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Saudi Journal of Biological Sciences logoLink to Saudi Journal of Biological Sciences
. 2022 May 22;29(7):103316. doi: 10.1016/j.sjbs.2022.103316

Toxic effects of chlorpyrifos on the growth, hematology, and different organs histopathology of Nile tilapia, Oreochromis niloticus

Mohammad Amzad Hossain 1,1,⁎, Lipi Sutradhar 1,1, Tumpa Rani Sarker 1, Shuva Saha 1, Mohammed Mahbub Iqbal 1
PMCID: PMC9614567  PMID: 36313386

Abstract

Chlorpyrifos is a widely applied insecticide that permeates on most waterways and affects aquatic organisms. The growth performances, hematological and histological impacts on Nile tilapia, Oreochromis niloticus following a 60 day of exposure to varying concentrations of chlorpyrifos 20 EC (T1 08 µgL−1, T2 16 µgL−1, and T3 32 µgL−1) were compared to a control Tc 0 µgL−1. The 96-hour LC50 of chlorpyrifos 20 EC was calculated as 46.80 μgL−1. The water quality parameters were recorded regularly. The value of dissolved O2 and NH3 stayed rather steady, although temperature varied considerably. It was revealed that as chlorpyrifos levels go up, the percentage of weight gain (WG %), specific growth rates (SGR), and survival rate decreases. The control group Tc had the highest percentages of SGR weight (1.16 ± 0.58) and the T3 group had the lowest percentages of SGR weight (0.25 ± 0.77). The hematological assessment showed significant differences of hemoglobin concentration, white blood cell counts and red blood cell numbers between chlorpyrifos treatment and control group (P < 0.05). Histological alterations in the liver, gills, and muscle tissues reported to be worse for T3 as compared to others. There were no statistical differences in GSI, HSI values between control and treatment groups. The chlorpyrifos 20 EC was shown to be highly toxic to O. niloticus at sub-lethal dosages.

Keywords: Pesticide, Organophosphate, Fish, Toxicity, Hemoglobin, SGR, HSI

1. Introduction

The Nile tilapia, Oreochromis niloticus, is one of the highly cultured commercial fish species in Asian territory (Syed et al., 2022). It is considered as a promising tropical aquaculture species, especially for low-income countries and proven to play a vital role in rural nutrition and economy of Afro-Asian realms (Chan et al., 2019). This fish showed excellent capacity to grow well in a wide range of thermal and environmental states (Edwin et al., 2019). As it can withstand physical and chemical changes, and toxins in water, it’s been often employed as a bioindicator of water pollution (Ihsan et al., 2019). Tilapia is now one of the most refereed model animals in aquatic toxicology investigation (Yan et al., 2020).

Chlorpyrifos, an organophosphate derivative is widely used insect repellent in global commercial Agro-farming practices (Hasanuzzaman et al., 2018, Silva and Samayawardhena, 2002) and it is counted as India's second most popular means of synthetic pest control (Stalin et al., 2019). It is extensively used in Bangladesh due to low price, availability in different formulations and effectiveness in little amount (Ali et al., 2020, Ihsan et al., 2018). Most of the insecticides used in crops are eventually expelled into nearby water bodies through canal, rains, and farm runoff. While pesticides serve a vital role in increasing land productivity and food quality for the world's rising population, particularly in developing nations, their presence in agricultural drainage poses a major threat to all aquatic ecosystem components (Abhilash and Singh, 2009). The minimal durability of such substances in aquatic ecosystems has prompted worries about their ability to harm non-target organisms, particularly fish (Ali et al., 2009). Different formulations of Chlorpyrifos are mostly used pesticides in Bangladesh and their incursion in aquatic habitat and life forms are still a mystery.

