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 remains incompletely characterized in freshwater fish.

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

Methods:
Acute toxicity was evaluated using a 96-hour semi-static bioassay in accordance with OECD Test Guideline 203. The median lethal concentration (96-h 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. Behavioral responses, hematological parameters, antioxidant enzyme activities (SOD, CAT, GPx), lipid peroxidation (MDA), and histopathological alterations in gill, liver, and kidney tissues were assessed. Data were analyzed using ANOVA or Kruskal–Wallis tests as appropriate.

Results:
The 96-h LC50 of urea was 11.8 g/L (95% CI: 11.1–12.6 g/L), indicating moderate intrinsic acute 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). Histopathological lesion scores differed significantly among groups (P<0.01; ε² range: 0.66–0.75).

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

Key words: Acute toxicity; LC50; Oreochromis niloticus; Sub-lethal effects; Urea fertilizer.