Acute effect of tetracycline on systemic stress markers and organ histopathology in juvenile Nile tilapia, Oreochromis niloticus

Abstract

Antibiotic contamination in aquaculture systems frequently encounter severe threat to aquatic life including fish. This study aimed to evaluate acute toxicological effects of tetracycline in juvenile Nile tilapia, Oreochromis niloticus. We exposed ten fish in each tank with nominal tetracycline concentration of 0 μg/L, 10 μg/L, 100 μg/L, 500 μg/L, 1000 μg/L, and 5000 μg/L, under semi-static condition for 96 h. After the exposure periods, six fish per treatment (n = 6) were sampled to evaluate blood glucose, hemoglobin, frequencies of erythrocyte cellular and nuclear abnormalities, and histopathology of gills, intestine, liver, and kidney. Tetracycline induced clear dose-dependent effects in studied fish. Blood glucose increased significantly (p < 0.05) from 100 μg/L and onward, indicating acute metabolic stress, whereas hemoglobin decreased significantly at 500 and 5000 μg/L indicating compromised oxygen transport. Erythrocyte cellular and nuclear abnormalities rose in a threshold manner; twin, tear-drop, elongated, binucleated cells and nuclear buds became significantly more frequent at ≥ 100 μg/L and intensified at higher doses. Gill pathology such as congestion, lamellar degeneration and fusion, epithelial hypertrophy/hyperplasia was appeared from medium doses and was most pronounced at 5000 μg/L. Significant reduction in villus length, vacuolation of enterocyte and fusion of brush-border of intestine was observed at 500 μg/L and higher doses. Liver lesions such as vacuolation, hemorrhage, melanomacrophase centers, and necrosis were intensified with doses. Besides, renal alterations such as mild glomerular expansion at 100 μg/L, dilation of Bowman’s space and tubular changes at 500 μg/L and above concentrations were observed. Our findings highlight sensitive early-warning biomarkers and underscore the need for chronic exposure studies and residual analysis for risk assessments of antibiotic usage in aquaculture.

Graphical Abstract

Highlights

• •Increased blood glucose levels in higher tetracycline doses, indicating metabolic stress in tilapia.
• •Decreased haemoglobin levels in higher tetracycline doses, suggesting impaired oxygen transport.
• •Erythrocyte abnormalities, including binucleated cells, increase with tetracycline concentration.
• •Increased histopathological changes in gills, liver, and kidney intensify with higher tetracycline doses indicating impaired physiology.

1. Introduction

Antibiotics are widely used in human and veterinary medicine and are increasingly detected in aquatic environments because of agricultural runoff, aquaculture practices and inadequate wastewater treatment [1], [2]. Moreover, the global use of tetracycline-class antibiotics in aquaculture practice has resulted in extensive occurrence of these compounds in aquatic systems [3], [4], [5] (Table 1). Due to their chemical stability, low biodegradability, and strong affinity for binding to organic matter and sediments, they persist and are frequently detected in surface waters, sediments, and biota, thus posing ecological and health concerns globally (Amangelsin et al., 2023; [6]). The environmental ubiquity raises concerns for non-target organisms, particularly farmed and wild fishes that are routinely exposed to antibiotic residues [7], [5].

Table 1.

Global occurrences of tetracycline in various aquatic systems.

Locations	Mean prevalence	References
Yellow river estuary, China	1.37 ng/L	Wang et al., [8]
Yangtze River (drinking water), China	11.16 ng/L (dry season)	Wang et al., [9]
Msunduzi River, Eastern South Africa	158.42–1290.43 ng/L	Addis et al., [10]
Rural rivers, Brazil	44.1 ng/L (OTC)	Monteiro et al., [11]
Honghu lake, China	965.7 ng/L	Chang et al., [12]
Surface and groundwater near animal farms,Tehran, Iran	5.4–8.1 ng/L	Javid et al., [13]
Marine aquaculture farms surrounding Zhoushan and Taizhou, East China	0.074–0.520 ng/L (mean 0.248 ng/L)	Zhang et al., [14]
Ponds, Jiangsu, China	0.93 µg/L (TC) and 6.87 µg/L (OTC) (pond water) 6.44 µg/L (farm effluents)	Wei et al., [15]
Naerincheon River across Hongcheon, Gangwon province, Korea	254.82 µg/L (water) 75.70 µg/kg (sediment)	Awad et al., [16]