Pesticides are exposed directly to humans and terrestrial animals in a variety of ways, including chronic exposure, domestic exposure, and intake of pesticide-polluted water and food (Ullah et al., 2018). Insecticides penetrate to fish via absorption across skin, uptake during respiration in gills and importantly through food or drinking contaminated water (Dawood et al., 2020). Insecticides being accumulated in edible aquatic organisms are finally consumed by human beings and then cause public health risk (Petchoy and Pung, 2017). Fish that are exposed to environmental contaminants, would experience functional disturbances in respiration, feeding, growth and reproductive capacities. Therefore, the impacts of chlorpyrifos on fish would be traced by histological, hematological and growth metric analysis. The hematology of fish serves as an appropriate means to assess the stress and associated health issues generated by biotic and abiotic mediators of the environment (Adhikari et al., 2004, Martins et al., 2011). Hematological indices such as red blood cells count, white blood cells count, hemoglobin level, and many other features were being used to diagnose fish health and as indications for alterations during stressful toxic conditions (Kumar et al., 2011). Muñoz et al., (2015) suggested the histopathology of vital organs in fish as a bio-monitoring method for accessing aquatic pollution. Histopathology could be used as a biomarker to track the environment or look at specific cellular response in the key organs. Current study intended to examine the effects of pesticide chlorpyrifos 20 EC on growth, blood counts and different organ levels of Nile tilapia.

2. Materials and methods

2.1. Designing the experiment and acclimatization of fish

The experiment was conducted for a period of 60 days from November 17, 2020, to January 16, 2021. Nile tilapia juveniles were acquired from nearby BRAC hatchery and carried out by oxygenated poly bags. Afterwards, fish were stocked in glass aquaria and reared for 7 days with continuous aeration to acclimatize under laboratory conditions. Then, fish with equal preliminary weight (3.60 ± 0.85 g) were allotted in 12 glass aquaria (60 L). Four groups of triplicate glass aquaria were indiscriminately disseminated with 25 fish in each aquarium. Each glass aquarium was set up with air stones for aeration. The treatment groups were assigned based on the computation of LC50 (section 2.2) and presented in Table 1. Fish were fed with commercial formulated diets three times (08:00 h, 14:00 h and 22.00 h) a day at 2–3% of their body weight. Uneaten feed was removed everyday through siphoning and siphoned water was then filtered and added to the treatment tank.

Table 1.

Designing the dosages for different treatment units following the 96-hour LC50.

Treatment Dose of Chlorpyrifos (µgL−1) Stocking density (no./treatment) Replication
TC (Control) 0 (0 % of LC50) 25 3
T1 8 (17 % of LC50) 25 3
T2 16 (34 % of LC50) 25 3
T3 32 (68 % of LC50) 25 3
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2.2. Toxicity test and preparing dosages for exposure

A prior 96-hour acute toxicity test had been conducted by using differential concentrations (0, 8,16, 24, 32, 36, 40, 48, 56, 62 µgL−1) of chlorpyrifos 20EC. Each of the treatment units had been employed with 40 L of tape water, well installed aeration system and 20 fish in each following the OECD standard protocol guidelines for aquatic toxicity assessment (EPA, 2021, OECD, 2009). The obtained mortality rates were plotted against 10 base logarithms of chlorpyrifos concentration in SPSS to do PROBIT analysis at P < 0.05 (Finney, 1971).

2.3. Acquisition of water quality factors

Temperature, dissolved oxygen, and pH was monitored and determined each week by using automated YSI Professional Plus (Pro Plus) Multiparameter Instrument (Brannum Lane, USA). The concentration of NH3 level was calculated by using a ready to use commercial Keen kit from USA.

2.4. Growth measurement and bleeding

Sampling was done on a weekly basis to define the growth of O. niloticus and to correct the feeding rate. At the end of the 60 days trial, body weight and total length of all fishes were recorded for growth analysis. Additionally, three fishes from each treatment unit were randomly selected, anesthetized with MS222 and blood sample was collected by using 1 mL heparinized syringes (Jimi syringes and medical devices Ltd, Bangladesh). Collected 300–500 µL blood were kept in an EDTA tube for further analysis. The complete blood counting was conducted by placing the EDTA tube on automated Hematology analyzer ELite Erba 3.0 (Erba Lachema S.R.O.). Again, the same fishes were sacrificed and, liver and gonads were separated carefully. Finally, weight of above organs was recorded for analysis of hepatosomatic index (HSI) and gonadosomatic index (GSI). A piece of gill, liver and muscle were collected gently and preserved in neutral buffered formalin (NBF) for further histological analysis.

