More innovative practices have replaced extensive traditional aquaculture practices in recent decades. These practices involve raising aquatic animals at high stocking densities, which increases the crowding stress and lowers immunity in reared animals. This, in turn, increases the risk of the emergence of new diseases, leading to financial difficulties and unsustainable fish farming (1). Among numerous devastating emerging fish diseases, bacterial infections are one of the important culprits for successful aquafarming (2). Ideally, fish farmers treat bacterial pathogens through medicated feed supplemented with desired levels of antibiotics for a prescribed period (3–5). Fish farmers often apply high doses of antibiotics for extended duration due to a lack of awareness on the impact of antibiotics (3), hence such a practice jeopardizes the safety of reared animals and end consumers. This injudicious use of antibiotics affects international trade and the aquatic environments (6). Research on the antibiotics that stay in fish muscle after long-term administration to treat bacterial diseases is the most important thing that necessitates the safe and wise use of antibiotics and the safety of aquatic products.

Oxytetracycline (OTC), an important antibiotic from the tetracycline group authorized for aquaculture use, is effective against the treatment of numerous bacterial pathogens (7), which works on the principle of inhibition of protein synthesis for bacterial lysis (8). However, studies have shown that it can hurt the fish liver and kidneys, change the microbiome of the gut, damage mitochondria, and make membrane lipid peroxidation worse (9, 10). Therefore, ensuring the biosafety of OTC in cultured aquatic animals is critical before advocating its widespread use. Environmental conditions significantly influence the efficacy of antimicrobials (11), with factors such as water temperature affecting fish metabolism due to their poikilothermic nature. Compliance with regional antimicrobial regulations, drug depletion studies, and dose-response analyses under specific environmental conditions are essential. Despite established guidelines for temperate fish species, there is a lack of information on OTC safety, efficacy, and residue accumulation in tropical regions (11). Recent studies have looked at how safe OTC is for fish species like rohu (12), snubnose pompano (13), Nile tilapia (14), striped catfish (15), mrigal (16), and catla (17).

Asian stinging catfish Heteropneustes fossilis is among the most extensively farmed freshwater omnivorous catfish of the Heteropneustidae family. Highly valued for its culinary appeal, low-fat content, high protein content, and medicinal properties (18, 19), this species thrives in challenging environments such as muddy waters with low oxygen levels. Its ability to tolerate prolonged periods of low oxygen and high ammonia concentrations further enhances its suitability for aquaculture, including stagnant water bodies like swamps and wetlands (20). To date, no study has investigated the impact of prolonged use of oxytetracycline hydrochloride (OTC) at gradually increasing doses through feed in H. fossilis. This study aimed to conduct a 30-day indoor in-feed OTC feeding trial at varying doses ranging from the therapeutic dose of OTC (1x; 80 mg/kg fish biomass/day) to 10 times the therapeutic dose (10x: 800 mg/kg fish biomass/day) and to explore the effects of OTC on non-specific immunity, serum biochemistry, oxidative stress biomarkers, residue retention in the edible muscle and liver histopathological alterations. Besides, the minimum inhibitory concentration (MIC) of OTC against common bacterial fish pathogens was determined to develop appropriate strategies on the dose and dosage, ensuring its sparing and responsible use in catfish aquaculture.

OTC is a widely used and highly preferred tetracycline-based antibiotic that is effective against a wide range of bacteria that can infect fish and is legal to use in aquaculture around the world (5, 16, 29). The most common method for administering antibiotics to treat and manage bacterial diseases in aquaculture is through medicated feed at a therapeutic or recommended dose (7, 11, 30). In this study, the tested bacterial strains were infective and isolated from sick fish with no documented evidence of OTC use. The range of OTC’s MIC values (0.5-8.0 µg/mL) against the examined pathogens was low, corroborating the findings of Gaunt et al. (31) in Aeromonas spp., Edwardsiella spp. and Streptococcus spp., with MIC values less than 1.0 regardless of their isolation location and times. These observations on low MIC can help predict the likelihood of a successful therapeutic intervention (32, 33). Before approval, the antibiotics are tested for their effectiveness, biosafety, impact on the environment, and ability to leave residues in the muscle (9, 30, 34, 35). As there are no effective guidelines on the use of OTC on tropical Indian fish, the current work assessed the safety of its oral supplementation at the therapeutic dose as well as up to ten times the therapeutic dose and its effect on several parameters of H. fossilis. Dietary supplementation did not cause any mortalities in treatment groups, thus proving its safety even at the highest dose and extended feeding regime. These results were comparable with previous studies recorded in rohu (12), snub-nosed pompano (13), Nile tilapia (14), striped catfish (15) and mrigal (16). However, the results revealed growth retardation in the treatment groups. Earlier reports suggested adverse impacts of OTC on fish possibly due to impaired nutritional absorption and assimilation, internal vital organ injury, disrupted metabolic functions and gastrointestinal microbiome dysbacteriosis (35, 36). Also, no significant differences in growth in rohu and mrigal upon feeding graded doses of OTC for up to 30 days were documented (12, 16). On the contrary, some reports categorized OTC as a growth promoter in striped catfish (15), Nile tilapia (37), and channel catfish (38).

