ASSESSMENT OF BIOACCUMULATION OF CIPROFLOXACIN IN Catla catla AND Labeo rohita EXPOSED IN A LABORATORY-BASED SETUP

INTRODUCTION

In 2020, aquaculture accounted for 56% of the global aquatic food for direct human consumption (FAO, 2022). From 2000 until now, an average 5.7% increase in total fish production has been reported (Tacon, 2020). On the other hand, there is growing concern over the prevalence of pharmaceuticals in aquatic environments (De-Assis, 2021). Ciprofloxacin is the most prevalent antibiotic in the Bohai Sea's natural saltwater environment (Han et al., 2021).

Antibiotics exhibit varying absorption and bioaccumulation in tissues of aquatic organisms in response to changes in various environmental factors (Chen et al., 2018). The value of annual mean biomagnification factors for ciprofloxacin ranges from 0.18 to 2.25, which suggests that along planktonic food webs, it could undergo biomagnification (Tang et al., 2020). Fish taken from the aquatic environment have been found to contain fluorinated antibiotics. The metabolic stability of quinolone antibiotics may be significantly influenced by a fluorine substituent. By effectively preventing oxidative metabolism, the addition of a fluorine substituent improves metabolic stability and prolongs the antibiotic's residence time in organisms (Sun et al., 2020). Therefore, fish are suitable organisms for research on the monitoring and bioaccumulation of fluoroquinolones. Ciprofloxacin bioaccumulation has been seen in the liver, muscle, bile and plasma of wild fish (Zhang et al., 2021).

Freshwater species of fish are considered important in aquaculture; primarily cultured are cyprinids and carps (Tacon, 2020). Species such as C. catla is a zooplanktivorous and surface feeder (Kibenge, 2022), while L. rohita is an omniplanktivorous and a bottom feeder and is widely cultured and consumed across South Asia (Masood et al., 2022). The risks associated with ciprofloxacin residue in aquatic products and the aquatic ecological environment are significant and are receiving more and more attention (Liu et al., 2022). Despite their economic and nutritional importance, limited information is available on the bioaccumulation potential of ciprofloxacin in these species, which is a widely used antibiotic. Existing studies have largely focused on histopathological, hematological and antioxidant responses, while data on tissue-specific ciprofloxacin accumulation in major carps remain scarce.

Therefore, the present study aimed to evaluate the bioaccumulation of ciprofloxacin at different exposure concentrations in the liver, gills and muscle tissues of C. catla and L. rohita. This study focused on acute, experimentally high exposure to ciprofloxacin to ensure measurable detection of antibiotic residues in fish tissues after short-term exposure. By elucidating species and tissue-specific accumulation patterns, this study provides insight into the potential risks of antibiotic residues entering the aquatic food chain and supports the development of responsible antibiotic use strategies in sustainable aquaculture.

MATERIALS AND METHODS

Experimental design:The experiment was conducted in the toxicology laboratory of Fisheries Research Farm, Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, in April 2024, to assess ciprofloxacin bioaccumulation in C. catla and L. rohita. All the materials and methods used in this research were carried out following the ethical considerations, as approved by the ethical committee of the University of Agriculture, Faisalabad. 90-day-old healthy fish with an average body weight of approximately 20 g (L. rohita) and 22 g (C. catla) were brought from Satiana Fish Hatchery, Faisalabad. These fish were produced under standard nursery rearing conditions involving earthen ponds with supplemental feeding (typically a mixture of rice bran and oil cake) and optimal water quality parameters (temperature 25-30°C, adequate dissolved oxygen and low stocking density to promote faster growth).Fish were acclimatized for 14 days in 70 L glass aquaria, each containing 50 L of dechlorinated water. Ten fish were placed in each aquarium with continuous aeration.

Chemicals and reagents: Ciprofloxacin (obtained from Sigma-Aldrich), acetonitrile, dimethyl sulfoxide (DMSO), HCl, phosphate buffer and HPLC-grade water were used. A ciprofloxacin standard solution (1 mg/mL) was prepared in distilled water containing 0.03% NaOH. A stock solution was prepared by dissolving ciprofloxacin powder in DMSO and diluting to 1000 mL with distilled water. Working solutions were prepared from this stock to obtain the required treatment concentrations.