2.5. Tools for growth, hepatosomatic index (HSI) and gonadosomatic index (GSI)

The specific growth fish which was calculated by following formula-.

SGR (weight or Length) % = (log Wf– log Wi)/T × 100; Where, Wi = Initial body weight (g) or length (cm) and Wf = Final body weight (g) or length (cm); T = duration in days.

Fulton’s condition factor, K = Body Weight/ (Total length)3 (Htun-Han, 1978).

Hepatosomatic index, HSI= (weight of liver/weight of fish) × 100 (Chellappa et al., 1995).

Gonadosomatic index, GSI= (weight of liver/weight of fish) × 100 (Brooks et al., 1997).

2.6. Histological analysis

The NBF preserved samples were washed out on tap water and went through xylene cleaning and graded series alcoholic dehydration process. Then tissues were infiltrated in paraffin and resulting blocks were sectioned at 3–5 µm by using the Leica Microtome Machine (Wetzlar, Germany). The standard staining process was conducted by following the protocol from Van-Dyk & Pieterse, (2008). Tissues were visualized under Zeiss Primo Star 3.0 microscope and micrographs were taken by using Zeiss Core 3.0 software on Windows 10 installation.

2.7. Data analysis

The results were evaluated by applying the one-way analysis of variance tools in IBM SPSS v27 and Duncan's Multiple Range test (DMRT) to establish P < 0.05. Graph and visualization were performed in office 365 tools based on the data obtained from SPSS output.

3. Results

3.1. Acute toxicity test

The results from PROBIT analysis exhibited 96-hour LC50 of Chlorpyrifos at 46.80 μgL-1 (P < 0.05). A reference table has been constructed to reinforce the current finding, which described acute toxicity tests for different formulations of Chlorpyrifos on Oreochromis genus (Table 2).

Table 2.

Value of 96-h Chlorpyrifos LC50 in previous studies with Oreochromis genus.

Species Value of 96-hour LC50 (μgL−1) Formulation of chlorpyrifos References
O. niloticus 42.0 Chlorpyrifos 20% EC (Majumder and Kaviraj, 2019)
76 Chlorpyrifos 20% EC (Ihsan et al., 2018)
70 Not specified (El-Bouhy et al., 2016)
1023 Not specified (Girón-Pérez et al., 2006)
1500 Chlorpyrifos-methyl (Gül, 2005)
O. mossambicus 22 Chlorpyrifos 20% EC (Muttappa et al., 2015)
25.97 Chlorpyrifos 20% EC (Rao et al., 2003)
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3.2. Water quality parameters

The water quality parameters are summarized in Table 3. The concentration of dissolved O2, NH3 remained constant for all the treatments during the different phases of trial. However, temperature and pH were reported as more fluctuated parameters during the trial periods. The value of temperature was decreased from 30.75 ± 0.5 °C to 21 °C as trials progressed. Again, pH values were also oscillated on different time slots. The pH varied slightly between 6.32 ± 0.09 to 5.97 ± 0.15. The values of other water quality parameters were conducted stable at different time slots and treatment units. Dissolved oxygen level also showed a trend of rising after the first 14 days of trial for all treatment groups (Table 3).

Table 3.

Changes in water quality parameters of O. niloticus exposed to chlorpyrifos (20% EC) for 60 days.

