Introduction

The aquaculture industry has increasingly developed as a promising activity for seafood production and food security. Moreover, aquaculture contributes to the economic sector, particularly in low-income countries (FAO 2020). However, on the environmental level, the extremely high levels of toxicants and xenobiotics were found to affect the growth performance and digestive enzyme activity of aquatic species (Li et al. 2015). Low hygiene and environmental contamination of aquaculture could affect consumer health and aquaculture productivity (Eltholth et al. 2015).

Chlorpyrifos (CPF) is an organophosphorus insecticide, extensively used for pest control in the agriculture sector (Dawood et al. 2020; Racke 1993). The half-life of CPF in river water ranges from 24 to 126 days (Liu et al. 2001). CPF has reported a contaminating environment, causing neurodevelopmental and metabolism disorders (Gao et al. 2017; Greer et al. 2019; McClelland et al. 2018). For example, CPF alters lipid metabolites and serine lipase activity in the brain of rainbow trout (Oncorhynchus mykiss) (Greer et al. 2019). CPF disrupts the endocrine system, which alters thyroid hormones and the expression of their receptors (Holzer et al. 2017; Raibeemol and Chitra 2020). Also, CPF causes the degradation of extracellular DNA in aquatic organisms and impaired hemolymph biochemical parameters in the crayfish (Astacus leptodactylus) (Banaee et al. 2019; Jin et al. 2015; Yang et al. 2019). CPF has been found to disrupt the antioxidant systems and immune response genes in common carp (Cyprinus carpio L.) (Banaee et al. 2014; Hatami et al. 2019; Li et al. 2013; Xing et al. 2019; Zhang et al. 2017). In Nile tilapia (Oreochromis niloticus), CPF caused complex physiological changes that included cytokine expression, oxidative damage, and tissue alterations, and the reversibility of these changes was varied following recovery (De Anna et al. 2021; Zahran et al. 2018). There are trials to alleviate the toxicity of CPF (Abo-Al-Ela 2020); for example, Aswathi et al. (2019) investigated Pseudomonas nitroreducens AR-3 as a biological agent to degrade CPF in the environment. Other studies tried to use beneficial substances, such as mannanoligosaccharide, Spirulina platensis, and β-glucan to rescue the toxicity induced by CPF (Dawood et al. 2020; Mokhbatly et al. 2020).

Vitamin C (VC) is a powerful antioxidant that reduces genotoxicity caused by chemical agents (Abo-Al-Ela et al. 2017; Dawood et al. 2020; Guha and Khuda-Bukhsh 2002; Sharifinasab et al. 2016). VC is found to reverse the effect of herbicide-induced toxicity, such as oxidative damage in cell lines (Gehin et al. 2005). VC has a hepatoprotective effect by regulating the serum total cholesterol, aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), triglyceride, high-density lipoprotein cholesterol, and total protein levels during herbicide intoxication (Otuechere et al. 2012). Dietary VC significantly improved the growth performance, survival, and metabolic enzyme activities (e.g., alanine aminotransferase in the liver and muscle and ALP activity in the liver) and bioaccumulation endosulfan in spotted murrel, Channa punctatus (Sarma et al. 2009). Markedly, dietary vitamin C relieved CPF-induced toxicity in Clarias batrachus by enhancing the growth rate, blood immune, and biochemical responses (Narra et al. 2015).

In the current study, we aimed to determine the effect of CPF on inflammation and immunomodulation processes and histoarchitecture of the liver, gills, and intestine tissues, as well as we hypothesized that VC could curb or mitigate the harmful effect of CPF, if any, using Nile tilapia as an aquatic animal model. We studied different physiological parameters, trying to obtain a clear vision for the involved processes.

Materials and methods

The experiments of the current study were approved by the Faculty of Agriculture, Kafrelsheikh University, Egypt.

Diet preparation

The basal diet was formulated to contain 30.9% crude protein and 7.1% ether extract. Two sets of diets were formulated by supplementing the basal diet with VC at 0.8 mg/kg of diet (l-ascorbic acid, Sigma-Aldrich) (Table 1). All ingredients were ground and mixed by the laboratory meat grinder, and finally, pellets with a plate of 2-mm die were produced. The formulated diets were dried in an oven at 50 °C until their moisture level reached less than 8%, then left to cool down, and preserved at 4 °C. The actual concentration of VC in the formulated diet was 0.62 mg/kg, which was in agreement with the recommendation of Barros et al. (2014) and Falcon et al. (2007). VC analysis was done using high-performance liquid chromatography (HPLC), according to AOAC (1998).