Fish are particularly vulnerable to antibiotics at multiple levels of biological systems, ranging from molecular and cellular endpoints to tissue and whole-animal responses [17]. Tetracycline can exert toxic effects through several pathways, may induce acute toxicity at high doses and sub-lethal effects at environmentally relevant concentrations [18]. Acute or chronic tetracycline exposure can disrupt antioxidants defense mechanisms by suppressing enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and by elevating lipid peroxidation (LPO), thereby inducing oxidative stress in fish species [19]. Studies have also reported disruption of metabolic processes, immunotoxicity and genotoxicity, and have been linked with histopathological lesions in gills, liver, and kidney tissues in many species [20], [21], [22]. Collectively, these mechanistic endpoints such as oxidative stress, immunotoxicity, genotoxicity, and tissue pathology, underscore that tetracyclines may compromise fish health and aquaculture productivity well below lethal thresholds [23], [24].

Nile tilapia (Oreochromis niloticus) is one of most important aquaculture species worldwide, accounting for substantial portion of inland fish production [25]. In 2020, global tilapia production from aquaculture reached approximately 6.1 million tons, overshadowing capture production (about 0.9 million tons) and highlighting its critical role in freshwater aquafarming systems [26]. Beyond production volumes, O. niloticus excels as an excellent model organism in ecotoxicological studies due to its robust physiology, rapid growth, omnivorous diet, and adaptability to variable environmental conditions [27], [28]. Additionally, its resilience to pollutants has made it a valuable model for combined toxicity and bioconcentration testing under OECD compliant protocols [29].

Acute toxicity tests (96 h exposures) following standardized OECD protocols [30] test guideline no. 203, remain a cornerstone for hazard classification and provide reproducible median lethal concentration (LC-50) [31]; however, mortality alone often underestimates sublethal impacts that can compromise fish physiology and overall fitness and ultimately aquaculture productivity [32]. Therefore, integration of physiological markers such as blood glucose (stress indicator), hemoglobin (indicative of oxygen carrying capacity), erythrocyte nuclear and cellular abnormalities (genotoxic and cytotoxic markers) [33], [34], [35], and histopathological assessment of gill, liver, kidney, and intestine may strengthen ecological interpretation and increases sensitivity for early effect detection upon exposed to range of stressors and toxicants [32]. Specifically, these biomarkers have been widely used in impact assessment of antibiotic exposure and fish health [36].

Previous studies on tetracyclines report species and dose dependent effects including oxidative stress, altered metabolic markers, genotoxic and tissue lesions in exposed fish [37], [38], [39]. Some authors have examined toxicological endpoints such as transcriptomic analyses including gill pathology [40], genotoxicity [41], antioxidant capacity and level of immune gene expression [19] specific to tilapia. Nonetheless, reported outcomes vary with route of exposure (water-borne vs dietary), concentrations, exposure duration, and life stages, yet combined multi-organ pathology and hematology under acute exposure (standardized OECD protocols-203) remain limited. To address this knowledge gap, the present study performed an acute toxicity test to quantify the toxicological effects of waterborne tetracycline on juvenile O. niloticus. We measured blood-biochemical indices (i.e. glucose & hemoglobin), erythrocyte cellular and nuclear abnormalities, and semi-quantitative histopathological assessment of major organs including gills, liver, kidney, and intestine. This multilevel biomarker approach increases mechanistic resolution, allowing us to determine the acute hazard of tetracycline and detect early physiological and genotoxic signals. Moreover, this investigation may provide data that can inform risk assessment and management of antibiotic contamination in aquaculture industries.

2. Materials and methods

2.1. Chemicals

Tetracycline hydrochloride (TC) powder (purity > 99 %) was procured from FUJIFILM Wako Pure Chemical Corporation, Japan. A stock solution was prepared by dissolving 5 g tetracycline in 300 mL distilled water and homogenized by magnetic stirrer. The solution was kept in a double capped glass vial at 4º C throughout the experimental periods.