It is reported that OTC-supplemented feed either enhances or reduces the non-specific immune responses (39). The diminished activities of the tested innate immune parameters like RBA, MPO, and lysozyme indicated immunosuppression, particularly at the higher OTC doses (5x-10x groups), supporting reports of Hand et al. (40). However, in agreement with the present results, the innate immunity indices of sea bream increased upon OTC administration at 75 mg/kg biomass/day for 10 days, which was retained up to three weeks after cessation of OTC dosing and then returned to normal levels (41) and the therapeutic dose demarcated significant enhancement in non-specific immunity parameters such as RBA and MPO. The present results on compromised immunity during the feeding trials suggest that the impact on immune responses is species-specific as the fish immunity varies with rearing conditions, fish species, antimicrobial dose, water temperature, route of delivery, and other conditions that affect drug absorption (36).

With the gradual increase in dose and duration of OTC administration, serum glucose increased, suggesting an aberrant metabolic activity and, consequently, stress-related events in fish similar to earlier studies (12, 15). The albumin level increased in the lower dose groups but declined in the higher dose groups, indicating biphasic activities and altered physiological status. The current finding reported reduced AST activity after an initial hike in the treatment groups, while ALT activity increased significantly in all treatment groups. The results indicated liver injury and dysfunction (42, 43), attributable to oxidative stress. Furthermore, elevated ALT levels in serum are suggestive of hepatitis arising from the toxicity of dietary OTC in H. fossilis at higher doses and the extensive burden on hepatic integrity. Concurrent with the present findings, Manna et al. (15) reported serum AST and ALT upsurge in catfish-fed graded doses (1x - 10x) of OTC. In another study, biphasic activities of AST and ALT were documented, with the highest levels in lower OTC-dosed fish than at the higher OTC-dosed fish (44). The increased concentration of these liver functional enzymes at lower doses and initial treatment times and then decreased concentration at higher doses and prolonged treatment may hypothesized to be the responses of H. fossilis is more likely due to terminal stages of feeding graded doses of OTC. Serum ALP was lower in fish-fed-graded OTC doses than in the control. The ALP levels decreased significantly in the 5x and 10x groups on the 30th day of administration, which indicated impaired phosphorus metabolism in OTC-fed catfish. With dose discontinuation, the levels of AST almost stabilized. On the other hand, the ALT and ALP levels were either higher or lower, indicating severe liver injury.

The SOD and CAT are vital for the detoxification of harmful reactive oxygen species, protecting cells, and maintaining homeostasis (45, 46). The MDA levels are considered an indicator of lipid peroxidation caused by oxidative stress and induced by exposure of fish to nutrients, pollutants, antimicrobials, etc. (45, 46). The present work documented a significant dose- and time-dependent increase in SOD, CAT, and MDA in comparison to control, revealing an impairment in the antioxidant systems of H. fossilis as a result of oxidative stress. Comparable results were obtained in pearl gentian grouper (10) and tilapia (14, 35) during the oral supplementation of OTC at varied doses. These results intricated an interplay between oxidative stress and drug toxicity on the vulnerability of aquatic organisms and highlighted the importance of monitoring antioxidant defences as a crucial aspect of fish health assessment.

While temperate fish species have well-documented data on residues (30, 39, 47), little is known about the depletion of OTC in tropical fish species. In recent years, reports on OTC residues in tropical fish species, including rohu (12), striped catfish (15), Nile tilapia (14), mrigal (16), and snub-nosed pompano (13) are expanding. The data on antibiotic residues in edible muscle tissues of each fish species is critical for the preparation of food safety guidelines (13), and comprehensive documentation of food safety rules for medication withdrawal times in aquaculture is required. The regulatory firms have set the MRL for OTC in edible fish flesh as 200 µg/kg (48) and 100 µg/kg (49). In this study, the residue levels in the edible flesh of H. fossilis were found to be significantly different based on dose and duration of administration. The therapeutic dose group documented residues below the MRL, indicating their compliance with the regulatory guidelines (48, 49). In contrast, OTC residues were above the MRL in higher-dosed groups. The residue accretion in various tissues led to metabolic stress, impaired liver function, oxidative stress and alterations in the liver histoarchitecture. These findings suggested that the use of a therapeutic dose of OTC even for an extended duration poses minimal threat to catfish health or human consumers.

The liver is the best organ for histopathological investigations to assess the drug effects (35, 50), as it is imperative for fish growth and metabolism (51). The histoarchitecture of the liver was severely impacted by OTC dose and duration and was directly correlated with the results of oxidative stress and liver function biomarkers. The hepatic parenchyma of the higher-dosed groups showed severe congestion, haemorrhage, and vacuolation, corroborating several earlier studies that reported hepatic vacuolization upon OTC administration in varied fish species (13, 15, 50–53). Vacuole formation may be a consequence of severe damage and degeneration as the liver is the main detoxification organ. It is, therefore, important to take these hepatotoxic effects into account, particularly if the fish are fed higher doses of antibiotics or longer durations.

In this study, OTC did not enhance fish growth, even when administered over an extended period. Notably, the therapeutic OTC dose (80 mg/kg biomass/day) did not negatively impact the non-specific immunological indices, enzymatic activities, stress biomarkers and residue accretion. However, administering OTC at higher doses (5x and 10x) had significant adverse effects on innate immunity, biochemical and oxidative stress markers, and liver histopathology. Therefore, to maintain optimal immunological and physiological indices in H. fossilis, a therapeutic dose of 80 mg/kg biomass/day may be adhered to for the treatment of bacterial infections. Additionally, higher doses led to a significant accumulation of OTC residues in the edible muscle of H. fossilis, which persisted even on day 10 of discontinuation of administration. Considering the negative effects of antibiotics, future research should focus on alternatives or other next-generation antibiotics to better control bacterial infections. This strategy will minimize the impact of antibiotics on cultured fish and other aquatic flora and fauna, as well as mitigate the issue of antimicrobial resistance and public health.