Parameters Treatments DAY_0 DAY-15 DAY-30 DAY-60
Temperature Tc (0 µgL−1) 29.92 ± 0.15d 27.57 ± 2.18c 24.22 ± 0.32b 21.12 ± 0.25a
T1 (8 µgL−1) 30.5 ± 0.57d 26.3 ± 1.67c 24.62 ± 1.21b 21 ± 0.00a
T2 (16 µgL−1) 30.25 ± 0.5d 28.3 ± 1.13c 25.35 ± 1.17b 21.5 ± 0.57a
T3 (32 µgL−1) 30.75 ± 0.5d 27.07 ± 1.45c 25.42 ± 1.02b 21 ± 0.00a
pH Tc (0 µgL−1) 6.27 ± 0.05a 6.3 ± 0.00a 6.08 ± 0.33ab 5.97 ± 0.15a
T1 (8 µgL−1) 6.25 ± 0.057a 6.25 ± 0.05a 6.13 ± 0.33a 6.15 ± 0.23a
T2 (16 µgL−1) 6.32 ± 0.09c 6.27 ± 0.09bc 6.06 ± 0.17a 6.1 ± 0.11ab
T3 (32 µgL−1) 6.3 ± 0.08b 6.32 ± 0.09b 6.03 ± 0.10a 6.15 ± 0.23ab
Dissolved O2 Tc (0 µgL−1) 5.82 ± 0.23a 6.42 ± 0.47b 6.7 ± 0.14b 6.85 ± 0.05b
T1 (8 µgL−1) 6.25 ± 0.17a 6.8 ± 0.42b 6.63 ± 0.23ab 6.76 ± 0.20b
T2 (16 µgL−1) 6.07 ± 0.15a 6.55 ± 0.1a 6.28 ± 0.50a 6.15 ± 0.23a
T3 (32 µgL−1) 6.15 ± 0.1a 6.52 ± 0.26ab 6.54 ± 0.42ab 6.88 ± 0.13b
NH3 Tc (0 µgL−1) 0.013 ± 0.12a 0.020 ± 0.25a 0.020 ± 0.28a 0.012 ± 0.06a
T1 (8 µgL−1) 0.018 ± 0.12a 0.015 ± 0.09a 0.013 ± 0.20a 0.012 ± 0.08a
T2 (16 µgL−1) 0.013 ± 0.12a 0.017 ± 0.16a 0.014 ± 0.15a 0.014 ± 0.07a
T3 (32 µgL−1) 0.020 ± 0.08a 0.022 ± 0.32a 0.012 ± 0.20a 0.012 ± 0.10a
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3.3. Growth parameters

Growth performances of O. niloticus have been organized in Table 4. It showed that the percentage of weight gain (WG %), specific growth rate (SGR), and survival rate gradually declined with the increase in concentration of chlorpyrifos. The highest weight gain (g) found in the control group was 3.59 ± 1.69 g while lowest in T3 was 0.78 ± 1.83 g followed by T2, T3 was 1.92 ± 2.72 g and 1.25 ± 1.86 g respectively (P < 0.05). Again, significant differences were observed between control and T1 groups in terms of final length (cm), final weight (g), length gain %, weight gain %, average daily length gain, and average daily weight gain. The highest percentages of SGR (weight) were demonstrated in control group Tc (1.16 ± 0.58) and the lowest in T3 group (0.25 ± 0.77), while differences between T1 and T2 were statistically non-significant (P < 0.05) (Table 4). The SGR (length) percentages also showed a similar pattern of variation between different treatment groups. There was significant variation in mortality rate between the treatments and control group (Fig. 1). Mortality rate was recorded as maximum 53% in T3 group, followed by 35 % in T2. It has been well indicated that higher mortality rates are associated with the higher concentration of toxicant.

Table 4.

Changes in growth parameters of O. niloticus exposed to chlorpyrifos (20% EC) for 60 days. The results showed significant difference at P < 0.05.