Table 1 Basal diet and proximate chemical composition (on a dry matter basis)
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Experimental design and procedure

Fish were collected from a commercial farm and transferred to the Fish Nutrition Laboratory, Baltim Research Station, National Institute of Oceanography and Fisheries (NIOF), Egypt. Following the acclimatization period (1 week, fed with basal diet), Nile tilapia (31.35 ± 0.25 g) were allocated into 12 glass aquaria (60 L) (10 fish per aquarium). The aquaria were distributed randomly into four groups (3 aquaria each) where the first and second groups were fed the basal diet without exposure to waterborne CPF (97% purity, Sigma-Aldrich), named control CPF, respectively, while the third and fourth groups were fed dietary VC without and with exposure to waterborne CPF, named VC and CPF/VC, respectively. The glass aquaria were continuously supplied with aeration, and fish were reared under natural day and dark cycle (approximately 12:12 h). The aquaria were siphoned daily to collect feces and remaining feed. The trial was done under laboratory conditions where the average values for water dissolved oxygen (6.5 ± 0.5 mg/L), temperature (25.1 ± 1.4 °C), total ammonia (0.23 ± 0.03 mg/L), and pH (7.1 ± 0.8) were registered and showed optimum levels for tilapia rearing.

For the waterborne CPF exposure, a stock of the CPF solution at a concentration of 15 μg/L was prepared and added to the CPF-exposed groups’ aquaria. The investigated level of CPF in the present study was determined by following Oruc (2012). The inclusion of CPF solution was added daily to the rearing water to keep the final concentration at 15 μg/L. In the beginning, the whole water in each aquarium was filled from the stock CPF solution (60 L/aquarium). Afterward, one-third of the aquarium water was replaced daily with the previously prepared CPF solution. Fish were fed with the prepared diets at a rate of 2% of their total biomass twice daily for 30 days. The concentration of CPF was regularly checked, which recorded a 14.58 ± 0.24 μg/L, according to Zalat et al. (2013). The fish were reared at 25.1 ± 1.4 °C, pH 7.1 ± 0.8, dissolved oxygen 6.5 ± 0.5 mg/L, and total ammonia 0.23 ± 0.03 mg/L.

Sampling

Considering the initial weight (IW) and the final weight (FW), the following equations were applied to calculate the growth performance of Nile tilapia after 30 days of CPF exposure:

Specific growth rate (SGR %) = 100 × (ln FW − ln IW)/30 days; Weight gain (%) = ((FW-IW)/IW)×100; Feed conversion ratio (FCR) = feed given/weight gain ; Survival rate (%) = (number at the end/number at the beginning) ×100.

Blood samples were collected after 15 and 30 days of CPF exposure for hematological and biochemical analysis. However, the liver, gills, and intestine tissues were collected at the end of the trial (30 days) for histological and gene expression tests. After 15 and 30 days, the fish were anaesthetized using 150 mg/L MS222 (Argent Laboratories, Redmond, Washington). Blood samples were collected from 3 fish per aquarium using EDTA-coated vials and non-coated vials for serum separation. Blood samples were left for 2 h to clot, then centrifuged at 3500×g for 15 min at 4 °C. The collected serum was kept at − 20 °C for further analysis. Liver, gills, and intestine tissues were collected from 3 fish per aquarium using Bouin’s solution for 18 h, then transferred to alcohol 70% for histopathological study. Besides, pieces of the liver tissue were preserved and immediately kept in liquid nitrogen then frozen at − 80 °C for gene expression analysis.

Blood analysis

Red blood cells (RBCs) and white blood cells (WBCs) were counted immediately with a hemocytometer using Natt and Herrick’s solution (Houston 1990). For differential leucocyte count, blood films were prepared and stained according to Lucky (1977), and cells were counted following the method by Jain (1986). The hemoglobin (Hb) concentration was determined using a spectrophotometer (Model RA 1000, Technicon Corporation, USA) at 540 nm (Blaxhall and Daisley 1973).

