Sodium chloride (NaCl), often referred to as the “aspirin of aquaculture,” plays a vital role in fish farming due to its wide range of applications (El-Gawad  et al., 2016). It has been effectively used for treating fungal infections in fish and eggs, managing bacterial diseases (Fathollahi et al., 2020) and controlling parasitic infections such as proliferative kidney disease caused by Tetracapsuloides bryosalmonae (Enevova et al., 2018). Additionally, NaCl supports critical processes such as fertilization, reproduction and early development, primarily by reducing stress and enhancing osmoregulatory function in aquatic animals (Burgdorf-Moisuk et al., 2011).
       
In terms of survival, NaCl is essential for maintaining homeostasis in fish, particularly under stressful or fluctuating environmental conditions. NaCl baths at concentrations of 30.0-60.0 g/L have been shown to improve weight gain, relative growth rate and specific growth rate in Oreochromis niloticus (Elrahman et al., 2016). Moreover, for safe transportation, concentrations ranging from 4.0-10.0 g/L for up to 5 hours are recommended for this species (Tavares-Dias, 2022), further emphasizing its practical relevance in aquaculture management.
       
Beyond physiological effects, NaCl can also influence fish behavior, which serves as a sensitive and immediate indicator of environmental stress. Behavioral adaptability is a crucial survival strategy in animals, complementing slower physiological adjustments (Salvanes et al., 2013; Sewall, 2015; Tseng et al., 2020). Fish, being ectothermic, are especially responsive to external changes, with factors like temperature, salinity and chemical exposure leading to notable alterations in behavior such as changes in swimming patterns, aggression and respiratory rates (Hansen et al., 2016; Olusanya and van Zyll de Jong, 2018; Tong et al., 2020; Alfonso et al., 2021; Keen et al., 2017). Sub-lethal effects have been well-documented in species like Tilapia mossambica and Oreochromis niloticus, highlighting behavior as a critical endpoint in toxicological studies (Shrivastava et al., 2011; Khalil and Emeash, 2018; Fattah et al., 2020).
       
Oreochromis niloticus (Nile tilapia) is widely used in aquaculture due to its hardy nature, broad salinity tolerance and fast growth. Its economic value and adaptability make it a suitable model organism for investigating the physiological and behavioral effects of environmental factors such as salinity.
       
The present study aims to evaluate the behavioral effects of sodium chloride exposure in Oreochromis niloticus, with a focus on understanding how varying concentrations may alter fish activity, stress response and adaptability. These findings are expected to contribute significantly to the development of behavioral biomarkers for stress in aquaculture settings and support sustainable fish health management practices.

Collection and maintenance of experimental fishes
 
The fish utilized were tilapia, Oreochromis niloticus (with mean weight of 14.5±2.54 g), acquired from a commercial fish farm. These fish were carefully examined for any signs of pathogenic infection and transferred into a 500 L of capacity tank for 15 days. Prior to the transfer, the acclimation tanks were cleaned with 0.1% KMnO₄ to eliminate any fungal or dermal infections. During this acclimation period the fish were fed with commercial ration and water changes were performed three times per week to ensure optimal conditions. The original day-night light cycle remained uninterrupted and temperature between the range 25 to 30^oC. Throughout the acclimatization period, the tank remained aerated and fish mortality remained below 10%.
 
Experimental design
 
The study was conducted in the Department of Zoology at Mar Thoma College for Women, Perumbavoor, during January-February 2025. In the present study, healthy Oreochromis niloticus were used. After 15 days acclimatization, in the stocking tank, fish used for study was transferred to 20 litre circular plastic troughs (6 fish/trough) and closed with circular nets. The troughs containing equal volumes of water were employed, with consistent maintenance of water quality, temperature and aeration across the tanks. Experimental groups, namely, L (0.8 g/L of sodium chloride) and H (1.6 g/L of sodium chloride), along with control groups were maintained for 10 and 60 days each as triplicate sets. The dose selected was much below the recommended safe limits prescribed for usage. Fish of similar size and weight (20.16±4.26 g and mean length of 9.44±0.91 cm) were selected. They were provided with the same quantity of food at regular intervals. After acclimatization, the behavioral assessment of tilapia was carried out for 5 days continuously during the days of exposure:   
5-10 days (10-day period) 
55-60 days (60-day period) 
For 3 sections (2 hour each):
Morning (6-8 am) 
Noon and (11 am- 1 pm) 
Evening (4-6 pm)
       
Behaviours were noted visually by using a note book for recording behavior, a stop watch and video camera (Altmann, 1974) according to predetermined criteria (Stephan et al., 2004; Hale et al., 2006; Ferey and Miller, 1972; Scott et al., 2003). Recorded values were averaged and subjected to statistical analysis.
       