Parameters Tc (0 µgL−1) T1 (8 µgL−1) T2 (16 µgL−1) T3 (32 µgL−1)
Initial length (cm) 5.82 ± 0.95a 5.84 ± 0.57a 5.85 ± 0.31a 5.87 ± 0.70a
Initial weight (g) 3.77 ± 1.26a 3.73 ± 0.61a 3.32 ± 0.40a 3.58 ± 0.95a
Final length (cm) 7.77 ± 0.61b 7.21 ± 1.23ab 6.82 ± 0.80a 6.57 ± 0.76a
Final weight (g) 7.37 ± 1.51b 5.65 ± 2.73ab 4.58 ± 1.61a 4.36 ± 1.71a
Length gain (cm) 1.95 ± 0.87b 1.36 ± 0.93ab 0.96 ± 1.04a 0.70 ± 1.02a
Weight gain (g) 3.59 ± 1.69b 1.92 ± 2.72ab 1.25 ± 1.86a 0.78 ± 1.83a
Length gain % 36.28 ± 21.30b 23.16 ± 15.70ab 17.23 ± 18.02a 13.29 ± 18.65a
Weight gain % 111.35 ± 63.64b 53.57 ± 71.63ab 42.46 ± 59.93a 28.00 ± 57.90a
Specific growth rate (%, weight) 1.16 ± 0.58b 0.56 ± 0.72ab 0.44 ± 0.76ab 0.25 ± 0.77a
Percentage of Specific growth rate (%, Length) 201.93 ± 7.82b 193.34 ± 16.96ab 188.43 ± 12.22a 184.82 ± 11.72a
Average daily length gain 0.032 ± 0.014b 0.022 ± 0.015ab 0.016 ± 0.017a 0.011 ± 0.017a
Average daily weight gain 0.059 ± 0.028b 0.032 ± 0.045ab 0.020 ± 0.031a 0.013 ± 0.039a
Fulton’s condition factor 1.55 ± 0.13c 1.46 ± 0.26b 1.39 ± 0.16a 1.47 ± 0.20b
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Fig. 1.

Fig. 1

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Mortality rate of O. niloticus exposed to chlorpyrifos 20% EC for 60 days.

3.4. Hematological parameters

The hematological properties of O. niloticus exposed to different doses of chlorpyrifos 20EC are tabulated in Table 5. The results showed a significant drop in the counts of hemoglobin concentration, WBC, monocytes, RBC, HCT with the increased concentration of chlorpyrifos exposure (P < 0.05). In contrast, the Total White blood cells (WBC) were reported as maximum in T3 (83233.33 ± 11718.93 cells/mm3) and minimum on Tc (45883.33 ± 5150.32 cells/mm3) (P < 0.05). Lymphocytes and Monocytes counts were described statistically stable in each treatment group. Total plate count was lowest in T1 (150666.7 ± 11547.01 cells/mm3) and highest in T3 (230666.7 ± 10016.65 cells/mm3). Besides, Procalcitonin (PCT) accounted as highest in T1 (0.12 ± 0.00 ng/mL) and lowest in T3 (0.18 ± 0.00 ng/mL). The mean corpuscular hemoglobin concentration was highest in T2 (34.7 ± 4.203 g/dL) and lowest in control Tc (28.45 ± 0.75 g/dL). Hematocrit concentration also remained statistically highest for control group in compared to the treatment groups. A strong tendency of rising procalcitonin has been allocated among the treatment group. However, blood glucose level was reported to be stable for all the treatment and control group.

Table 5.

Changes in hematological parameters of O. niloticus exposed to chlorpyrifos 20EC for 60 days.

Parameters Tc (0 µgL−1) T1 (8 µgL−1) T2 (16 µgL−1) T3 (32 µgL−1)
Haemoglobin (Hg) (g/dL) 4.43 ± 0.50a 2.96 ± 0.11a 3.36 ± 1.51a 2.83 ± 0.11a
Total White blood cells (WBC) (cells /mm3) 45883.33 ± 5150.32a 55966.67 ± 26633.31ab 51266.67 ± 6824.46a 83233.33 ± 11718.93b
Lymphocytes (%) 94.18 ± 0.59a 96.06 ± 0.12a 95.5 ± 2.5a 96 ± 0a
Monocytes (%) 1.78 ± 0.71a 0.95 ± 0.05a 1.5 ± 0.5a 1.0 ± 0.0a
Total Plate counts (cells /mm3) 219333.3 ±9073.77b 150666.7 ±11547.01a 217166.7 ±719094.07ab 230666.7 ±10016.65ab
Mean platelet volume (MPV) (fL) 7.45 ± 0.25c 7.85 ± 0.25a 7.55 ± 0.15ab 7.7 ± 0.1ab
Procalcitonin (PCT) (ng/mL) 0.12 ± 0.00a 0.16 ± 0.0b 0.17 ± 0.01bc 0.18 ± 0.00c
Red Blood Cell Count (cells *103/mm3) 1.19 ± 0.15b 0.74 ± 0.05a 0.93 ± 0.38ab 0.8 ± 0.02ab
Haematocrit (HCT) 15.45 ± 1.35b 9.7 ± 0.3a 9.826 ± 0.551a 9.3 ± 0.1a
Mean corpuscular volume (MCV) (fL) 130.5 ± 5.1a 133.2 ± 5a 121.1 ± 3a 114.96 ± 0.5.37a
Mean corpuscular haemoglobin (MCH) (pg/cell) 37.05 ± 0.45ab 39.9 ± 2.7b 36.68 ± 1.206a 36.2 ± 0.8a
Mean corpuscular haemoglobin concentration (MCHC) (g/dL) 28.45 ± 0.75a 29.95 ± 0.95a 34.7 ± 4.203b 31.7 ± 0.2ab
Red cell distribution width (RDW)(fL) 17 ± 1.4c 13 ± 1.1b 12.2 ± 0.5ab 11.15 ± 0.35a
Glucose (mg/dL) 196.70 ± 5.06a 194.63 ± 7.0a 189.70 ± 2.95a 184.03 ± 4.22a
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3.5. Hepatosomatic index (HSI) and gonadosomatic index (GSI)