2.2. Tilapia acquisition and maintenance

Healthy and non-diseased 120 tilapia fingerlings of almost uniform size and weight (9.44 ± 0.27 cm and 13.32 ± 1.63 g respectively) were obtained from Sarnolota Fish Hatchery, Mymensingh. The fingerlings were handled and transported carefully to minimize stress. They were then placed in a large 1500 L rectangular cemented tank equipped with continuous water flow in the Laboratory of Fish Ecophysiology at Bangladesh Agricultural University, Mymensingh. The fish were acclimated to laboratory conditions for 15 days at an ambient temperature of 30 ± 1 °C with natural photoperiods (14 h light:10 h darkness). During the conditioning period, they were fed twice daily (at 9:00 am and 5:00 pm) with commercial tilapia feed at 5 % of their body weight.

2.3. Design of acute toxicity test

The experiment was conducted according to the general guidelines for fish acute toxicity tests - 203, described by organization for economic co-operation and development (OECD, 2025). After acclimatization periods, sixty fingerlings were selected for acute toxicological assay. Ten fish with a total biomass of ∼135 g in each glass tank (100 L) were exposed to five treatment groups having series of tetracycline concentrations such as 10, 100, 500, 1000, and 5000 µg/L with an additional control group without test substance. The exposure solutions were completely renewed and test tanks were carefully cleaned after 48 h of exposure. The experiment was conducted in a semi-static manner to reduce loss of tetracycline concentration from photodegradation and environmental decomposition. All the tanks received adequate oxygenation through air-pumps throughout the test periods. Water quality parameters such as dissolved oxygen, pH and total alkalinity were checked each day (see supplementary table 1). Test fish were not provided any feed during the exposure test. The visible behavioral alterations and mortality were checked at 6 h of intervals. After the completion of the exposure (96 h), fish were sampled and several biomarkers such as blood and histopathology were assessed. All the experimental actions including fish handling, chemical treatments, investigations were approved and followed by the guidelines of ‘Animal Welfare and Ethics Committee,’ Bangladesh Agricultural University (License No.: BAU-FoF/2022/002).

2.4. Blood-biochemical indices

At the end of the experimental periods (96 h), six fish (n = 6) from each group were carefully sampled using anesthesia to minimize handling stress. The blood samples were drawn from the caudal peduncle and immediately collected into vials containing 20 mM EDTA. Hemoglobin (g/dL) and glucose (mg/dL) levels were examined instantly using an Easy Mate ET232 analyzer (Bioptic Technology Inc. Taiwan). The remaining blood samples was instantly stored at 4°C for subsequent morphological assessments.

2.5. Erythrocyte cellular and nuclear structures

Erythrocyte cellular (ECAs) and nuclear abnormalities (ENAs) were assessed based on established protocols [42]. For each treatment, blood samples from six fish (n = 6) were smeared onto glass slides, air-dried, ethanol fixed, Giemsa stained, and mounted in DPX. Slides were examined at 400 times magnification using a digital microscope (Micros, MCX100) equipped with a camera (AmScope, MA1000). From each fish, six slides were prepared, and 2000 erythrocytes were counted randomly per slide. Morphological anomalies (e.g. twin, teardrop, fusion) and nuclear aberrations (e.g. nuclear bridges, buds, notches) were quantified following the classification criteria described in prior studies [43], [44], [45].

2.6. Histopathology of gills, intestines, liver, and kidney

Six fish (n = 6) from per group were dissected immediately to remove the entire intestine, gills, liver, and kidney following established protocol [46]. Tissues were fixed in Bouins solution for 24 h, then transferred to labeled containers with 70 % ethanol and stored at 4°C. Histological process were performed following standard procedures; dehydration via graded alcohols, paraffin embedding, microtome sectioning, staining, and DPX mounting [46]. Histological imaging was performed using a microscope (Micros, MCX-100). Quantitative morphometric parameters were evaluated by scoring and categorizing each slide.

2.7. Data analysis

Quantitative data are presented as mean ± standard deviation (SD) in both tables and figures. Semi-quantitative assessment of histopathological alterations was conducted using a categorical scoring system as described by Mishra and Mohanty [47]. Prevalence of each parameter was classified according to the proportion of affected sections; no abnormalities (0 % of sections), mild abnormalities (< 10 % of sections), moderate abnormalities (10 – 50 % of sections) and severe abnormalities (> 50 % of sections), and displayed as bar plot with error bars. Data were analyzed using IBM SPSS statistical package (version 31) and one-way analysis of variance (ANOVA) was performed to estimate statistically significant difference at p < 0.05. Graphical presentation of data was performed using python programming (www.python.org; version 3.11).