Serum total protein and albumin were determined according to Doumas et al. (1981) and Doumas et al. (1972). Globulin content was calculated mathematically. Activities of AST, ALP, and ALT were determined calorimetrically at wavelength 540 nm (Reitman and Frankel 1957). Serum creatinine, uric acid, urea, and bilirubin were determined following the protocol by Heinegård and Tiderström (1973) and Coulombe and Favreau (1963).

The phagocytic functional assay of leukocytes was analyzed according to Abo-Al-Ela et al. (2017) and Cai et al. (2004), which the phagocytic activity equals the percentages of leukocytes that engulfed bacteria, and phagocytic index equals the total number of bacterial cells phagocytized divided on the total number of phagocytic cells.

The lysozyme activity of sera was assayed, which the concentration of serum lysozyme (μg/mL) was obtained using a standard curve prepared from the lysozyme activity of a lyophilized hen egg-white according to Demers and Bayne (1997) and Abo-Al-Ela et al. (2017).

Gene expression

Total RNA was extracted from 50 mg of liver tissue using Trizol (iNtRON Biotechnology, Inc., Korea) according to the manufacturer’s manual. The total RNA of the samples was first extracted using Trizol (iNtRON Biotechnology). The quality and quantity of the extracted RNA were assessed by Nanodrop (Uv-Vis spectrophotometer Q5000/Quawell, USA). Then, complementary DNA (cDNA) was synthesized using the SensiFAST™ cDNA synthesis kit (Bioline, UK) according to the manufacturer’s protocol.

Gene-specific primer sequences were used for the antioxidant genes catalase (cat) and glutathione peroxidase (gpx), heat shock protein 70 (hsp70), caspase-3 (casp3), the immune-related genes: interleukin 1β (il1β), interleukin 8 (il8), and interferon-gamma (ifnγ) genes, and β-actin as a housekeeping gene (Table 2). The quantitative real-time PCR (Stratagene MX3000P) was used for gene expression.

Table 2 Primers used for real-time PCR analysis
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SYBR green was used to quantify the gene expression using qRT-PCR (SensiFast SYBR Lo-Rox kit, Bioline). The thermocycling conditions for the reaction were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 min at 60 °C, and finally 5 min at 85 °C (except for ifnγ, which was at 61 °C) for 1 min. Gene expression was corrected using the β-actin as a reference gene. After verification of PCR efficiency to be close to 100%, the gene expression data were calculated using the 2−ΔΔCt method according to Livak and Schmittgen (2001).

Histopathology

The histopathological examination was adopted according to Abumandour and Gewaily (2016). Nine fish were randomly selected from each treatment (3 fish per aquarium). After deep anesthesia using 40% ethyl alcohol, the abdomen was dissected to obtain samples from the gills, intestine, and liver. The collected tissues were cut into pieces of approximately 0.5 cm3 and fixed in Bouin’s solution for 18–24 h. The fixed samples were then dehydrated in ascending grades of alcohol (70%, 80%, 90%, and 100%, respectively), cleared with xylene, and embedded in paraffin wax. Sections with a thickness of 5 μm were obtained with Leica rotatory microtome (RM 20352035; Leica Microsystems, Wetzlar, Germany) and stained with hematoxylin and eosin. The tissue sections were examined using a BX50/BXFLA microscope (Olympus, Tokyo, Japan).

Statistical analysis

Data were presented as means with the standard error of means, and they were analyzed by a one-way ANOVA method using SPSS 22.0 (SPSS version 22, SPSS Inc., Il, USA). Duncan’s multiple range test was used to determine differences among treatments with a significance set at P < 0.05.

Results

Growth performance and feed utilization

The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) were significantly increased in the VC group compared to the CPF group (P<0.05) without significant differences with the other groups (P>0.05). CPF significantly decreased Nile tilapia’s growth performances, where the WG was decreased by approximately 6% compared to control (Table 3). However, VC and CPF/VC groups showed relatively increased WG by 11–16.5% and 17–23% compared to the control and CPF groups, respectively. It is noticed that the SGR recorded the highest values in VC and CPF/VC groups, while the lowest value was in the CPF group. Feed intake (FI) was significantly increased in all treated groups compared to control. However, the feed conversion ratio (FCR) of the CPF group achieved the highest significant record. CPF markedly decreased the survival rate of Nile tilapia by 10% than those in control. However, VC greatly rescued fish’s survivability, which achieved an increase of more than 3% in the VC group compared to control.

