
==== Front
Comp Immunol Rep
Comp Immunol Rep
Comparative Immunology Reports
2950-3116
Elsevier

S2950-3116(24)00035-1
10.1016/j.cirep.2024.200168
200168
Article
Health status biomarkers and hemato-biochemical indices in Nile tilápia
Bavia Lorena a
da Silva Ana Paula a
Carneiro Milena Carvalho b
Kmecick Melyssa a
Pozzan Roberta a
Esquivel-Muelbert Juan c
Isaac Lourdes b
Prodocimo Maritana Mela maritana.mela@ufpr.br
a⁎
a Ecotoxicology and Molecular Biology Laboratory, Department of Cell Biology, Federal University of Paraná, Brazil
b Department of Immunology, Institute of Biomedical Science, University of de São Paulo, São Paulo, SP, Brazil
c School of Natural Sciences, Macquarie University, New South Wales, Australia
⁎ Corresponding author at: Department of Cell Biology, Sector of Life Sciences, Federal University of Paraná, Cel. Francisco H. dos Santos Avenue, 19031, Brazil. maritana.mela@ufpr.br
10 9 2024
12 2024
10 9 2024
7 2001681 8 2024
2 9 2024
7 9 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Highlights

• Blood analysis is a valuable tool to assess fish health.

• Baseline levels in Tilapia are relevant for environmental biomonitoring programs.

• Baseline parameters can facilitate data comparison after experimental conditions.

• Alternative pathways can be used as a biomarker after pollutant exposure.

• Acute and chronic exposure to pollutants alter blood analysis parameters.

Considering the aquatic environment, fish are notorious bioaccumulators of persistent pollutants, and that Oreochromis niloticus (Nile tilapia) is a species of great economic interest and an excellent model for ecotoxicological experimental studies. Because of this, establishing baseline reference parameters for this healthy animal is extremely valuable for comparison. Thus, our aim is to establish reference values for Nile tilapia for comparative purposes for: (i) functional activation of the alternative pathway of the complement system (ACH50), (ii) some serum biochemical parameters of liver, kidney and muscle tissue integrity, and (iii) hematological profile of Nile tilapia under controlled conditions. Juvenile specimens of O. niloticus were obtained from the fish farming and acclimatized for 15 days. Blood samples were collected without and with anticoagulants (heparin and EDTA) and submitted to hematological assays for leukogram and erythrogram evaluation. Serum was used for the alternative complement and biochemical assays. A mean of 137.6 U/mL was found for ACH50, 52.3 U/mL for alanine aminotransferase, 81.7 U/mL for aspartate aminotransferase, 30.3 U/mL for alkaline phosphatase, 4.3 g/dL for albumin, 8.4 mg/dL for urea, 1.9 mg/dL for uric acid, 3654 U/L for total creatine kinase, 1329.9 U/L for creatine kinase MB fraction, 260.2 U/L for lactic dehydrogenase, 227.2 mg/dL for glucose, 5.6 g/dL for total protein, 252.7 mg/dL for triglycerides, 169.5 md/dL for cholesterol, 19.1 mg/dL for high-density lipoprotein, and 12.5 mg/dL for low-density lipoprotein. For all hematological parameters we found a difference between the lack and presence of the anticoagulants, being the lack and heparin the conditions that presented more stability of the cell numbers along the time. Thus, in this study we establish baseline health parameters for Nile tilapia and it will be applied as an essential and comparative tool for monitoring fish health and physiological fitness at various experimental levels, such as bioassays and environmental biomonitoring.

Graphical abstract

Image, graphical abstract

Keywords

Health parameters
Alternative complement pathway
Serum measurements
Hematology
Teleost fish
Biomarkers
==== Body
pmcIntroduction

Due to their sensitivity to environmental contamination and their link with fish health status, blood biomarkers are of great interest for environmental risk assessment studies [1]. However, the lack of knowledge about the basal parameters can lead to data misinterpretation and false diagnostics. Therefore, the determination of reference values is very important for the integration of these biomarkers in ecotoxicological studies. Determining reference values may help to better understand the biological impact of contaminants during biomonitoring studies and improve data interpretation. Among the main baseline parameters for verifying the general health of fish specimens exposed to environmental adverse conditions are: the evaluation of the immune system by activity of the complement system; evaluation of serum biochemical parameters of tissue integrity and metabolism; and as well as the evaluation of hematological parameters.