There was no statistical difference for HSI and GSI values between the treatments and control group. However, an apparent increasing trend of HSI has been reported as the doses of Chlorpyrifos 20 EC increased (Fig. 2).

Fig. 2.

Fig. 2

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Hepatosomatic index (HSI) and gonadosomatic (GSI) index of O. niloticus.

3.6. Histopathology of gill, liver, and muscle tissues

The structure of the gill of control fish having neat order of primary and secondary lamella, secondary lamella, pillar cell, mucous cell and no abnormalities were detected (Fig. 3.A). Necrosis of epithelial cells, epithelial lifting, lamellar fusion, and ruptured secondary lamellae and swelling were the most prevalent alterations in the treatment units with varied concentrations of Chlorpyrifos 20EC. Fish treated in T1 exhibited swelling of primary lamellar epithelium, damaged gill filament (Fig. 3.B). In the T2 group, necrotic secondary lamellae, diffusion of mucous cells and epithelial lifting were noted (Fig. 3.C). Fish in the T3 group showed fusion and shortening of secondary lamellae, and epithelial necrosis (Fig. 3.D).

Fig. 3.

Fig. 3

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Longitudinal histological sections through the gill of Nile tilapia, Oreochromis niloticusA. Control 0 μgL-1 Chlorpyrifos (0 % of LC50); B.T1 8 μgL-1 Chlorpyrifos (17% of LC50); C. T2 16 μgL-1 Chlorpyrifos (34% of LC50); D. T3 32 μgL-1 Chlorpyrifos (68% of LC50) exposure for 60 days. (PL-Primary lamella, SL-Secondary lamella, PC-Pillar cell, MC-Mucous cell, SSL-shortening secondary gill lamella, PLE-primary lamellar epithelium, DF- damage of filament, EN-epithelial necrosis, CSL-congestion of secondary gill lamella, DMC-diffusion of mucous cell, EL-epithelial lifting, LLE-lifting of lamellar epithelium, AN-acute necrosis).

The continuous mass of large hexagonal hepatic cells, amid sinusoids and centrally located prominent nuclei were registered in control group (Fig. 4.A), while liver in T1 group showed focal necrosis, degenerative nuclei, granular degeneration, and vascular dilation (Fig. 4.B). The liver in T2 also witnessed widespread necrosis and vascular dilation (Fig. 4.C). The T3 group were characterized with severe nuclei congestion, acute necrosis, vacuoles formation and dilation of sinusoids (Fig. 4.D).

Fig. 4.

Fig. 4

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Histopathological alterations of liver in Nile tilapia, Oreochromis niloticusA. Control 0 μgL-1 Chlorpyrifos (0 % of LC50); B.T1 8 μgL-1 Chlorpyrifos (17% of LC50); C. T2 16 μgL-1 Chlorpyrifos (34% of LC50); D. T3 32 μgL-1 Chlorpyrifos (68% of LC50) exposure for 60 days. (H-hepatocytes, Nu-nuclei, SS-Sinusoids, DH-degenerative hepatocytes, GD-Granular degeneration, VD-vascular dilation, N-necrosis, V-Vacuoles, AN/CN-acute/chronic necrosis).