3. Results

3.1. Blood biochemical indices

Blood glucose and hemoglobin levels of exposed fish at different concentrations are depicted in Fig. 1 (panel A & B). As illustrated in Fig. 1A, glucose levels showed a clear upward trend with increasing concentrations of tetracycline. The 100 µg/L, 500 µg/L, 1000 µg/L, and 5000 µg/L groups exhibited significantly higher (p < 0.05) glucose level compared to the control and 10 µg/L groups. Conversely, hemoglobin remained statistically unchanged in 10 µg/L to 500 µg/L groups compared to control. However, 1000 µg/L, and 5000 µg/L concentration groups experienced a significant decline (p < 0.05) underscoring a concentration dependent decrease in serum hemoglobin at higher treatment doses of tetracycline (Fig. 1B).

Fig. 1.

Level of (A) Glucose, and (B) hemoglobin in fish exposed to different concentrations of tetracycline. Different letters in the superscript designate statistically significant difference (p < 0.05) among treatments.

3.2. Erythrocyte cellular and nuclear abnormalities

Frequencies of observed erythrocyte cellular morphological changes such as twin, fusion, tear drop, and elongated across six treatments are presented in Fig. 2 (panel A-D). Twin shaped cells increased significantly (p < 0.05) from 100 µg/L and onward compared to control and 10 µg/L groups (Fig. 2A). Likewise, fusion increased significantly (p < 0.05) only at the highest exposures such as 1000 µg/L groups, while all lower treatments (0–500 µg/L) showed no significant difference from the control group (Fig. 2B). Tear drop followed a similar trend such as frequencies in 500 µg/L, 1000 µg/L, and 5000 µg/L treatments were significantly higher (p < 0.05) than those observed in control through 10 µg/L and 100 µg/L groups (Fig. 2C). Lastly, elongated cells showed significantly higher frequency at 500 µg/L and above in contrast to control, 10 µg/L, and 100 µg/L groups (Fig. 2D). These patterns demonstrate a clear concentration-dependent increase in abnormal erythrocyte morphologies, with significant elevations emerging at medium concentrations of tetracycline and intensifying at higher levels.

Fig. 2.

Frequencies of erythrocyte cellular abnormalities in fish exposed to several treatments. Twin (panel A), fusion (panel B), tear-drop (panel C) and elongated (panel D). Different letters in the superscript designate statistically significant difference (p < 0.05) among treatments.

Frequencies of observed erythrocyte nuclear abnormalities (ENAs) such as bi-nucleated cells, nuclear buds and karyopyknosis are presented in Fig. 3 (panel A-C). Frequency of bi-nucleated cells significantly increased (p < 0.05) from 100 µg/L and onward; 100 µg/L, 500 µg/L, 1000 µg/L, and 5000 µg/L concentrations had significantly higher occurrences than control (0 µg/L) and 10 µg/L groups (Fig. 3A). Nuclear buds showed a similar pattern, with 1000 µg/L and 5000 µg/L concentrations exhibited significantly higher frequencies than the lower concentrations including 0 µg/L to 100 µg/L (Fig. 3B). Karyopyknosis followed the same pattern, only the 1000 µg/L and 5000 µg/L groups exhibited significantly greater (p < 0.05) frequencies than the lower three levels (Fig. 3C). These findings indicate a concentration-dependent increase in erythrocytic nuclear anomalies in tilapia exposed to varying levels of tetracycline concentrations.

Fig. 3.

Frequencies of erythrocyte nuclear abnormalities in fish exposed to tetracycline treatments. Bi-nucleated (panel A), nuclear-bud (panel B) and karyopyknosis (panel C). Different letters in the superscript designate statistically significant difference (p < 0.05) among treatments.

3.3. Histopathological changes in gills

A semi-quantitative assessment of histological changes in the gills is summarized in Fig. 5. Congestion in primary lamellae was absent in the control and 10 µg/L groups, but appeared as mild changes in the 500 µg/L and 1000 µg/L concentration groups and moderate (48 %) changes at the highest concentration of 5000 µg/L. Degeneration in secondary lamellae became evident as mild (8 %) changes from 1000 µg/L groups. However, degeneration in primary lamellae was first observed in 100 µg/L, peaked with moderate (44 %) changes in 500 µg/L groups, then slightly deceased in 1000 µg/L and above groups. In the case of fusion in secondary lamellae, it was absent in control and 100 µg/L, but mild fusion emerged in the 10 µg/L, 500 µg/L, 1000 µg/L, and 5000 µg/L groups. Hypertrophy and hyperplasia in primary lamellae were only observed at the 5000 µg/L concentration with mild (8 %) changes. The sample image of histopathological changes in gills is illustrated in Fig. 4.