Like in mammals, the immune system of teleost fish can be divided into innate and adaptive. The first comprises the first line of defense against pathogens and external agents, and the second is responsible for immunological specificity and memory [2]. The innate immune system is composed of several immune cells [macrophages, monocytes, mast cells, granulocytes and natural killer cells (NK)], soluble factors (lysozyme) including inflammatory mediators (cytokines), and the complement system which participates in both the innate and adaptive immune response. The adaptive immune system is composed of several cells such as monocytes, macrophages, dendritic cells, and in particular of helper T, cytotoxic T and B lymphocytes [3]. The Complement System is composed of more than 40 proteins present in plasma or on cell surfaces. These proteins can be activated by three pathways: (i) the classical pathway activated after the interaction between the C1q and the IgM or IgG bound to antigen; (ii) the alternative pathway activated spontaneously; (iii) the lectin pathway activated after the binding of collectins and ficolins to carbohydrates present on the surfaces of pathogens [2].

Great progress in the molecular identification and functional characterization of the components of the Nile tilapia (Oreochromis niloticus) complement system has been observed in the last decade [4]. Although functional assays are widely used to study the effects of nutritional factors on the complement system in fish, little is known about the effects of environmental factors on this system [2]. Fig. 1 illustrates the possible defense mechanisms of O. niloticus, including the activation of the complement system.Fig. 1 Immune system of Nile tilapia. The immune system response can be divided into innate and adaptive. The innate immunity comprises the first line of defense against pathogens and it is composed by physical natural barriers (e.g., tight junctions in the skin, epithelial and mucous membrane surfaces including nares, gills, and gut, and mucus itself); Natural Killer cells, granulocytes, macrophages, and monocytes; and innate humoral components (e.g., defensins, cytokines, lysozyme, and complement proteins). Adaptive immunity is highly specific and depends on immunoglobulins and cytokines (adaptive soluble components) and a cellular response involving T-dependent, T and B cells responses. It is important to mention that although the complement system is presented here as part of the innate response, it acts as a bridge linking the innate and adaptive responses, and may be activated in the adaptive response. This figure was created with BioRender.

Fig 1

Serum biochemical parameters in fish are considered sensitive markers of fish health and may detect early hazardous effects of external agents [5]. These biochemical indicators, such as the activities of various enzymes and metabolites, may uncover potential adverse effects on the liver, kidney, muscle, and metabolism functions. Thus, these are relevant parameters for testing in aquatic organisms that are in contact with contaminants. Transaminases, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), play a significant role in protein and amino acid metabolism [6]. Alkaline phosphatase (AP) is a hydrolase that removes the phosphate group from proteins and nucleotides and plays an important role in fish innate immunity [7]. Whereas lactate dehydrogenase (LDH) acts on energetic metabolism [8]. All these enzymes are markers of liver function. Creatinine and uric acid are indicators of glomerular filtration rate, and together with urea are used as biomarkers of kidney function [9]. In addition to liver and kidney biomarkers, there are indicators of muscle damage that may be associated with environmental conditions, such as creatine kinase (CK) [10]. Glucose, total protein, cholesterol, and triglyceride, are parameters often assessed to detect the health and nutritional status of fish [1]. Alterations in the glucose levels could potentially be associated with kidneys and liver damage, as well as nutritional deficiencies [11]. Changes in the level of serum total protein and albumin can be an indicator of liver damage [12]. Lipids are an important energy source and also play a significant role in signal transduction pathways and molecular recognition processes [13]. Therefore, changes in lipid metabolism could impair crucial pathways. Besides, cholesterol is also a precursor of steroid hormones [14].

Hematological analyses provide substantial information about the physiological aspects of animal welfare assessment, including the activation status of the immune system [15]. Hematological profiles are among the most important indicators of the toxic compounds in aquatic environments and have an important role in determining the physiological response of fish to environmental conditions [16]. Peripheral blood tests involve measurement of various red and white blood cells indices and are often supplemented by biochemical analyses. To use blood parameters as biomarkers, it is necessary to know their standard values and reference interval for a given fish species [17]. However, the ranges of normal values of the key hematological parameters are still undefined for some fish species, especially when using different types of anticoagulants and analysis times.