Table 3 Growth performance of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days
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Blood parameters

The Hb concentration showed a remarkable decline in the CPF and CPF/VC groups, particularly after 30 days post-exposure (Table 4). However, the VC group recorded high values of Hb on the 15th and 30th days of treatment. There was no great change in RBC count, except for some increases in the VC group. The packed cell volume (PCV) displayed decreases in the CPF and CPF/VC groups but increased in the VC group. Slight differences were recorded in the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) among groups, except for the CPF/VC group, which showed decreased levels on the 15th day, but returned to normal levels on the 30th day (Table 4). WBCs were lower in CPF and CPF/VC groups than in the other treated groups, primarily due to decreases in lymphocyte and monocyte population.

Table 4 Hematological parameters of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days
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The creatinine level was high in the CPF group, while it returned to a normal CPF/VC group (Table 5). In the VC group, creatinine was the lowest among other groups even than that of the control. Urea and bilirubin were high in the CPF group, but their levels were declined in the CPF/VC group and much lower values in the VC group. Globulin and albumin were higher in VC and CPF/VC groups than in CPF and control groups, reflected in the total protein levels. ALP, AST, and ALT showed substantial increases in the CPF group and the treatment duration; however, their levels were significantly improved in the CPF/VC group. ALP, AST, and ALT levels were low in the VC group but within the normal range.

Table 5 Blood biochemical parameters of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days
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VC induced increases in the lysozyme activity, while CPF reduced it (Table 6). However, VC succeeded in keeping lysozyme activity at a closer level of control. The same was observed in the phagocytic activity and phagocytic index, but phagocytic activity was low in the CPF/VC group on the 15th day of treatment; however, the activity was retained 30th day of treatment.

Table 6 Lysozyme and phagocytic activities of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days
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Gene expression

The expression of antioxidative genes (gpx and cat) in the liver of Nile tilapia was upregulated in the CPF, and CPF/VC groups, where the highest upregulation of gpx was in the VC group and cat was in the CPF/VC group (Fig. 1). In the CPF group, the expression of gpx was upregulated and the expression of cat was similar to the control.

Fig. 1
figure 1

Relative expression of antioxidative genes (catalase (cat) and glutathione peroxidase (gpx)) in the liver of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days. Values are expressed as mean ± SE from triplicate groups. Bars with different letters are significantly different from those of control group (P<0.05)

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The highest expression of hsp70 and casp3 was recorded in the CPF group. However, the expression of these genes was kept at levels close to the control in the VC and CPF/VC groups (Fig. 2). Regarding the expression of immune-related genes (ifnγ, il8, and il1β), they were significantly upregulated in the liver of CPF treated fish; however, VC markedly reduced their expression in the CPF/VC group but remained at levels higher than that of the control (Fig. 3).

Fig. 2
figure 2

Relative expression of (A) heat shock protein 70 (hsp70) (B) caspase 3 (casp3) genes in the liver of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days. Values are expressed as mean ± SE from triplicate groups. Bars with different letters are significantly different from those of control group (P<0.05)

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Fig. 3
figure 3

Relative expression of immune-related genes (ifnγ, il8, and il1β) in the liver of Nile tilapia exposed to chlorpyrifos (CPF), supplemented with or without dietary vitamin C (VC) for 30 days. Values are expressed as mean ± SE from triplicate groups. Bars with different letters are significantly different from those of control group (P<0.05)

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Histopathology

Gills

In the control and VC-treated groups, the gills appeared normal. The lining epithelium of primary and secondary filaments was normal, with the presence of mucous cells (Fig. 4 A and C). CPF resulted in histopathological alterations, which displayed congestion of afferent blood vessels and the degeneration of the primary filaments’ epithelial lining. Telangiectasia (dilated apical end of secondary filaments) was also prominent in the CPF group (Fig. 4B). In the VC group, gills showed an improvement of the degenerative changes induced by CPF, and also, the degeneration and telangiectasia were not detected (Fig. 4D).

