Evaluation of hematological parameters in freshwater fish species, with special emphasis on Catla catla exposed to chlorpyrifos

Introduction

The intensification of agricultural practices to meet the increasing global demand for food has led to a substantial rise in the use of chemical pesticides, many of which ultimately enter non-target ecosystems, particularly freshwater bodies. Among these agrochemicals, organophosphate pesticides continue to be widely used in developing countries because of their high efficacy against insect pests and relatively low persistence compared to organochlorines. However, repeated applications, surface runoff, leaching, spray drift and irrigation return flows facilitate the transport of these compounds into rivers, reservoirs, ponds and dams, where they pose serious risks to aquatic organisms (Alengebawy et al. 2021; Deb & Das, 2013) [5,14] . Chlorpyrifos is a broad-spectrum organophosphate insecticide extensively applied in agriculture to control a wide range of crop pests. Its primary toxic mode of action involves inhibition of acetylcholinesterase, leading to disruption of nerve impulse transmission. Although chlorpyrifos is intended for terrestrial use, numerous studies have documented its frequent detection in surface waters and sediments, particularly in regions dominated by intensive agriculture (Abrantes et al. 2010; Hossain et al. 2022) [3,15] . Even at sublethal and environmentally relevant concentrations, chlorpyrifos has been shown to induce physiological, biochemical, hematological and histopathological alterations in fish, thereby compromising growth, immunity and survival (Majumder & Kaviraj, 2019; Deb & Das, 2013) [18,14] . Fish are widely recognized as sensitive bio-indicators of aquatic pollution because they occupy higher trophic levels, integrate contaminant exposure over time and respond to toxic stress at multiple levels of biological organization. Hematological parameters such as hemoglobin content, erythrocyte and leucocyte counts, packed cell volume and erythrocyte indices are particularly valuable biomarkers, as they reflect both the health status of fish and the functional integrity of hematopoietic tissues (Blaxhall & Daisley, 1973; Barton et al. 2002) [9,8] . Alterations in these parameters often occur earlier than overt signs of toxicity or mortality, making them reliable early-warning indicators of pesticide stress. Indian major carps play a crucial role in inland fisheries and aquaculture, contributing significantly to food security and rural livelihoods. Among them, Catla catla is an economically important and widely cultured freshwater species in India. Due to its ecological relevance, sensitivity to pollutants and commercial importance, C. catla has been extensively used in toxicological studies to assess the impacts of pesticides and other contaminants (Abhijith et al. 2012; Kavitha et al. 2010) [2,16] . However, region-specific data on the hematological effects of chronic sublethal chlorpyrifos exposure remain limited, particularly for freshwater systems associated with reservoirs and dams used for fisheries and irrigation. In this context, the present study was undertaken to evaluate the hematological responses of freshwater fish species, with special emphasis on Catla catla, following chronic exposure to sublethal concentrations of chlorpyrifos under controlled laboratory conditions. By integrating multiple hematological endpoints, the study aims to provide insight into the physiological stress and hematopoietic disruptions induced by chlorpyrifos, thereby contributing to ecological risk assessment and supporting the use of hematological biomarkers in monitoring pesticide contamination in freshwater ecosystems.

Methodology

The present investigation was conducted during 2024-25 in a controlled laboratory facility at Bilaspur, Chhattisgarh, under the supervision of the School of Sciences, MATS University, Raipur, following a Completely Randomized Design (CRD). The study aimed to evaluate the toxicological effects of the organophosphate pesticide chlorpyrifos on selected freshwater fish species, with special emphasis on hematological responses. Chlorpyrifos (technical grade, ~95% purity) was procured from a certified supplier and stock solutions were prepared using analyticalgrade methanol. Healthy juvenile Catla catla were acclimatized under laboratory conditions and exposed to sublethal concentrations of chlorpyrifos (4.0, 8.0, 16.0, 32.0 and 64.0 ppb) along with a control for 60 days using a static-renewal bioassay. Fish were maintained in FRP tanks with dechlorinated water under controlled conditions (temperature 26±2 °C, pH 7.2-7.6, dissolved oxygen > 6 mg L⁻¹, photoperiod 12L:12D) and fed a commercial pellet diet (≈30% protein) at 3% body weight per day. At the end of exposure, fish were anesthetized with MS-222 and blood samples were collected by cardiac puncture using heparinized syringes. Hemoglobin content, total erythrocyte count, total leucocyte count and packed cell volume were estimated following standard hematological methods, while mean corpuscular volume, mean corpuscular hemoglobin and erythrocyte sedimentation rate were calculated using established formulae. Data were expressed as mean±SD and analyzed statistically using one-way ANOVA under a Completely Randomized Design, followed by standard error at p ≤ 0.05 to determine significant treatment effects.