Acute exposure: Control groups with no antibiotic exposure and treatment groups were made. For L. rohita, the concentration of ciprofloxacin given in T₁ was 7.7 mg/L and in T₂ was 18 mg/L. Similarly, C. catla in T₁ was exposed to a 5.7 mg/L concentration, while the concentration of ciprofloxacin in T₂ was 13 mg/L. The experimental concentrations were selected as sublethal exposure levels following the standard toxicological practice of using fractions of reported 96-h LC₅₀values for acute fish exposure studies. Similar mg/L-level ciprofloxacin exposures have also been used in laboratory zebrafish (Danio rerio) toxicity assessment (Ding et al., 2017). Ambili et al. (2013) investigated the toxicity of oxytetracycline in Labeo rohita using mg/L-level exposure concentrations, with a sublethal concentration selected for evaluating physiological and biochemical responses under controlled laboratory conditions.

These concentrations were selected to ensure measurable bioaccumulation over the short duration of the acute trial. These concentrations are several orders of magnitude higher than environmentally relevant levels, which are typically in the ng/L range. Environmentally relevant concentrations are used mostly in chronic trials and are unlikely to produce detectable accumulation during short-term exposure.

Therefore, higher doses were necessary to assess the bioaccumulation potential of ciprofloxacin under controlled acute conditions. Sampling was done after 24, 48, 72 and 96 hours, respectively. At the end of the trial, fish were dissected and liver, gills and muscles were carefully separated, placed in polyethylene bags and stored at –20°C until further analysis. The method for sample preparation and chromatographic analysis was adapted from He et al. (2016) with slight modifications.

Sample preparation: Approximately 4 g of homogenized tissue was weighed and taken into 50 ml polypropylene centrifuge tubes. A solution of acidified acetonitrile was prepared by mixing acetonitrile with 18% HCl at a ratio of 2500:20 v/v and 20 ml was added to the samples. The mixture was allowed to stand for 15 min at 4°C. The samples were then vortexed for 8 minutes and centrifuged at 10,000 g for 15 minutes.

The supernatant was collected and transferred to a clean tube and 8 mL of hexane was added for defatting. The mixture was vortexed thoroughly and allowed to separate into two phases. The upper hexane layer was discarded and the lower acetonitrile phase was collected. The extract was then evaporated to near dryness under a gentle stream of nitrogen at room temperature. The residue was reconstituted in an appropriate volume of mobile phase, filtered through a 0.22 µm membrane filter and transferred to HPLC vials for analysis.

Chromatographic analysis: Using a 10 μL injection volume, an HPLC separation system consisting of C-18 (4.6 mm×250 mm, 5 μm) was operated at 30°C. At an emission wavelength of 452 nm and an excitation wavelength of 280 nm, the fluorescence detector was run. The mobile phase consisted of acetonitrile and phosphate buffer (pH 2.5) (20:80 v/v) at a flow rate of 1.0 ml/min.

The analytical method was validated following He et al. (2016). In that study, the Limit of Detection (LOD) and Limit of Quantification (LOQ) for ciprofloxacin were determined based on signal-to-noise ratios of 3:1 and 10:1, respectively and recovery values from spiked fish tissues were within acceptable limits. The same extraction and chromatographic conditions were applied in the present study, ensuring reliable quantification.

Determination of physico-chemical parameters: Using methods of A.P.H.A. (2012), physicochemical parameters of water were monitored regularly. The temperature was measured with a digital thermometer (Jenway 100 models) by dipping it for 30 seconds. The pH was determined by using the pH micro-process meter (HANNA HI-9023). Dissolved oxygen (DO) levels were measured with a DO meter (HANNA H1-9147). Total hardness was determined by EDTA titration using Erichrome Black-T as an indicator. The following equations for the calculation of physicochemical parameters were adopted from A.P.H.A. (2012):

Total hardness (mgL^-1) = 

Where A = mg CaCO₃ is equivalent to 1.0 ml EDTA titrant

Carbon dioxide was estimated by titration with sodium carbonate and phenolphthalein as an indicator.

Carbon dioxide (mgL^-1) = 

Calcium was estimated using EDTA titration with ammonium purpurate as an indicator.

