Assessment of Haematological Alterations in Labeo rohita (Rohu) Exposed to Ibuprofen

Fingerlings of L. rohita (9±2 g, 8.5±1.5 cm) were obtained from Kallidaikurichi and Thanjavur Private Fish Farms, Tamil Nadu, India. They were acclimatized for 20 days in FRP tanks under laboratory conditions with continuous aeration and routine water exchange. Fish were fed a commercial diet (Coppens) with regular tank cleaning to remove waste. Water quality parameters were maintained per APHA (2017) standards: temperature 30.45±0.10°C, pH 6.42±0.08, dissolved oxygen 6.51±0.6 mg/L, total alkalinity 352±1.2 mg/L, and total hardness 131±1.5 mg/L as CaCO3.

Ten fish were exposed to different concentrations of the pharmacological agent IBP (CAS number 15687-27-1, > 98% purity) purchased from Sigma-Aldrich in a 35-liter trough holding 10 L of water for each test concentration. Throughout the testing phase, the experimental organisms were not fed. Each concentrations were done in triplicate manner. Wide range finding test for IBP was calculated to analyse the LC50 value for acute toxicity. The range for the concentrations (100, 10, 1, 0.1 and 0.01 ppm) were estimated by the OECD guidelines.

Blood samples were collected on days 1-4 of exposure by pelvic fin puncture using a heparinized syringe (21-gauge needle) and stored in EDTA vials to prevent clotting. The blood samples were analyzed according to Blaxhall and Daisley (1973). Packed cell volume (PCV) was measured using the micro-hematocrit method, while haemoglobin (Hb) levels were assessed with Sigma Diagnostics kits using the cyanohemoglobin technique. RBC and WBC counts were determined with a Neubauer haemocytometer (Dacie and Lewis, 2006), and lymphocyte differentials were analyzed on May-Grunwald-Giemsa-stained blood smears (Mirale, 1982). Erythrocyte indices (MCHC, MCH, MCV) were calculated using RBC count, Hb, and PCV following Dacie and Lewis (2006).

The probit method (Finney, 1971) was used to analyze acute toxicity data concerning quantal responses (mortality). A two-way ANOVA was performed on the haematological parameters with Tukey post hoc test using SPSS version 20 to compare means and identify significant differences at the 5% probability level.

LC50 of IBP was found to be 1.54 ppm by using the method of Log Probit Analysis software. The Commission of the European Communities (1996) established EU guidelines (93-67-EEC) that divide chemicals into various categories based on their LC50 (for aquatic animals, less than 1 mg/l is extremely poisonous, 1–10 mg/l is toxic, and 10-100 mg/l is hazardous). This result indicates that IBP is severely toxic for L. rohita. The toxicity however varies across species, with LC50 values reported as 0.38 ppm for Clarias gariepinus (Malarvizhi et al., 2012), 173 ppm for Lepomis macrochirus, and 132.6 ppm for Daphnia magna (Islas-Flores et al., 2017), reflecting species-specific and exposure-related differences.

The blood parameters such as WBC, RBC, HCT, MCV, MCH and MCHC were analysed for the drug IBP. There were significant differences (p<0.05) in all parameters of the treated fish compared to the control group in terms of both concentration and duration. In case of IBP, WBC shows the higher value in 96 h at concentration of 3.092 mgl-1 and lower in 48h at 0.386 mgl-1. The higher value for RBC was attained in 1.546 mgl-1 concentration and the lower was in 96h at 0.386 mgl-1 concentration. Whereas in case of MCH and MCHC higher level occurs in the 48 h at 6.184 mgl-1 and lower level occurs in 24 h at 1.546 mgl-1 and 0.386 mgl-1. In HCT, the higher concentration (6.184 mgl-1) was at 24 h showed higher value and lower concentration (0.386 mgl-1) showed lower value. And in MCV, it showed higher value at (1.546 mgl-1) at 96 hour and lower value occurred in concentration of 6.184 mgl-1 at 48 h which was mentioned in the Table I. The Table I indicates the four days parameters (WBC, RBC, HCT, MCV, MCH and MCHC) with the accordance to five different concentrations (6.184, 3.092, 1.546, 0.773 and 0.386 mgl-1).