Results

The hematological data of Catla catla exposed to different sublethal concentrations of chlorpyrifos for 60 days are presented in Table 1 andFig 1-7. A clear dose-dependent alteration was observed in most hematological indices, indicating the physiological stress and hematopoietic disruption caused by prolonged pesticide exposure.

The hemoglobin content ranged from 3.25±0.03 g/L in the control (T 1 ) to a minimum of 1.90±0.03 g/L in the highest concentration (T 6 , 64.0 ppb), with a mean value of 2.54±0.04 g/L. The decline in Hb levels with increasing chlorpyrifos concentration indicates anemic conditions possibly due to the destruction of erythrocytes or inhibition of erythropoiesis under pesticide stress. Reduced Hb may also reflect lowered oxygen-carrying capacity, impacting metabolic efficiency.

Total Erythrocyte Count (TEC, ×10⁶/mm³)

The total erythrocyte count decreased progressively from 3.57±0.16 ×10⁶/mm³ in the control to 2.35±0.01 ×10⁶/mm³ in T 6 , with a mean of 2.97±0.14 ×10⁶/mm³. The reduction in erythrocyte count suggests destruction or lysis of red blood cells and suppression of hematopoietic activity due to toxic interference of chlorpyrifos with hematological tissues.

Total Leucocyte Count (TLC, ×10⁶/mm³)

In contrast, the total leucocyte count showed a progressive increase from 6.71±0.38 ×10⁶/mm³ (control) to a maximum of 9.38±0.26 ×10⁶/mm³ (T 6 ), with a mean of 7.96±0.33 ×10⁶/mm³. This leukocytosis indicates an immune response to pesticide-induced stress and possible tissue damage, reflecting activation of defense mechanisms to counter toxic effects.

The packed cell volume showed a decreasing trend, ranging from 44.94±3.48% in the control to 30.93±1.39% in the highest treatment (T 6 ), with an overall mean of 36.54±1.75%. The marked reduction in PCV at higher concentrations indicates hemodilution or erythrocyte destruction due to pesticide toxicity, which impairs blood oxygen transport and viscosity balance.

MCV values increased from 1.42±0.04% in the control to 2.92±0.15% in T 6 , with an average of 2.09±0.08%. The rise in MCV suggests the formation of larger but fewer red blood cells, possibly as a compensatory response to anemia, reflecting macrocytosis caused by erythrocyte swelling under toxic stress conditions.

MCH showed minimal variation among treatments, ranging between 8.23±0.29% (T 5 ) and 8.76±0.30% (T 4 ), with a mean of 8.48±0.24%. This parameter remained nearly constant, indicating that while erythrocyte size and number changed, the hemoglobin concentration per cell remained relatively stable, suggesting partial maintenance of erythrocyte hemoglobin content despite chlorpyrifos exposure.

Erythrocyte Sedimentation Rate (ESR, 1st hr)

The ESR increased significantly from 2.71±0.17 mm/hr in the control to 7.36±0.18 mm/hr at the highest concentration (T 6 ), with an overall mean of 5.28±0.25 mm/hr. The elevated ESR values indicate inflammatory or pathological conditions, possibly due to alterations in plasma protein composition and cellular damage resulting from chlorpyrifos stress.