Fig. 5.

Semi-quantitative assessment of histopathological changes in gills of fish exposed to tetracycline. No abnormalities (0 % of sections), mild abnormalities (< 10 % of sections), moderate abnormalities (10 – 50 % of sections) and severe abnormalities (> 50 % of sections).

Fig. 4.

Several histological changes of gills: (a) normal gills, (b) degeneration in primary lamellae, (c) fusion in secondary lamellae, (d) congestion in primary lamellae; degeneration in secondary lamellae, (e) hypertrophy and hyperplasia in primary lamellae.

3.4. Histopathological changes in intestine

Semi-quantitative assessment of histopathological changes in the intestine across groups are presented in Fig. 6. In the lowest does (10 µg/L), mild epithelial displacement from the lamina propria and onset of brush boarder fusion were observed, while vacuolation of enterocyte remained minimal. At the concentration of 100 µg/L, epithelial displacement persisted and vacuolation began to appear, although the brush boarder was largely intact. At medium dose (500 µg/L), epithelial architecture was largely maintained, but vacuolation became more pronounced (about 8 %). However, with the increase in tetracycline concentration epithelial displacement and vacuolation become bordering to severe. Fusion of brush border was consistently observed in 500 µg/L and above concentrations with mild changes.

Fig. 6.

Semi-quantitative assessment of histopathological changes in intestine of fish exposed to tetracycline. No abnormalities (0 % of sections), mild abnormalities (< 10 % of sections), moderate abnormalities (10 – 50 % of sections) and severe abnormalities (> 50 % of sections).

Besides pathological changes, morphology of intestine was also affected. Histomorphological changes in intestine are depicted in Table 2. Quantitative measurements of intestinal morphology revealed that villus length and width was significantly reduced in all treated groups compared to control. However, total thickness of intestinal wall remained statistically unchanged across all groups, indicating preservation of overall mucosal structure. The thickness of muscular layer showed significant (p < 0.05) thickening at the 1000 µg/L and above concentration compared to 10 µg/L and 100 µg/L groups, while other treatments did not differ significantly. Crypt depth varied irregularly, with the lowest concentration group (10 µg/L) exhibited a significant reduction relative to control, no clear dose-dependent trend was evident. Fattening of mucosal fold showed no significant alterations in any treatments. Photomicrographs of histopathological changes of intestine are displayed in Fig. 7.

Table 2.

Histomorphological changes of intestine in fish exposed to tetracycline.

Parameters	Tetracycline concentrations (µg/L)
	0	10	100	500	1000	5000
Villus length (µm)	283.88 ± 13.78b	288.77 ± 17.68b	215.09 ± 15.66a	201.46 ± 12.12a	214.75 ± 13.34a	200.15 ± 11.58a
Villus width (µm)	109.29 ± 17.41b	97.76 ± 19.95ab	94.51 ± 15.17ab	90.57 ± 12.53ab	85.17 ± 10.55a	86.80 ± 8.94a
Thickness of the wall (µm)	25.06 ± 5.07a	22.79 ± 6.06a	20.68 ± 5.54a	24.97 ± 6.17a	26.04 ± 6.79a	21.42 ± 5.11a
Thickness of muscular (µm)	27.52 ± 7.05ab	27.60 ± 5.61ab	21.17 ± 7.23a	27.05 ± 6.42ab	31.09 ± 5.97b	23.65 ± 5.32ab
Crypt depth (µm)	33.47 ± 9.46b	23.87 ± 5.98a	32.20 ± 7.09ab	25.20 ± 5.74ab	26.20 ± 5.74ab	24.57 ± 5.26a
Fattening of mucosal fold (μm)	78.65 ± 11.59a	81.77 ± 10.12a	95.34 ± 13.88a	84.11 ± 14.62a	91.72 ± 19.78a	80.17 ± 5.54a

Fig. 7.