Fish are ubiquitous in almost all aquatic environments, comprising a critical component of the community exposed to contaminants. Furthermore, the physiology of fish is generally well-understood, allowing for targeted studies on internal levels of tissue contamination and early adverse effects. However, there is a considerable variation among their basic physiological features, and their response to environmental contaminants [18]. The Nile tilapia, is a fish native to Africa and the southwestern Middle East [2]. Due to its high nutritional qualities, quick development rate and furthermore responds promptly to environmental alterations [19], tilapia is considered an important species in ecotoxicology studies [20].

Considering the significance of the mentioned parameters, our aim is to establish the basal immunological, basal hematological profile and basal physiological parameters of O. niloticus to expand our analysis to experimental conditions that evaluate the effects of persistent organic pollutant exposure. Our objective gains strength when we realize that in the coming years, we will have fewer environments free of environmental pollutants, reinforcing the importance of acquiring baseline parameters for future comparisons. Fig. 2 summarizes all the parameters analyzed in this work, as well as their importance as a biomarker.Fig. 2 Blood biomarkers. Blood biomarkers are great candidates for assessing fish health globally. Blood biomarkers targeting hematological parameters reflect physiological characteristics dependent on iron (erythrocytes, hemoglobin, and hematocrit), blood coagulation (thrombocytes) and immunological status (immune cells and complement system). Furthermore, they can indicate the organ function and tissue integrity [liver: alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and lactate dehydrogenase (LDH); kidney: urea, uric acid, creatinine, and LDH; muscle: creatine kinase (CK) and LDH]. And they show the metabolic condition of the fish (total proteins, triglycerides, cholesterol, glucose). This figure was created with BioRender.

Fig 2

Material and methods

Experimental fish and acclimatization

Twenty juvenile specimens of O. niloticus were obtained from the Panamá fish farming (municipality of Paulo Lopes, Santa Catarina, Brazil) and transported to the laboratory of the Federal University of Paraná (UFPR). Before the experiment, the animals were distributed in 150 L tanks with a flow system containing a submerged pump and controlled water conditions (dissolved O2, temperature, pH, nitrite and ammonia). The filtration system was composed of quartzite as a biological filter, activated carbon and zeolite for the removal of nitrite and ammonia, including a Perlon™ type blanket for mechanical filtration of residues. The fish were acclimatized for 15 days before the collection of biological material. All experimental steps were carried out on the Ethics Committee on the Use of Animals of the Biological Sciences Sector, of the Federal University of Paraná (CEUA-BIO – UFPR/Curitiba/Brazil) at number 1527. At the end of the experiment, ten fish were used for complement system and biochemical analysis and the other half for hematological analysis.

Euthanasia and blood collection

The animals were anesthetized with MS 222 (0.12% ethil-ester-3-aminobenzoic acid in water, Sigma/Aldrich®), and total weight and length were measured (weight 179.4 g ± 31.4; length 17.1 cm ± 2.3). To obtain blood samples, fish were caught in a small scoop net and then quickly taken out from water held firmly on a bench with a cloth covering the head and blood samples from each fish were withdrawn from the caudal vein. To obtain the serum, the blood samples were drained using a 1 mL plastic syringe without anticoagulant. Afterward the collected blood remained at room temperature for one to two hours to clot, they were centrifuged for 5 min at 4 °C at 3000 × g, and stored at −80 °C until biochemical and complement assays. Serum samples were used for biochemical assays and for the hemolytic assay of the alternative pathway. For hematological assays, three blood collections were performed on the same fish, using a 1 mL syringe (with or without anticoagulant) for determination of the leukogram and erythrogram. The first collection was obtained without anticoagulant, the second with heparin (sodium heparin, 5,000 IU/mL, Cristália Produtos Químicos Farmacêuticos Ltda) and the third with EDTA (0.5 M, pH 8.0, Thermo Fisher Scientific). After collection, fish were sacrificed by medullary section and the sex confirmed by visual analysis.