Fig. 4
figure 4

Histomicrograph of Nile tilapia gills shows the histological structure of the control group (A) and other treated groups, CPF (B), VC (C), and CPF/VC (D). In A and C, the gills show normal histological structures including primary filaments (PF), secondary filaments (SF), and mucous cells (black arrow head) between the secondary filaments. (B) Telangiectasia (black arrow) in the secondary filaments, and congestion of blood vessels of primary filaments (labeled with C) are observed. (D) the gills show a relatively normal structure in addition to leukocytic infiltration (white arrow head) (H&E; bar = 100 μm)

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Intestine

The intestinal histology appeared normal, where the intestinal villi were lined by enterocytes (simple columnar epithelium), and submucosa, muscularis, and serosa showed a normal appearance in control (Fig. 5A) and CPF/VC (Fig. 5D) groups. The CPF group showed degeneration and sloughing of the apical part of the intestinal villi with lymphocytic infiltration (Fig. 5B). In the VC group, the intestinal villi were long, branched, and crowded (Fig. 5C).

Fig. 5
figure 5

Histomicrograph of Nile tilapia intestine shows the histological structure of the control group (A) and other treated groups, CPF (B), VC (C), and CPF/VC (D). In A and C, the intestinal villi (V), lamina propria sub mucosa (LP), tunica muscularis (M), and tunica serosa (S) show normal histological structures; in addition to an overcrowding and branching of the higher villi (C). (B) A degeneration and sloughing of the apical part of the intestinal villi epithelium (arrow) and leukocytic infiltration (arrow head) are observed. (D) The intestinal villi are intact and appeared normal (H&E; bar = 100 μm)

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Liver

In the control group, the hepatopancreas showed a cord-like appearance of polyhedral hepatocytes and was separated by endothelial cell–lined blood sinusoids. The hepatopancreas contained a normal exocrine pancreatic tissue, and the afferent portal vein was surrounded by the exocrine pancreatic cover (Fig. 6 A and C). The CPF group displayed a hepatotoxic effect in the form of aneurysm and congestion of hepatic vein and degeneration and vacuolation of hepatocytes with pyknotic nuclei (Fig. 6B). VC restored the normal structure of the hepatopancreas (hepatic parenchyma and the pancreatic cells). The melanomacrophages were prominent at the periphery of pancreatic tissue (Fig. 6D).

Fig. 6
figure 6

Histomicrograph of Nile tilapia hepatopancreas shows the histological structure of the control group (A) and other treated groups, CPF (B), VC (C), and CPF/VC (D). In A and C, hepatopancreas shows cord-like appearance of polyhedral hepatocyte (H) and pancreatic cells that make pancreatic cover around afferent hepatic vein (P). (B) A degeneration of hepatocytes and pyknosis of their nuclei, aneurysm, and congestion of hepatic vein are observed. (D) Hepatopancreas retained its normal structure either in the hepatic (H) or pancreatic (P) parts (H&E; bar = 100 μm)

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Discussion

The CPF group showed the highest FI and FCR, while the FBW, WG, and SGR recorded the lowest values among the other treated groups. FCR is usually low in young animals, while increases in older animals; however, young Nile tilapia were used in the current study. This may occur due to herbicides’ detrimental effect on the metabolism of nutrients (Dawood 2021). To illustrate, fish larvae exposed to CPF had underdeveloped guts and a reduced thyroid hormone level (T3) (Holzer et al. 2017). Other studies indicated that environmentally relevant CPF exposure modulated lipid metabolites and serine lipase activity in the trout brain (Greer et al. 2019). Besides, broken integrity of the gut barrier and altered microbiota was observed in CPF-exposed animals that led to the increased entry of lipopolysaccharide into the body, which ultimately resulted in low-grade inflammation and insulin resistance (Liang et al. 2019). The changes in gut microbiota composition included increases in opportunistic pathogens and short-chain fatty acid–producing bacteria (Fang et al. 2018), affecting health status and growth. In this study, VC significantly improved the growth performance (e.g., SGR and FBW) in both VC and CPF/VC groups with values more than that in control. This is consistent with the capacity of VC to increase SGR, WG, and feed efficiency in fish (El Basuini et al. 2017; Zhou et al. 2012). Similarly, Narra et al. (2015) stated that dietary vitamin C enhanced the growth performance of Clarias batrachus exposed to CPF toxicity.