Histological changes in the intestine: (a) normal; VL = villus length, VW = villus width, TW = thickness of wall, TM = thickness of muscular, CD = crypt depth, MF = fattening of mucosal fold, (b) DEL = displacement of epithelium from lamina propria, (c) EV = vacuolation of enterocytes, (d) F = fusion of brush border.

3.5. Histopathological changes in liver

Semiquantitative assessment of histological alterations of liver are illustrated in Fig. 8. Histological evaluation of liver tissue in different treatments revealed a progressive pattern of liver injury. Blood congestion with mild changes emerged in 1000 µg/L and 5000 µg/L concentrations and with moderate hemorrhage (∼ 41 %) at 500 µg/L. Cytoplasmic vacuolation appeared mildly at 10 µg/L to 500 µg/L groups. Melanomacrophase centers were minimally present even at lower doses such as 10 µg/L and 100 µg/L, increased at 500 µg/L and above groups and were most prominent (∼ 48 %) in the highest concentration of 5000 µg/L group. Necrosis was detected starting from 100 µg/L, continuing through all higher concentrations with a peak at 5000 µg/L with moderate severity but borderline to severe abnormality (∼ 49 %). Collectively, these findings indicate a clear dose dependent increase of liver pathology, with early onset of some lesions at low exposures. Samples of histopathological changes of liver are represented in Fig. 9.

Fig. 8.

Semi-quantitative assessment of histopathological changes in liver of fish exposed to tetracycline. No abnormalities (0 % of sections), mild abnormalities (< 10 % of sections), moderate abnormalities (10 – 50 % of sections) and severe abnormalities (> 50 % of sections).

Fig. 9.

Histopathological changes of liver: (a) Normal liver, (b) Cytoplasmic vacuolation, (c) hemorrhage, (d) melano-macrophage center, (e) necrosis, and (f) blood congestion.

3.6. Histopathological changes in kidney

Semi-quantitative assessment of renal alteration is showed in Fig. 10. Histological examination revealed clear indications of renal injury that exaggerated with increasing concentrations. Mild glomerular expansion was observed at 100 µg/L and become intensified at highest concentration (5000 µg/L) of tetracycline, with a moderate pathological severity scoring about 40 % of the sections. Dilation of Bowman’s space detected at 500 µg/L, increased in moderate severity (about 42 %) at 1000 µg/L, and remained evident at the highest dose. Notably, enlargement of renal tubule diameter was absent at lowest dose (10 µg/L) and intermediate doses (100–500 µg/L) but become apparent starting at 1000 µg/L and persisted at the highest concentration of tetracycline. These findings reflect a dose-dependent progression suggesting compromised nephron integrity. Sample images of renal lesions are presented in Fig. 11.

Fig. 10.

Semi-quantitative assessment of histopathological changes in kidney of fish exposed to tetracycline. No abnormalities (0 % of sections), mild abnormalities (< 10 % of sections), moderate abnormalities (10 – 50 % of sections) and severe abnormalities (> 50 % of sections).

Fig. 11.

Histopathological changes of kidney: (a) normal, (b) glomerular expansion; dilation of Bowman's space, (c) increasing in the diameter of renal tubules.

4. Discussion

The present study demonstrates that waterborne tetracycline produced clear concentration-dependent physiological, cytological, and histological effects in juvenile Nile tilapia, O. niloticus. Moreover, it has emerged evident as progressive hyperglycemia, decreased hemoglobin, elevated frequencies of erythrocytic cellular and nuclear abnormalities and dose-dependent histopathological lesions in gill, intestine, liver, and kidney. These findings altogether assume an integrated stress response that impinges on respiration, blood integrity, nutrient absorption, and detoxification pathways. In contrast to the present findings, some authors have reported compromised physiology and mortality of tilapia, for example, Dey et al., [24] reported that complete mortality in tilapia fries exposed to 3500 and 7000 mg/L, and significant histopathological changes in gills, intestine, liver, and kidney at 700 mg/L., which also correspond to the present investigation. However, present study found histopathological lesions in multiple organs studied well below these concentrations, may varied due to difference in exposure time and length (i.e. chronic vs acute). Besides, significant changes in liver enzymes (e.g. ALT, AST, and LDH) and kidney markers (e.g. creatinine and urea), DNA damage, hematological parameters, immune indices, and antioxidant enzymes were reported at doses of 80 and 160 mg/kg body-weight of tilapia [38].