Hemolytic functional activity mediated by complement alternative pathway

To functionally evaluate the activity of the alternative pathway of the complement system (ACH50), we used rabbit erythrocytes in sterile Alsever (100 mM dextrose, 90 mM NaCl, 20 mM sodium citrate) solution (CCSLabor). The erythrocytes were washed three times in HEPES-EGTA-Mg buffer (composition: 100 mM HEPES, 10 mM EGTA, 10 mM MgCl2, 150 mM NaCl, 0.1% gelatin, pH7.0) and centrifuged at 300 × g for 10 min at 4°C, and the cell concentration was adjusted to 3%, in the same buffer. A volume of 50 µL of the erythrocyte's suspension was added to 100 µL of O. niloticus serum, serially diluted in a 3/4 ratio. As a blank, or 0% hemolysis, 100 µL of HEPES-EGTA-Mg buffer was added to the red blood cell suspension. The tubes were incubated at 24 °C for 30 min. The reaction was stopped by adding 1 mL of ice-cold saline to each tube and then centrifuged at 300 × g for 7 min at 4 °C. The optical density (OD) was measured at 414 nm. The total hemolysis is determined by optical reading of the supernatant of the same red blood cell suspension added to 1100 µL of distilled water. The reciprocal of the serum dilution inducing 50% red blood cell lysis designated the ACH50 titer and the results are presented as ACH50 units per mL. The method was adapted from Sunyer et al. [21], Wu et al. [22], and Moreno-Indias et al. [23]. To calculate the activity, we use the following formula:

% lysis for each dilution (or Y) = (ODs - ODneg) / (OD 100% hemolysis - OD 0% hemolysis)

Where ODs = OD of sample supernatant, ODneg = OD of negative control of the sample (the same dilution), OD100% = OD of the lysis of the water, OD0% = OD of spontaneous lysis in the buffer. We constructed a curve for each individual with a percentage of lysis for each serum dilution from the same specimen. To construct the curve, the Y values were treated according to the Von Krogh transformation. Then, the Y / (1 - Y) values and the reciprocal of the serum dilutions were plotted on a log-log scale to obtain the ACH50 unit per mL. Serum dilutions resulting in more than 90% or less than 15% lysis will be excluded from the calculation.

Biochemical parameters

Serum samples were assayed for ALT (cat. K035), AST (cat. K034), AP (cat. K224), bilirubin (cat. K005), albumin (cat. K040), urea (cat. K047), creatinine (cat. K016), uric acid (cat. K139), CK (cat. K0160), creatine kinase MB fraction (CK-MB, cat. K069), LDH (cat. K014), glucose (cat. K082), total proteins (cat. K031), triglycerides (cat. K117), cholesterol (cat. K083), high density lipoprotein (HDL, cat. K071), low density lipoprotein (LDL, cat. K088), using commercially available enzymatic assay kits (Bioclin Quibasa, Química Básica Ltda. Belo Horizonte, M.G., Brazil) following the manufacturer's instructions. Biocontrol N (human normal levels, cat. K073) and biocontrol P (human pathological levels, cat. K074) from Bioclin Quibasa were used as internal controls in all biochemical assays as previously adapted by Bavia et al. [24]. To calculate the determination of the serum concentration of CK, CK-MB and LDH, we used a mathematical correction, because the control values were 4 × below the expected value, possibly due to the reduction in the assay volume (from tube to plate of 96 wells). However, the proportion between the ratio of values obtained for biocontrol N and biocontrol P remained constant for all tests and repetitions. In this way, we used as a correction factor the multiplication of the value obtained by the biocontrol N/ biocontrol P ratio with the value obtained for each of the parameters.

For the initial tests of the serum biochemical parameters, we followed the methodology used by Bavia et al. [24], performing the assay with the total volume of serum (as indicated by the manufacturer) concomitantly with the test of the reduced serum volume (10 or 5 µL). The same was done with the internal controls of normal levels (biocontrol N) and pathological levels (biocontrol P), which were provided by the manufacturer and have known reference values (as indicated in Table 1). All parameters presented values within those expected for controls, except for bilirubin, that presented negative results. Only the values with the reduced serum volume are presented in Table 1.Table 1 Biochemical parameters of Oreochromis niloticus.