Blood parameters, linked to energy, respiration, and defense, serve as physiological indicators of environmental changes (Nwani et al., 2014; Iheanacho et al., 2017). The various haematological parameters sensitive to pollutants and help assess an organism’s physiological status (Adhikari et al., 2014).

In our study, haematological responses showed an increase in WBC and MCHC under lethal IBP concentrations throughout the study. The elevation in WBC count, linked to immune regulation across organisms, indicates a general immune response and protective reaction to IBP (Saravanan et al., 2011). The toxicant’s immune-stimulatory effect likely triggered lymphocyte release as a defense mechanism, contributing to the elevated WBC count in fish. Additionally, the study noted an increase in MCHC

Values are the Mean ±SD, (p<0.05), two-way ANOVA with Tukey post hoc test. Significantly different from control group. Where MCV is Mean Corpuscular Volume, MCH is Mean Corpuscular haemoglobin and MCHC is Mean Corpuscular Haemoglobin Concentration. The a, b and c of the values indicates the subset of the values in which each value comes under a different category (Tukey test).

which may suggest the onset of spherocytosis (Umamaheshwari and Senthilnathan, 2014). Similar observations of elevated MCHC levels were seen in M. cephalus (Grey Mullet) exposed to pesticides (Francesco et al., 2012). Furthermore, a slight decrease in the HCT parameter, moving from higher to lower IBP concentrations, indicates anemia (Thakur and Sahai, 1987). Comparable reductions in RBC count, HCT, and Hb levels have been reported in carps exposed to various toxicants (Lavanya et al., 2011). The accumulation of toxicants in the gill area may lead to haemolysis due to gill structure damage and compromised osmoregulation, potentially causing RBC reduction (Saravanan et al., 2011). In line with these findings, the present study observed a reduction in RBC count under lethal IBP exposure, likely due to IBP’s suppression of RBC production.

MCV assesses the size or condition of RBCs and indicates whether cell division during erythropoiesis is normal or abnormal. Conversely, MCH indicates the average haemoglobin content in each red blood cell within a blood sample. In our drug IBP toxicity test, the MCV and MCH showed the decrease in mean count throughout the study period which indicates the hypochromic microcytic anaemia. MCH values typically align with MCV values (Kumar and Banerjee, 2016). The substantial increase in mean RBC, along with a decrease in MCV and MCH observed in this study, may indicate erythrocyte damage and gill tissue damage, leading to impaired oxygen transport and hindered gaseous exchange (Burgos-Aceves et al., 2019).

The present study highlights the adverse effects of ibuprofen exposure on the haematological profile of L. rohita. The significant reduction in RBC count, haemoglobin levels, and haematocrit indicates impaired oxygen transport and potential anaemia, while the increase in WBC count and altered leukocyte distribution suggest an immune response to chemical stress. These findings provide strong evidence that pharmaceutical contaminants like IBP can severely impact the physiological health of fish, leading to potential ecological consequences. Given the widespread presence of IBP in aquatic environments, this study emphasizes the urgent need for monitoring and regulating pharmaceutical pollutants in water bodies.

The authors expressed their gratitude to the Dean of TNJFU-Fisheries College and Research Institute, Thoothukudi as well as the Professor and Head, Assistant Professors of the Department of the Aquatic Environment Management at the same institution for their indispensable support, encouragement and provision of all necessary facilities which were crucial for the successful completion of this research work.

This study was financially supported by TNJFU- Fisheries College and Research Institute- Thoothukudi.

This study was conducted based on the approval of the ethical committee of TNJFU, Nagapattinam, Tamil Nadu, India.

The authors have declared no conflict of interest.