In the current investigation, the progressive rise in blood glucose with increasing tetracycline concentration might be due to stress response mediated by catecholamine and corticosteroids mobilizing energy reserves which is recognized as an adaptive response to toxicant exposure [23]. Elevated glucose level therefore likely reflects generalized metabolic stress and increased energetic demands for detoxification and repair [33], [35]. Although this study minimized handling stress and performed sufficient acclimation, stress derived from releasing and catching of fish may be a factor increasing cortisol and glucose levels [48]. Moreover, short-term food deprivation can also change circulating glucose in teleost. Thus, fasting during the experiment can confound interpretation from tetracycline exposure related hyperglycaemia [49]. Concomitantly, the observed decline in hemoglobin at 1000 µg/L and 5000 µg/L dose indicate possible impairment of oxygen-carrying capacity, which could compromise aerobic metabolism and compound the effects of hyperglycemia [50]. Report suggests that elevated glucose and reduced hemoglobin together may lead to concurrent metabolic and respiratory compromise that can reduce aerobic performance and increase susceptibility to additional stressors [41], [51]. The concentration dependent increase in erythrocytic cellular abnormalities (i.e. twin, teardrop, fusion) and nuclear anomalies (i.e. binucleated cells, karyopyknosis) reported in the present experiment assumes cytotoxic and genotoxic insult in the blood of fish exposed to antibiotic [41], [52]. Morphological and nuclear erythrocyte changes may also reflect membrane damage, impaired erythropoiesis, chromatin condensation and chromosome instability often driven by oxidative stress and DNA damage [40]. However, direct assays such as comet and micronucleus test are therefore required to confirm genotoxicity [53]. The emergence of several nuclear aberrations at 500 µg/L and higher concentrations in this study suggest a threshold above which cellular repair mechanisms might be overwhelmed, a pattern seen in other antibiotic-exposed fish studies [21].

Histopathological changes observed in gills such as congestion, lamellar degeneration and fusion in responses to waterborne toxicants are claimed to responsible for reducing effective respiratory surface area [54], [55], [56], [57]. Such structural alteration may reduce the open water space between lamellae and decreases the effective respiratory surface area available for gas exchange, thereby can directly impairing oxygen uptake [58]. Besides, loss of lamellar surface and fusion are functionally important because they impair gas exchange surface area and disrupt ionic/osmoregulatory balance, thereby contributing systemic hypoxic stress in fishes [22], [59]. Epithelial hypertrophy and hyperplasia observed at the highest dose (5000 µg/L) may represent a compensatory epithelial response in protecting the gill surface, but this response can paradoxically increase diffusion distance and further reduces respiratory efficiency [60]. The functional significance of these histological changes is well supported by earlier experimental work showing correlations between lamellar fusion/hyperplasia and reduce respiratory performance and altered blood parameters in contaminated conditions [58], [59].

Intestinal observations such as enterocyte vacuolation, microvillus/brush-border damage and reduced villus dimensions presume loss of absorptive surface area and possible impairments of nutrient uptake [61], [62]. These histomorphological changes are therefore assumed to impair transepithelial nutrient uptake because the enterocyte brush border and villus architecture could concentrate digestive enzymes and transporters that mediate carbohydrate, peptide, and lipid absorption [63]. Study reported that enterocyte vacuolation and microvillus damage specifically can reduce the effectiveness of absorptive membrane and can disrupt brush-border enzyme activity, decreasing digestive efficacy even when gross intestinal wall thickness appears preserved [64]. Moreover, xenobiotics induced injury of gut commonly includes oxidative stress, enterocyte apoptosis or necrosis and altered epithelial renewal, all of which may drive villus shortening and microvillus blunting [65]. Intestinal epithelial degeneration following xenobiotic exposure has been associated with reduced feed efficiency and altered gut microbial communities in multiple fish species [66], [67]. Since gut integrity is tightly linked to immune status and nutrient assimilation, even transient morphological damages can have outsized effects on growth and disease resilient if exposure is repeated [68].

The liver showed dose dependent cytoplasmic vacuolation, hemorrhage, necrosis and increased melanomacrophase centers (MMCs) in the present study, indicates metabolic disruption and activation of innate phagocytic pathways [21], [69]. MMCs are a broadly recognized histological biomarker of pollutant exposure and immune activation in fish, and increases in MMCs frequency or size have been correlated with contaminant load in numerous earlier studies [24], [69]. Hepatic necrosis and hemorrhage at higher exposures are supposed to compromise detoxification and resulting accumulation of toxic effects as reported for antibiotic exposure [22], [40].