Calcium (mgL^-1) = 

Magnesium was calculated as:

A-B = C, Mg (mgL^-1) = , Where A = Total hardness, B = Calcium×2.5

Total ammonia was determined by spectrophotometry using Nessler’s reagent, measured at 420 nm with a 1 cm light path. A standard calibration curve was constructed under identical temperature and reaction time conditions.

Statistical analysis: Three replicates were used for each treatment, and all tissue analyses were performed in triplicate. A factorial experimental design under Completely Randomized Design (CRD) was used, with tissue bioaccumulation as the dependent variable, while duration and treatment were the independent variables. Two-way Analysis of Variance (ANOVA) was applied to the obtained bioaccumulation values using Statistix 8.1 (Analytical Software, USA) to determine statistical differences and similarities among variables, followed by Tukey’s HSD post-hoc test to compare treatment means. Different superscript letters indicate a significance level of p ≤ 0.05. Physico-chemical parameters of control, T₁ and T₂ media were also analyzed through correlation studies. Results were shown as Mean ± S.D.

RESULTS

Effect of time duration and ciprofloxacin exposure levels on bioaccumulation: The exposure duration and their interaction significantly (p ≤ 0.01) influenced ciprofloxacin bioaccumulation in all organs of both species (Table 1). Low baseline levels were observed in control fish that may be attributed to prior environmental exposure in hatchery conditions. The tissue concentrations of ciprofloxacin increased from day 1 to day 4 in all three organs of L. rohita and C. catla, indicating increased absolute accumulation with increasing exposure concentration as represented in Fig. 1.

Table 1. Two-way ANOVA summary for ciprofloxacin bioaccumulation in different organs of L. rohita and C. catla.

^** indicates statistical significance at p ≤ 0.001.

Fig. 1. Mean bioaccumulation (µg/g) of ciprofloxacin in different organs of L. rohita and C. catla after 96-hrs exposure. Values represent Mean ± S.D and different letters indicate significant differences among treatments (p ≤ 0.05).

Pattern of ciprofloxacin bioaccumulation in fish tissues: In this study, three experimental groups of tested fish exhibited significant variations in the bioaccumulation of ciprofloxacin in different organs (liver, muscles and gills) throughout a 96-hour exposure period. The ciprofloxacin bioaccumulation pattern as noted above was identical for both low and high doses of ciprofloxacin for 96 hours for both fish in the following order: liver>gills˃muscles. The highest accumulation of ciprofloxacin was observed in the fish liver of both treated groups, with standard accumulation values in fish in the control group, as represented in Tables 2-7 and the Tukey test also revealed that all 3 treatment means and 4 duration means for each of these organs of both fish were significantly (P ≤ 0.05) different from one another, as represented by different superscript letters for these values.

 Table 2. Bioaccumulation of ciprofloxacin (µg/g) in the liver of L. rohita during 96-hrs exposure

 Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences.

 Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.Table 3. Bioaccumulation of ciprofloxacin (µg/g) in the gills of L. rohita during 96-hrs exposure.

Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences. Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.

 Table 4. Bioaccumulation of ciprofloxacin (µg/g) in the muscles of L. rohita during 96-hrs exposure

Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences. Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.

 Table 5. Bioaccumulation of ciprofloxacin (µg/g) in the liver of C. catla during 96-hrs exposure.

Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences. Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.

 Table 6. Bioaccumulation of ciprofloxacin (µg/g) in the gills of C. catla during 96-hrs exposure.

Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences. Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.

 Table 7. Bioaccumulation of ciprofloxacin (µg/g) in the muscles of C. catla during 96-hrs exposure.

Different superscript letters within Duration ^* Treatment combinations indicate significant differences according to Tukey’s HSD test (p ≤ 0.05), whereas shared letters indicate non-significant differences. Superscripts in the final row and final column represent treatment and duration main effects, respectively. The final cell represents the overall grand mean.