Discussion

Hematological parameters are widely recognized as sensitive biomarkers for assessing physiological stress and toxicological impacts in fish, as they reflect the functional status of hematopoietic tissues and the overall health of the organism (Blaxhall & Daisley, 1973; Barton et al. 2002) [9,8] . In the present investigation, Catla catla exposed to increasing sub-lethal concentrations of chlorpyrifos for 60 days exhibited pronounced, dose-dependent alterations in key hematological indices, clearly indicating pesticideinduced stress and hematopoietic dysfunction. The significant reduction in hemoglobin (Hb) content observed with increasing chlorpyrifos concentration suggests the induction of anemia in treated fish. Similar declines in Hb have been reported in Catla catla and other teleosts exposed to organophosphate pesticides, including methyl parathion and chlorpyrifos (Abhijith et al. 2012; Lakshman et al. 2013) [2,17] . The decrease in Hb may be attributed to erythrocyte destruction, inhibition of hemoglobin synthesis, or impaired iron metabolism caused by pesticide-induced oxidative stress. Reduced hemoglobin levels ultimately compromise oxygen transport efficiency, leading to impaired aerobic metabolism and reduced physiological performance (Barton, 2002) [7] . A parallel decline in total erythrocyte count (TEC) further supports the occurrence of pesticide-induced anemia. The reduction in erythrocyte numbers may result from hemolysis, suppression of erythropoiesis in hematopoietic organs such as kidney and spleen, or damage to erythrocyte membranes due to lipid peroxidation (Dautremepuits et al. 2004; Deb & Das, 2013) [13,14] . Similar reductions in TEC have been documented in Labeo rohita, Oreochromis mossambicus and Catla catla following exposure to organophosphate and pyrethroid pesticides, confirming the hematotoxic nature of these compounds (Ali & Rani, 2009; Abhijith et al. 2012) [2] . In contrast to erythrocytic parameters, total leucocyte count (TLC) showed a marked increase with rising chlorpyrifos concentrations. This leukocytosis reflects activation of the immune system in response to pesticide-induced tissue damage and physiological stress. Increased TLC has been widely interpreted as a defensive or adaptive response, enhancing the organism's ability to counteract toxic injury and infection (Akinrotimi et al. 2009; Cericato et al. 2004) [4, 10] . Similar findings have been reported in fish exposed to chlorpyrifos and acephate, where elevated leucocyte counts were associated with stress-mediated immunological activation (Kulkarni & Bhilave, 2015; Hossain et al. 2022) [15] . The observed reduction in packed cell volume (PCV) further corroborates the anemic condition induced by chlorpyrifos exposure. Decreased PCV values are indicative of reduced erythrocyte mass or hemodilution and have been consistently reported in pesticide-exposed fish species (Abdallah & Abdallah, 2008; Lakshman et al. 2013) [1,17] . Lower PCV values impair blood viscosity and oxygen transport capacity, thereby reducing endurance and growth potential in fish. Interestingly, mean corpuscular volume (MCV) increased significantly with pesticide concentration, indicating macrocytosis. This enlargement of erythrocytes may represent a compensatory response to anemia, where fewer but larger erythrocytes are produced to partially offset reduced oxygen-carrying capacity (Dacie & Lewis, 1991) [11] . Similar increases in MCV have been reported in fish exposed to organophosphate pesticides and heavy metals, reflecting erythrocyte swelling and membrane damage under toxic stress (Akinrotimi et al. 2009; Das et al. 2004) [4,12] . Mean corpuscular hemoglobin (MCH) remained relatively stable across treatments, suggesting that despite reductions in erythrocyte number and Hb concentration, the hemoglobin content per individual erythrocyte was maintained. This stability may indicate partial physiological compensation aimed at sustaining oxygen transport at the cellular level during prolonged exposure (Blaxhall & Daisley, 1973) [9] . The significant elevation in erythrocyte sedimentation rate (ESR) with increasing chlorpyrifos concentration is indicative of inflammatory and pathological conditions. Elevated ESR values are commonly associated with alterations in plasma protein composition, tissue injury and systemic inflammation (Barton et al. 2002) [7] . Similar increases in ESR have been reported in fish exposed to toxicants, reflecting chronic stress and cellular damage (Dautremepuits et al. 2004; Hossain et al. 2022) [13,15] . Overall, the present findings clearly demonstrate that chronic sub-lethal exposure to chlorpyrifos induces severe hematological disturbances in Catla catla, characterized by anemia, leukocytosis, macrocytosis and inflammatory responses. These alterations are consistent with previous studies on organophosphate toxicity and confirm the usefulness of hematological parameters as early warning indicators of pesticide-induced stress in freshwater fish. The observed hematological impairments may ultimately reduce growth, survival and fitness of fish populations in contaminated aquatic ecosystems.