The control fish showed normal muscle fiber as in Fig. 5.A; on the other hand, degeneration of muscle fiber, splitting of muscle fiber and vacuoles in different rates were reported in T1 and T2 (Fig. 5.B-C). Extensive necrosis and vacuole formation were detected in T3 (Fig. 5.D).

Fig. 5.

Fig. 5

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Muscle tissue section of Nile tilapia, Oreochromis niloticusA. Control 0 μgL-1 Chlorpyrifos (0 % of LC50); B.T1 8 μgL-1 Chlorpyrifos (17% of LC50); C. T2 16 μgL-1 Chlorpyrifos (34% of LC50); D. T3 32 μgL-1 Chlorpyrifos (68% of LC50) exposure for 60 days. (DMF-Degeneration of muscle fiber, V-vacuoles, SMF-splitting of muscle N-necrosis).

4. Discussion

Basic physico-chemical parameters such as conductivity of water, dissolved oxygen level, pH, and water temperature were kept essentially consistent during the time of exposure following the standard parameters of Bhatnagar and Devi, 2013, Chapman et al., 2016. As the trials progressed, the temperature dropped from 30 °C to 21 °C. This can be explained by the drop in temperature during November to December, accompanied by the cycle of winter in Bangladesh.

The lethal concentration index LC50 is one of the widely used sensitivity tests to quantify chemical toxicants on animals (Sharmin et al., 2021). Current findings suggest that most of the commercial formulations of Chlorpyrifos imposed strong toxicity at very low doses for fish. The 96-hour LC50 for Chlorpyrifos 20% EC ranged between 22 and 76 μgL-1 (Ihsan et al., 2018, Muttappa et al., 2015). The highest concentrations were noted as 1023–1500 μgL-1, although formulation of pesticides was not specified (Girón-Pérez et al., 2006, Gül, 2005). A stable survival rate of 100 % has been reported for control units which were within the condition for testing toxicity in aquatic organisms according to APHA, (1999). The differences in LC50 are attributed to type of species used, their size and age, physicochemical properties of the rearing environment and formulation of pesticide administered.

Fishes exposed to organophosphate insecticides are reported to experience behavioral abnormalities, and increased death rate due to metabolic disturbances linked with toxicants (Dembélé et al., 2000). Tilapia guineensis was subjected to higher mortality rate with increased concentration and treatment duration of chlorpyrifos (Chindah et al., 2005). Mortality of stinging catfish Heteropneustes fossilis rose at higher concentrations of Envoy 50 (Akter et al., 2020) and with exposure to sublethal concentration of Pb toxicant (Hussain et al., 2021). Chlorpyrifos has been shown to have considerable impacts on growth of O. niloticus (Majumder and Kaviraj, 2019). Negative growth issues were detected in Tandanus tandanu treated with Chlorpyrifos (Huynh and Nugegoda, 2012). Reduced body weight might be caused by poor food intake and liberation of extensive energy to compensate for toxic related stress while rearing (Adel et al., 2017, Majumder and Kaviraj, 2019). Poor body biomass and increased mortality rate in the treatment group compared to the control group also act as supportive to the above findings.

Hematology results corresponds to a better measurement of fish health and environmental status (Eissa and Abou-ElGheit, 2014) and they can be substantially manipulated by animal’s age, size, physiology, diet component and surrounding hydrology (Parrino et al., 2018). The hematology counts in current research demonstrated significant alternation between the animals in control group and treatment groups. A study by Singh and Srivastava, (2010) revealed there was an increased count of total leukocyte in Labeo rohita exposed to 28 days of organophosphate pesticide profenofos. In contrast, it had also been noted that hematology remains non affected in Caspian brown trout treated for 20 days with Chlorpyrifos (Adel et al., 2017) and common carp for three-week chlorpyrifos (Jaffer et al., 2017). A noticeable variation was reported in the hemoglobin concentration and white blood cells of O. niloticus associated with 30 days exposure to Chlorpyrifos (Abdo et al., 2021). Again, O. niloticus treated with Sumithion considerably yields lower red blood cell and hemoglobin levels, with increased white blood cell counts (Sharmin et al., 2021). Reduced mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were detected in fish treated with Ronster for three weeks (Oluah et al., 2020). Increased level of blood procalcitonin could be regarded as a biomarker for indicating respiratory tract inflammation (Gilbert, 2011).