Bioconcentration factor: As a steady state was not achieved during the exposure period, bioconcentration factors (BCF) were calculated using 96-hour tissue concentrations. Both 96-h endpoint BCF and time-weighted average BCF (TWA-BCF) values were determined using the following relationship:

BCF = 

Where, = Concentration of ciprofloxacin in fish tissues (µg/g)

 = Exposure concentration in water (µg/L)

 TWA-BCF values were derived from concentrations measured at uniform 24-h intervals; therefore, the arithmetic mean of tissue concentrations was considered equivalent to the time-weighted average. The BCF values, as shown in Table 8, were higher in the T₁ group than in the T₂ group, suggesting reduced bioaccumulation efficiency at elevated concentrations for each of the organs of L. rohita and C. catla. This suggests possible saturation of uptake mechanisms and enhanced elimination at elevated concentrations.

 A limitation of the present study is that steady-state conditions were not achieved within the 96-h exposure period, as tissue concentrations of ciprofloxacin continued to increase. Consequently, bioconcentration factors (BCF) were calculated using 96-h endpoint concentrations and time-weighted average BCFs (TWA-BCF) rather than steady-state values. Additionally, uptake (k₁) and depuration (k₂) rate constants could not be reliably estimated because no depuration phase was conducted. These limitations should be considered when interpreting the bioaccumulation dynamics and longer-term studies incorporating both uptake and depuration phases are recommended for a comprehensive toxicokinetic assessment, as described by Chen et al. (2019) based on a mass balance model (Mackay and Fraser, 2000).

 Table 8. Bioconcentration factors (L/kg) of ciprofloxacin in different tissues of L. rohita and C. catla at the 96-hrs endpoint and as time-weighted average (TWA-BCF) values over the 96-hrs exposure period

Physico-chemical parameters of different media for L. rohita and C. catla: Tables 9 and 10 represent the mean values of each parameter noted at the specified interval for the respective media. Key findings suggested that T₂ significantly (P ≤ 0.05) influences physicochemical characteristics of water compared to the control and T₁, which could have direct implications for fish physiology and survival. In both L. rohita and C. catla, acute exposure to ciprofloxacin resulted in increased temperature, pH, total hardness, carbon dioxide, magnesium and total ammonia from control to T₁ and T₂ media, while dissolved oxygen and calcium levels showed a consistent decline.

 The observed changes followed a consistent dose-dependent pattern (Control≤T₁≤T₂) in both L. rohita and C. catla, suggesting that these variations are primarily associated with ciprofloxacin exposure. Correlation analysis, i.e., Pearson Correlation (Tables 11 and 12), further supports this interpretation, where ammonia, CO₂ and magnesium mostly showed significant (P ≤ 0.05) positive correlations with exposure duration and treatment levels, whereas dissolved oxygen and calcium exhibited significant (P ≤ 0.05) negative correlations.

Table 9. Mean physico-chemical parameters (Mean ± S.D) of control media, T₁ and T₂ for L. rohita during acute exposure to ciprofloxacin.

 Table 10. Mean physico-chemical parameters (Mean ± S.D) of control media, T₁ and T₂ for C. catla during acute exposure to ciprofloxacin.

 Table 11. Pearson’s correlation matrix among physico-chemical parameters and exposure duration in different media for L. rohita.

^ns = Non-significant (P ≥ 0.05); ^* = Significant (P ≤ 0.05); ^** = Highly significant (P ≤ 0.01). Values represent Pearson’s correlation coefficients.

Table 12. Pearson’s correlation matrix among physico-chemical parameters and exposure duration in different media for C. catla.

^ns = Non-significant (P ≥ 0.05); ^* = Significant (P ≤ 0.05); ^** = Highly significant (P ≤ 0.01). Values represent Pearson’s correlation coefficients.

 DISCUSSION

 Antibiotic residues are the major concern in the aquatic environment, whether marine or freshwater. According to Li et al. (2021), the concentrations of the majority of antibiotics in freshwater cultured products were at a medium or lower level, but they were slightly greater compared to those found in marine cultured products. In muscle tissues, the highest ciprofloxacin concentration was 1900 ng/g ww and it was above the maximum allowable residue of 100 ng/g ww.