The total leukocyte count and Hb concentration were low in CPF, and CPF/VC groups and CPF-exposed Labeo rohita (Ismail et al. 2018). The low Hb was attributed to the ability of CPF to decrease serum iron concentration (Goel et al. 2006). The elevated bilirubin in the CPF group likely resulted from Hb breakdown. Although the MCHC was not markedly changed among groups, there was a significant alteration between groups, indicating that VC and CPF do not affect the Hb inside cells. Thus, VC cannot restore the changes in blood cell as well as Hb levels; however, this might be explained by earlier reports indicating that VC could not modulate or has little effect on blood cell population and count (Abo-Al-Ela et al. 2017). However, pretreatment with VC could protect from the reduction in Hb in CPF-exposed animals (Goel et al. 2006). In the current study, the decrease in the total leukocytes was due to lower counts of the lymphocytes and monocytes. It has been found that CPF induces the overproduction of tumor necrosis factor-alpha (tnfα), which in turn mediates apoptosis of lymphocytes and monocytes (Dreschers et al. 2013; Navaei-Nigjeh et al. 2015). Another potential explanation might be that increased insulin resistance resulted in low glucose uptake by such cells’ deposit of sustainable high plasma glucose recorded during the CPF exposure (Narra et al. 2015), leading to their apoptosis (Liang et al. 2019; Ndonwi et al. 2020).

The high levels of creatinine and urea in the CPF group indicate impaired kidney function (e.g., low clearance) or liver oxidative stress because urea is formed by deamination of amino acids in the liver. Fish fed dietary VC showed improved or near-normal creatinine and urea levels, indicating the protective role of VC against CPF-induced renal damage.

ALP, AST, and ALT were increased in the CPF group, and this was observed all over the exposure (30 days). The liver enzymes (AST and ALT) are involved in the protein metabolism; their high levels may be associated with the availability of more amino acids (e.g., tissue breakdown) or inflammation or hepatocellular damage (Sarma et al. 2009). Oxidative stress is another possible cause of tissue damage during intoxication with CPF, indicated by low activities of catalase, glutathione peroxidase, and glutathione (Mokhbatly et al. 2020; Yonar 2018; Zahran et al. 2018). A high level of ALP suggests the stimulation of a metabolic reaction to neutralize the deleterious effects of CPF (El-Nahhal et al. 2020). Similarly, the pesticide endosulfan reduced the enzymatic activities of various metabolic pathways in the spotted murrel, Channa punctatus (Sarma et al. 2009). Our findings showed that the activities of these enzymes were restored to normal levels in fish fed with VC.

Gamma-globulins constitute the major fraction of the globulin of total serum protein. Immunoglobulins are the most significant gamma-globulins (Abdel-Mageid et al. 2020). Globulin fraction of the blood was high in VC and CPF/VC groups, indicating an active immune response. This supports the notion of VC acting on leukocyte function but not population (Abo-Al-Ela et al. 2017).

Lysozyme is an enzyme present in different body fluids, such as blood, mucus secretion and egg whites, and other parts of eukaryotic organisms. It is a hydrolytic enzyme that cleaves the cell wall of Gram-positive and some Gram-negative bacteria (McKenzie and White 1991). Phagocytosis, a critical biological activity, is modulated by changes in the physiological status. These changes are reflected in the phagocytic activity and phagocytic index of phagocytic cells (Platt and Fineran 2015). CPF reduced the capacity and ability of phagocytic cells to engulf foreign bodies, consistent with previous studies (Girón-Pérez et al. 2006), possibly because of its effect on cells’ sensitivity to recognize foreign materials. VC is a powerful molecule that can potentiate the immune system, including lysozyme and phagocytic activities (Abo-Al-Ela 2019; Abo-Al-Ela et al. 2017; Dawood et al. 2018). It was noted that VC could mitigate the harmful effects of CPF on the lysozyme activity and phagocytic function.

