Jena, T., Chhatoi, S.K., Khandai, S.S., Mishra, R., Mohanty, A., Sharma, K.K. and Mohapatra, D. 2025. Sublethal urea exposure in Nile tilapia: morphological, behavioral, and histological alteration. Chemosphere 372, 144086; doi:10.1016/j.chemosphere.2025.144086

Liebel, S., Tomotake, M.E.M. and Oliveira Ribeiro, C.A. 2013. Fish histopathology as biomarker to evaluate water quality. Ecotoxicol. Environ. Contamination 8(2), 9–15; doi:10.5132/eec.2013.02.002

Maitra, S. and Nath, S. 2014. Toxic impacts of urea on the hematological parameters of air-breathing fish (Heteropneustes fossilis). Am-Eurasian. J. Agricult. Environ. Sci. 14(4), 336–342. Available via http://www.idosi.org/aejaes/jaes14(4)14/10.pd

Makaras, T., Razumienė, J., Gurevičienė, V., Sauliutė, G., Matviienko, N., Kozij, M. and Stankevičiūtė, M. 2025. Impact of urea nitrogen fertilizer on the physiology, behavior, and histology of juvenile rainbow trout. Fish Physiol. Biochem. 51(4), 115; doi:10.1007/s10695-025-01528-5

Menon, S.V., Kumar, A., Middha, S.K., Paital, B., Mathur, S., Johnson, R., Kademan, A., Usha, T., Hemavathi, K.N., Dayal, S., Ramalingam, N., Subaramaniyam, U., Sahoo, D.K. and Asthana, M. 2023. Water physicochemical factors and oxidative stress physiology in fish: a review. Front. Environ. Sci. 11, 1240813; doi:10.3389/fenvs.2023.1240813

Organization for Economic Co-operation and Development. 2019. Test No. 203: fish, acute toxicity test. Paris, France: OECD Publishing; doi:10.1787/9789264069961

Rand, G.M. 2020. Fundamentals of aquatic toxicology: effects, environmental fate, and risk assessment. Boca Raton, FL: CRC Press; doi: 10.1201/9781003075363

Sultana, S., Nasiruddin, M., Azadi, M. and Chowdhury, M. 2020. Toxicological effect and behavioral response of a predatory stinging catfish (Heteropneustes fossilis) exposed to three indigenous plant seed extracts. Bangladesh J. Zool. 48(2), 379–391; doi:10.3329/bjz.v48i2.52377