The behaviours included:
 
Food response
 
Time taken to complete the feed provided (seconds).
 
Rheo sensitivity
 
Rheological disturbances were created by moving a glass rod in water and fish movement was observed. The time taken to create an abnormal swimming pattern or imbalance (sec) was noted.
 
Acoustic sensitivity
 
As for acoustic responses, fish movement on tapping the tank surface with a pencil was done. The time taken (sec) for which the response lasted was noted.
 
Aggressiveness
 
Fish orienting itself and swimming towards another fish, nips, chases and defensive acts (fish responding to an aggressive act by other fish)- agonistic behaviour. The number of aggressive encounters during the observation hours was noted.
 
Surfacing behavior
 
Mean frequency of the fish rise to surface to gulp air (average of the number per 1 minute).
 
Number of midline crossing
 
The aquarium was divided by a midline externally and the numbers of midline crossing from fish through 1 minute were detected for each aquarium. During each observation period, it was repeated thrice and the average number taken.
       
One-way ANOVA was performed using R software to ascertain the effect of the different doses on various behaviours. Normality and homogeneity of variance were assessed formally using Shapiro-wilk and Levene’s tests, respectively. Follow up analyses were performed using Dunnett’s multiple comparisons between control and the treatment concentrations/durations (Hothorn et al., 2008).

Fig 1 shows that the feeding rate of O. niloticus. The feeding rate was higher in the NaCl-exposed groups compared to the control. Both the L and H groups required less time to complete feeding at both 10-day and 60-day exposure durations. The control group consistently took the longest time to complete feeding at both time points. The low concentration (L) group fed faster than the control but slower than the high-concentration group. The High concentration (H) group exhibited the fastest feeding behavior, completing the feed in approximately 15 seconds by day 60-almost one-fourth of the initial control value.


       
Fig 2 clearly shows that the control groups very sensitive to acoustic stimuli. But the L and H treatment groups gets restored soon after responding to an acoustic stimuli. When the control fishes remain sensitive for 33 sec to an acoustic response, L and H treatment groups are sensitive only for 32sec and 19 sec respectively. In the case of the 60-day period also a similar trend is noted (23 sec, 20 sec and 14 sec for control, L and H respectively).


       
Similar is the results obtained for rheo sensitivity (Fig 3). If 23 sec and 18 sec was the time taken to create an abnormal swimming pattern or imbalance in the fish in the 10-day period for control L and H groups respectively as against the control where 26 sec was used. 10 sec, 8 sec and 3 sec was taken during the 60-day period for Control, L and H respectively.


       
The frequency of aggressive behavior (Fig 4) noted in the control group was (1) lower than both the L and H groups (3) both in the 10-day period and 60-day period (3, 7, 10 for Control, L and H respectively). In case of surfacing behavior (Fig 6), higher frequency is observed in the L and H treatment groups when compared with the controls both in the 10-day period and 60-day period (1, 2, 3 and 1, 3, 6 for Control, L and H of 10-day and 60-day respectively). The number of midline line crossings (Fig 5) also followed a similar trend as the surfacing behavior. In the 10-day period, 2, 5, 7 midline crossings were noted in Control, L and H respectively and in the 60-day period, 7, 9, 18 midline crossings were noted in control, L and H respectively.


       
The frequency of aggressive behavior (Fig 4) noted in the control group was (1) lower than both the L and H groups (3) both in the 10-day period and 60-day period (3, 5, 9 for Control, L and H respectively).
       
The number of midline crossings (Fig 5) recorded over the 10-day period were 2, 5 and 7 in the control, L and H groups respectively, while after 60 days, the corresponding values increased to 7, 9 and 18 respectively.


       
In case of surfacing behavior (Fig 6), a similar trend as the midline line crossings was noted. Higher frequency is observed in the L and H treatment groups when compared with the controls both in the 10-day period and 60-day period (1, 2, 3 and 1, 3, 6 for Control, L and H of 10-day and 60-day respectively).


       
Across all six behavioral parameters-feeding, rheotaxis sensitivity (Rheo), acoustic sensitivity, aggression, Midline Crossing and Surfacing-there is a consistent dose-dependent and time-dependent effect observed when comparing low (L) and high (H) treatments against the control. At 10 days, significant reductions were seen in feeding particularly at higher doses, Conversely, aggression, midline crossings and surfacing showed a notable increase, especially in high-dose groups, suggesting stress-induced hyperactivity or anxiety-like responses. In case of acoustic and rheotaxis sensitivity, across all groups it seem to be higher in the control than both L and H groups. All p-values for these significant changes were well below 0.001, confirming strong statistical relevance (Table 1).