CPF induces a pro-inflammatory response (Liang et al. 2019), oxidative stress (Dawood et al. 2020; Mokhbatly et al. 2020), and genotoxicity (Ali et al. 2008), and modulates the expression of the involved genes. Immunity and oxidative stress are closely linked and modulate each other (Abdel-Mageid et al. 2020). Our results indicated an upregulation of the antioxidative genes gpx and cat, the oxidative related genes hsp70 and casp3, and the immune-related genes ifnγ, il8, and il1β in the liver of Nile tilapia. CPF-induced reactive oxygen species regulate immune response by triggering the antigen-presenting ability of head kidney in common carp (Zhang et al. 2017). In a previous study, the changes in the expression of some of these genes are accompanied by an increase in the antioxidant enzyme activity (i.e., malondialdehyde and glutathione) and a significant decrease in catalase, glutathione S-transferase, and superoxide dismutase in the liver and gills of fish exposed to CPF for 14 or 15 days (Zahran et al. 2018; Zhang et al. 2017). Besides, continuous expression of cytokines has an immunotoxic effect and could badly affect the immune function (Abo-Al-Ela 2018). The microRNA machinery regulates the physiological response in any given organism (Abo-Al-Ela and Burgos-Aceves 2020). CPF might have an indirect effect on gene expression through modulating microRNA machinery, which induced oxidative stress and modulated the miR-19a-AMPK axis in common carp (Zhang et al. 2019). The induction of the antioxidative and detoxification systems may respond to detoxify CPF; however, VC as an antioxidative molecule showed relative expression values to the control (Tripathi and Shasmal 2010). In similar supporting findings, VC could adjust the expression of genes involved in the antioxidant function of Nile tilapia exposed to toxic concentrations of cadmium (El-Sayed et al. 2016). Of note, the survivability of fish was decreased in the CPF group, presumably because of its immunotoxic and oxidative stress that could decrease fish’s ability to cope with biotic and abiotic stress.

The histoarchitecture of gills, intestine, and hepatopancreas presented altered structures. The gill is an important organ for respiration and osmoregulation and allows the entrance of toxicants into the body (Zahran et al. 2018). Degenerative changes and telangiectasia were prominent in the CPF-exposed group that indicate severe irritation of pesticides, as evidenced in previous studies (Banaee et al. 2013; Zahran et al. 2018). This might be linked to a significant increase of acid DNase activity in gills of mussel Mytilus galloprovincialis exposed to high concentrations CPF (10 and 100 μg/L) (Kovačić and Medić 2016). These results indicate a high DNA metabolism rate to degrade the apoptotic and damaged DNA (Evans and Aguilera 2003). A similar alteration was detected in the intestine with lymphocytic infiltration, possibly because of the opportunistic microorganisms present in the intestine lumen. The sloughing of the apical part of the intestinal villi may be attributed to the irritation, and the inflammatory effect resulted from CPF (Dawood et al. 2020, Mokhbatly et al. 2020) or CPF-induced reactive oxygen species production (Zhang et al. 2017).

The liver is the primary detoxification organ and is rapidly affected by xenobiotics exposure (Banaee 2020; El-Sayed et al. 2016; Zahran et al. 2018). Aneurysm and congestion of the hepatic vein and degeneration, nuclear pyknosis, and vacuolation of the hepatocytes were observed in the CPF group, similar to observation by Zahran et al. (2018) in Nile tilapia exposed for 14 days. Generally, the destructive action or necrosis is markedly associated with oxidative stress (e.g., reactive oxygen species) where lipid peroxidation is the main source of membrane bilayer susceptibility (Avci et al. 2005; Li et al. 2000).

Overall, VC significantly improved the histoarchitecture of the studied tissues and increased the melanomacrophages centers in the hepatopancreas, which indicate enhanced immunity (Abo-Al-Ela 2019; Abo-Al-Ela et al. 2017). Similar findings indicated the protective role of VC in fish exposed to different pesticides (El-Sayed et al. 2016; Sarma et al. 2009).

The acute toxicity of CPF is usually considered moderate. Despite this, the environmental exposures, particularly continuously and at levels below that acute cause toxicity, could result in health concerns and developmental disorders (Gao et al. 2017; McClelland et al. 2018). Food additives are the best potential tool that promotes immunity and improves physiological function in aquaculture (Ahmadifar et al. 2020; El Basuini et al. 2021; Zaineldin et al. 2020), and sustainable supplementation of aquatic organisms with beneficial elements, particularly VC, would help such animals to cope with bad environmental conditions and contaminations (Dawood and Koshio 2018).

Conclusion

In conclusion, we found that chlorpyrifos causes immunotoxicity, hematological and biochemical disturbances, and damage to tissues such as gills, intestine, and liver, which makes fish exposed to chlorpyrifos have a low resistance to external biotic stresses, and could present a higher susceptibility to infection. These findings should be addressed concerning pesticide safety evaluations in future studies. Our results demonstrate that vitamin C treatment ameliorated the CPF-induced toxicity in Nile tilapia.