 According to Han et al. (2021), in the marine aquaculture farms surrounding Laizhou Bay, Bohai Sea, fluoroquinolones were the most common antibiotics in organisms and culture sediments/biofilms. Ciprofloxacin was observed to accumulate more in the summer than in the winter. Zhang et al. (2020a) assessed that adsorption capacity to particles and comparatively high aqueous solubility are the reasons for the high bioaccumulation factors of fluoroquinolones in fish. Zhang et al. (2022) evaluated that the key factor impacting the bioaccumulation of antibiotics in organism samples is biotransformation. The ability of parent pollutants to accumulate in organisms is decreasing as their biotransformation becomes more advanced. Zhao et al. (2018) also confirmed that in organisms, enrofloxacin can be metabolized into ciprofloxacin and then in muscle, there was a significant positive correlation between enrofloxacin and its metabolite, ciprofloxacin (p ≤ 0.05). Consequently, the reduction in enrofloxacin's ability to accumulate is due to its biotransformation into ciprofloxacin.

 Tang et al. (2022) also determined that antibiotic metabolic biotransformation is a major factor in the biomagnification of antibiotics. Only ciprofloxacin exhibited notable trophic magnification (TMF=1.95) in comparison to other antibiotics. It was found that among omnivore fish, common carp, ciprofloxacin had the highest log BAF value (4.73±0.30) and each antibiotic's BAF in aquatic biota was affected by the route of exposure and bioaccumulation kinetics in addition to its pH-dependent distribution coefficient. Antibiotic bioaccumulation factors (BAFs) in benthic biota were found to be higher than in fish, suggesting that ingesting sediment could be a potential route of antibiotic exposure.

 This study found higher ciprofloxacin bioaccumulation in C. catla compared to L. rohita due to species differences and it was increased at high antibiotic concentrations. Additionally, physiological differences in uptake and excretion may enhance accumulation in C. catla. These findings are justified by the study of Chen et al. (2018) that one of the key variables influencing the distribution of antibiotics in various tissues of cultured fish is species. Grass carp had high antibiotic levels in fish tissue samples, indicating ciprofloxacin and enrofloxacin showed high transportability from the plasma into the tissues, while tilapia, crucian carp, mud carp and mugilidae had low antibiotic concentrations, indicating relatively low transportability from the plasma into the tissues. The distribution of antibiotics in fish tissues may also be influenced by environmental factors, animal excrement and nutrition. Furthermore, Zhang et al. (2020a) evaluated that besides the lipid contents in fish and the concentration of pollutants in water, the bioaccumulation of pollutants in fish tissues can be influenced by a wide range of factors, including the species of fish, size and weight.

 In the present study, it was found that bioaccumulation values were different from one another in each organ. The highest ciprofloxacin bioaccumulation after 96 hours (T₂) occurred in the liver of both L. rohita (45.95±0.74 µg/g) and C. catla (50.98±0.72 µg/g) due to high transformation rates, as it is the main metabolic organ, while the least bioaccumulation occurred in the muscles of both L. rohita (25.08±0.69 µg/g) and C. catla (31.21±0.94 µg/g) due to a lower uptake rate. As a result, the values of the time-weighted average bioconcentration factor (TWA-BCF) were higher for the liver of L. rohita (T₁ 4.91; T₂ 2.55) than for the muscles (T₁ 2.79; T₂ 1.39). Similarly, these values were higher for T₁ (8.39) and T₂ (3.92) of the liver than the muscles (T₁ 4.78; T₂ 2.40) of C. catla. Chen et al. (2018) and Chen et al. (2019) also assessed that the value of the bioconcentration factor for fluoroquinolones was highest for the liver (0.24-39.55 L/kg) and lowest for muscles (0.04-1.07 L/kg). This difference is most likely caused by high detoxification and digestion of the targeted antibiotics in the liver and bile, while the muscles have a low potential for bioaccumulation, high depuration rates and low uptake rates.

 The main finding of this study was that the observed pattern of ciprofloxacin bioaccumulation was as follows: liver˃gills˃muscles. Zhang et al. (2021) and Zhang et al. (2022) confirmed these results that in lipid-rich tissues (fish liver) or respiratory organs (fish gill), antibiotics showed a higher bioaccumulation capacity than in muscle. Xie et al. (2017) also reported the tissue distribution in seven fish species, including carps from Taihu Lake and observed that the concentration of ciprofloxacin in the liver (BAFs=2008 L/kg) was generally higher, followed by the gills (BAFs=811 L/kg) and muscle (BAFs=545 L/kg). This is because the liver functions as the main site where xenobiotic metabolism occurs.