       
By 60 days, most behavioral alterations intensified. Feeding continued to decline significantly over time, with the highest suppression observed at 60 days in the high-dose group (-27.25). Rheo sensitivity and acoustic sensitivity fell further, for H dose compared to L and control. Aggression also rises dramatically for both doses at 60 days, indicating progressive behavioral dysregulation. Midline crossings increased sharply, peaking at 60 days for the high group (+10.17), further reinforcing the pattern of hyperactivity. Surfacing showed minimal changes at lower doses but became strongly elevated at high doses by 60 days. Overall, the data suggest that chronic exposure amplifies behavioral disruptions in a dose-dependent manner (Table 2).

​
       
The present study suggests that the feeding rate is positively influenced by both the concentration and duration of sodium chloride exposure. Enhanced osmoregulatory demands at higher salinities would require the breakdown of energy-dense nutrients (Bœuf and Payan, 2001). This heightened energy requirement for osmoregulation, following environmental stress, which happens to be increased salinity levels due to NaCl exposure in the present study, triggers changes in physiological processes aimed at maintaining energy balance, to optimize food digestion and nutrient absorption (Psochiou et al., 2007,  Gheisvandi et al., 2015).
       
Both the acoustic and rheo response reveals that the NaCl treatment, facilitates stress management. The acoustic and rheo sensitivity data of the NaCl exposed group in comparison with control group reveals two things. The NaCl treatment, is seen to decrease the period of neural sensitivity of the exposed fish to sound; the greater the concentration of NaCl and greater the duration for which the fish is exposed, the lesser responsive is the fish to a sound stimuli. This finding is supported by the study of Fournet et al., (2019) which showed that for every one unit increase in salinity the probability of toadfish acoustic occurrence decreases by 16%.
       
The swimming pattern of control fish remained under equilibrium from longer duration when compared to NaCl exposed groups. In other words, swimming equilibrium was lost earlier by fish exposed to H concentration of NaCl than those in L group and Control for both 10 and 60 days period. This shows that sensitivity of the NaCl exposed fish again decreases and points the NaCl exposure for increased periods and for increased concentrations in the studied range which is way below recommended safe limits (4-10 g/L) lowers the coping mechanism of the organism. Studies of Stoessel et al., (2019) on Murray hardyhead suggests that the probability of loss of equilibrium increases with increasing salinity.
       
Aggression, surfacing and midline crossings exhibited a clear dose-dependent response. Lorenz et al., (2015) reported that aggression levels in fish may increase with rising salinity. According to Fondriest Environmental (2013), higher NaCl concentrations and prolonged exposure reduce dissolved oxygen levels, which may explain the increased surfacing behavior observed in the exposed fish as they attempt to gulp atmospheric air. The resulting oxygen deficiency could also trigger heightened aggression and midline crossings due to competition for limited oxygen resources, reflecting restlessness and stress.
       
Surfacing behavior and midline crossings could be indicative of respiratory or adaptive stress caused due to the increase in the level of sodium chloride. Fish in high salinity can elevate energy expenditure and induce metabolic stress, ultimately affecting growth, reproduction and overall fitness (Hochachka and Somero, 2002). As salinity increases, dissolved oxygen levels decrease exponentially, leading to significant impacts on fish metabolism, fish behavior, including swimming speed, foraging and habitat selection, (Fondriest Environmental, 2013; Wootton, 1990; Wu, 2002). Insufficient oxygen, particularly in hypoxic conditions, can induce stress, negatively impacting fish health and performance (Marium et. al, 2023). Similarly, Huang et al., (2021) observed that exposure to deltamethrin and cadmium induced prolonged hyperactivity in zebrafish, linked to metabolic and physiological disturbances such as altered oxygen consumption and ventilation.
       
NaCl is widely used in aquaculture as a prophylactic for disease management and moderate salinity (8-10 ppt) has been shown to support survival, growth and physiological homeostasis in many cultured species like Pangasianodon hypophthalmus (Lingam et al., 2025) and improved rohu (Jayanti) (Murmu et al., 2020). The present study emphasizes that even NaCl treatment within safe usage limits can still affect fish behavior and physiology.

Behavioral changes in apparently healthy fish might indicate a nonspecific response to stressors. However, these changes can still provide a valuable, quick and non-invasive way to predict and assess potential economic losses from disease before mortality occurs.

Extending sincere acknowledgement to Mrs. Supriya Susan Kurian, Librariran, Mar Thoma College for Women, Perumbavoor for assistance in literature search.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University, Animal Care Committee.
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.