Progressive renal lesions such as glomerular expansion, Bowmans’s space dilation, tubular changes with increasing tetracycline concentrations assume impaired filtration and excretion capacity that would slow elimination of tetracycline and metabolites and can disturb fluid/ionic homeostasis in fishes [69], [70]. Similar nephropathies have been observed in fish following therapeutic or environmental antibiotic exposure and are likely to impair ionic/osmotic balance and xenobiotics clearance [24]. Analytical measurement of tissue residues would therefore be valuable to correlate internal dose with lesion severity and to assess clearance kinetics relevant to withdrawal times in aquaculture [70].

Considering the disruption of metabolic markers and multi-organ histopathology, this study propose that tetracycline exposure may set to set off a chain of physiological events beginning with blood glucose-hemoglobin alteration and damage to cellular membrane and nuclear materials of erythrocytes, however suggesting more deliberate investigations with molecular and cellular pathways for further confirmations. This study suspect that the resulting genotoxic damage may compromises the integrity of red blood and the epithelial cells of the gills, which together can reduce the fish’s ability to take up oxygen from the water and to transfer to the bloodstream and resulting limited oxygen delivery, tissues experience metabolic strain may be reflected in elevated blood glucose.

Analytical verification of test solution that is comparison of nominal and measured concentrations in exposure water is vital to ensure that organisms are exposed to intended doses of chemicals (e.g. tetracycline) [14]. We note that tetracycline can be loosed in exposure media by photolysis, complexation with Ca^2+, Mg⁺², sorption to particulate matter etc. However, in the present study extraction and measurement of aqueous concentrations of tetracycline was not possible due to lack of access to validated liquid chromatography at the time of the study, nominal concentrations were therefore prepared under standardized procedure and documented to minimize errors [71]. To minimize photodegradation, expose tanks were partially wrapped with aluminum foil and our wet lab naturally inhibit direct sunlight. Acknowledging these limitations, we plan to verify water concentrations in future works in collaboration with analytical chemistry laboratory. Finally, individual fish were treated as experimental replicates rather than tanks; we note that OECD guidelines for fish acute toxicity [30] does not suggest tank level replications, we recognize potential for pseudo-replications; future studies will include tank-level replications to explicitly accounts for potential tank effects.

5. Conclusion

This study demonstrates that waterborne tetracycline produces clear, concentration-dependent sublethal effects in juvenile Oreochromis niloticus, indicating metabolic stress (elevated blood glucose), impaired oxygen transport (reduced hemoglobin), increased erythrocyte cytological/genotoxic abnormalities, and progressive histopathological lesions in gill, intestine, liver, and kidney. The integrated pattern of biomarkers assumes compromised respiration, nutrient absorption and detoxification capacity, ultimately provoking hepatic and renal overload and immune activation. Although limited by the short (96 h) exposure and absence of analytical residue verification and oxidative/genotoxic biochemical assays, the results flag important risks to fish health and aquaculture performance at sublethal antibiotic concentrations. Future work should prioritize chronic and recovery trials, measurement of tissue/water residues, inclusion of oxidative-stress and immune endpoints to fully inform risk assessment and management.

Funding acknowledgement

We gratefully acknowledge the financial support provided by the Bangladesh Agricultural University Research System (BAURES; Project No. 2025/18/BAU).

CRediT authorship contribution statement

Md Al-Emran: Writing – review & editing, Writing – original draft, Visualization, Supervision, Funding acquisition, Formal analysis, Conceptualization. Md Asad Ud Zahan Siddique: Writing – original draft, Investigation, Formal analysis, Data curation. Md Sayem Sheikh: Writing – original draft, Investigation, Formal analysis, Data curation. Md Ruhul Amin: Formal analysis, Data curation. Most. Rifah Tamanna: Investigation, Data curation. Purabi Karmoker Puja: Investigation, Data curation. Kumari Bristi Rani: Investigation, Data curation. AKM Afzal Hossain: Investigation, Data curation. Md Abu Rahad: Investigation, Data curation. Md Shahjahan: Writing – review & editing, Resources.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary material

Data availability

Data will be made available on request.