References

1. Abdallah MAM, Abdallah AMA. Biomonitoring study of heavy metals in biota and sediments in the southeastern coast of the Mediterranean Sea, Egypt. Environmental Monitoring and Assessment. 2008;146(1-3):139-145.
2. Abhijith BD, Ramesh M, Poopal RK. Sublethal toxicological evaluation of methyl parathion on some hematological and biochemical parameters in an Indian major carp Catla catla. Comparative Clinical Pathology. 2012;21(1):55-61.
3. Abrantes N, Pereira R, Gonçalves F. Occurrence of pesticides in water, sediments and fish tissues in a lake surrounded by agricultural lands: Concerning risks to humans and ecological receptors. Water, Air, & Soil Pollution. 2010;211(1-4):1-12.
4. Akinrotimi OA, Abu OMG, Ansa EJ, Edun OM, George OS. Haematological responses of Tilapia guineensis to acute stress. International Journal of Natural and Applied Sciences. 2009;5(4):338-343.
5. Alengebawy A, Abdel Rahman GH, Al-Jaouni SK, AlJahdali NS. Pesticides and their effects on human health and the environment: A review. Environmental Science and Pollution Research. 2021;28(13):20009-20026.
6. Ali HAJ, Rani VJ. Effect of phosalone on haematological indices in the tilapia Oreochromis mossambicus. Turkish Journal of Veterinary and Animal Sciences. 2009;33(5):407-411.
7. Barton BA. Stress in fishes: A diversity of responses with particular reference to changes in circulating corticosteroids. Integrative and Comparative Biology. 2002;42(3):517-525.
8. Barton BA, Morgan JD, Vijayan MM. Physiological and condition-related indicators of environmental stress in fish. In: Adams SM, editor. Biological indicators of aquatic ecosystem stress. Bethesda (MD): American Fisheries Society; 2002. p. 111-148.
9. Blaxhall PC, Daisley KW. Routine haematological methods for use with fish blood. Journal of Fish Biology. 1973;5(6):771-781.
10. Cericato L, Soso AB, Fagundes M, Conrad J, Lacerda LA, Terra S. Hematological changes in jundia (Rhamdia quelen) after acute and chronic stress. Aquaculture. 2004;237(1-4):229-236.
11. Dacie JV, Lewis SM. Practical haematology. 8th ed. London: Churchill Livingstone; 1991.
12. Das PC, Ayyappan S, Jena JK, Das BK. Nitrite toxicity in Cirrhinus mrigala: Sub-lethal effects on selected haematological parameters. Aquaculture. 2004;235(14):633-644.
13. Dautremepuits C, Paris-Palacios S, Betoulle S, Vernet G. Modulation in hepatic and head kidney parameters of carp induced by copper. Comparative Biochemistry and Physiology Part C. 2004;137(4):325-333.
14. Deb N, Das S. Chlorpyrifos toxicity in fish: A review. Current World Environment. 2013;8(1):77-84.
15. Hossain MA, Sutradhar L, Sarker TR, Saha S, Iqbal MM. Toxic effects of chlorpyrifos on the growth, hematology and histopathology of Nile tilapia (Oreochromis niloticus). Environmental Science and Pollution Research. 2022;29:57312-57328.
16. Kavitha C, Malarvizhi A, Kumaran SS, Ramesh M. Toxicological effects of arsenate exposure on hematological, biochemical and liver transaminases activity in an Indian major carp Catla catla. Food and Chemical Toxicology. 2010;48(10):2848-2854.
17. Lakshman M, Reddy PK, Rao JV. Impact of dichlorvos on hematological parameters of Oreochromis mossambicus. International Journal of Environmental Sciences. 2013;3(6):1912-1920.
18. Majumder R, Kaviraj A. Acute and sublethal effects of organophosphate insecticide chlorpyrifos on freshwater fish Oreochromis niloticus. Drug and Chemical Toxicology. 2019;42(5):487-495.