Increased HSI in Mullus barbatus had been documented due to heavy metals treatment and it could be associated with cellular deformities and an increase in hepatic enzyme degradation activities (Kucuksezgin et al., 2011). Majumder and Kaviraj, (2019) also noted elevated HSI value in chlorpyrifos intoxicated O. niloticus. As the rate of necrosis in liver cells increased current research following the pesticides exposure, the liver would have gained weight as extra mass in the form of fluid congestion.

Tissue histology, which is used as an indicator of pollution exposure, is a valuable method for determining the extent of pollution, especially for sublethal and chronic effects (Cengiz & Ünlü, 2003). Fish gills are sensitive to contaminants in water because of their placement and huge exterior area (Afshan et al., 2014, Stara et al., 2020). Pesticides have been proven to have comparable effects on fish gills in several studies (Cengiz and Unlu, 2006) as they execute a variety of tasks including breathing and digestion, osmoregulation, and excretion. Metal-contaminated Oreochromis niloticus and Lates niloticus from Lake Nasser, Egypt, showed shortening or fusion of gill lamellae, lamella destruction, increased vacuolation, and irregular gill lamellae (Younis et al., 2013). The effects of similar histopathology have been conducted while treating guppy Poecilia reticulata with chlorpyrifos (Silva and Samayawardhena, 2002). The liver functioned as principal organ in detoxifying metabolites and its histology would act as effective biomarkers in demonstrating the impact of toxicant at cellular level (Cuevas et al., 2016). Hepatic lesions were stated to related with increased toxicant levels (Sadiq et al., 2012). The Cory, Corydoras paleatus treated with methyl parathion developed swelling, bile stagnation, necrosis, atrophy, and vacuolization in hepatic cells (Fanta et al., 2003). Sarkar et al., (2005) reported hyperplasia, vacuolation, interrupted hepatocytes and necrosis, in Labeo rohita treated with cypermethrin. Enhanced degeneration, necrosis, and localized hemorrhage were detected on liver tissue of the same fish due to a two-week chlorpyrifos treatment (Farhan et al., 2021). Hepatic hypertrophy, displaced nuclei, necrotic cells sites and degenerative cells were reported in the same species in 7 days of carbofuran treatment (Américo-Pinheiro et al., 2020). The current findings are coherent with above investigations. It had been identified that increasing the amount of chlorpyrifos 20EC accelerated the level of degenerative process in liver cells. Muscle cell damage and vacuolation were detected in Oreochromis treated with Chromiam (Abbas and Ali, 2007). The exposure to chlorinated pesticides and PCBs also induced histological adjustments in the muscular tissue of freshwater fish Hoplias alabaricus (Miranda et al., 2008). In conclusion, the findings of the present histological investigations demonstrated a direct correlation between pesticide exposure and histopathological disorders as observed in several tissues.

5. Conclusion

Results of the current study clearly revealed that chlorpyrifos 20EC is highly toxic to aquatic vertebrates and had a detrimental impact on growth, hematological indicators, and somatic indices. The effect of toxicity is also proven in cellular level through distinctive histopathology in high dose treatment units.

Ethical approval

The Animals Ethics Committee from the Sylhet Agricultural University Research System (SAURES), Sylhet Agricultural University, Sylhet-3100, Bangladesh, approved the design and utilization of animals for the current project.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Thanks to UGC (University Grant Commission) and Sylhet Agricultural University Research System (SAURES), Bangladesh, for providing funds for research and student’s allowance in referring to budget allocation code 3632104. Authors acknowledge the NST (National Science and Technology) fellowship scheme, for providing their partial fundings in research and student stipend as well.

Footnotes

Peer review under responsibility of King Saud University.

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