Table 1Application	Concentration	
Assay	Mean ± SD	N control	Interval N control	P control	Interval P control	Unit	
Metabolism parameters - Liver function	
ALT	52.3 ± 51.0	17.51	15–25	80.2	68–128	U/mL	
AST	81.7 ± 65.2	27.3	18–34	82.7	84–158	U/mL	
AP	30.3 ± 18.0	106.6	102–152	209.3	202–302	U/mL	
Albumin	4.3 ± 0.5	3	3.1–3.9	4.1	4.2–5.2	g/dL	
Metabolism parameters - Kidney function	
Urea	8.4 ± 1.8	26.3	22–34	156.4	118–178	mg/dL	
Creatinine	Nd	2.8	1.4–2.1	7.2	4–6	mg/dL	
Uric acid	1.9 ± 1.0	3.9	3.5–4.3	9	8.4–10.2	mg/dL	
Metabolism parameters - Muscle function	
CK-NAC	3654* ± 4097	107*	70–112	475*	342–514	U/L	
CK-MB	1329.9* ± 1388	15.9*	16–30	40.4*	53–79	U/L	
Metabolism parameters - Liver, kidney, and muscle function	
LDH	260.2* ± 287.8	214.6*	213–331	474*	645–967	U/L	
Metabolism parameters - General	
Glucose	227.2 ± 61.6	66.4	62–76	273.1	251–307	mg/dL	
Total proteins	5.6 ± 0.7	6.5	5–6.2	8.1	6.9–8.5	g/dL	
Triglycerides	252.7 ± 159.4	82.5	57–71	411	373–465	mg/dL	
Cholesterol	169.5 ± 42.2	134.9	115–141	270.9	238–290	mg/dL	
HDL	19.1 ± 6.0	23.5	25–37	99.2	69–103	mg/dL	
LDL	12.5 ± 9.4	58.6	53–79	30.5	29–43	mg/dL	
Abbreviations: ALT (alanine aminotransferase), AST (aspartate aminotransferase), AP (alkaline phosphatase), CK-NAC (total creatine kinase), CK-MB (creatine kinase MB fraction), LDH (lactic dehydrogenase), HDL (high-density lipoprotein) e LDL (low-density lipoprotein). Nd (not detected). Note: The values presented in the table refer to assays with reduced volumes of serum. Serum from the same fish was used for all tests. * These values have been mathematically corrected.

Hematological profile

Hematological analyses were carried with the anticoagulants (10% ethylenediaminetetraacetate acid EDTA or Heparin 100 UI) and without anticoagulants, for comparison. Hemoglobin (Hb) estimation was done by using a digital EasyLife Hb meter [25]. The values were expressed in g/dL. The microhematocrit method was used to determine the hematocrit (Ht). Red blood cell (RBC), white blood cell (WBC) and thrombocyte counts were measured under light microscope with an improved Neubauer hemocytometer [26]. The blood was used to make blood extensions that were stained with MayGrunwald/Giemsa/Wright-MGGW for differential leukocyte counts [27]. The derived hematological indices of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using standard formulae as described by Ranzani-Paiva et al. [27]:

MCV =(Ht ÷ RBC in millions) × 10 µm3

MCH =(Hb in g ÷ RBC in millions) × 10 pg

MCHC =(Hb ÷ Ht) × 100 g per 100 mL

Data analysis

Normal distributions of the continuous variables were tested by Kolmogorov-Smirnov method. Difference between male and female in serum concentration of the alternative complement activity, serum biochemical assays and hematological profile were compared by t test not paired (parametric data) or Mann Whitney test (non parametric data). In addition, the different conditions of blood collection (without anticoagulant, heparin and EDTA) and the different time of analysis (0, 24 h, 48 h, 7 d and 14 d) were compared by ANOVA one way followed by Tukey's posttest (parametric data) or Kruskal-Wallis followed by Dunn's post test (non parametric data). Differences were considered significant when p ≤ 0.05 and all data were expressed as mean and standard deviation.

Results and discussion

In this study, we provided baseline health values for O. niloticus, under controlled conditions (e.g., temperature, food, and population density). Establishing a baseline value for fish, principally in reared conditions is problematic due to the many variables (especially environmental pollutants) that affect blood chemistry [5]. So, the determination of baseline parameters is of great importance for fish biology and pathology studies.

Functional alternative complement pathway assay

Exposure to certain pollutants, such as heavy metals, pesticides, and industrial chemicals, can lead to immunosuppression in fish. This means that the fish's immune system becomes weakened, making them more susceptible to infections and diseases. The hemolytic assay functionally evaluating the alternative pathway of the complement system is one of the important parameters for assessing the integrity of innate immunity [2].

From a total of ten serum samples, nine showed hemolytic activity. We found a mean of 137.6 U/mL ACH50 and no difference was found between male and female (Fig. 3). Since our protocol was based on two important studies, the value obtained is between to those described by Sunyer et al. [21] and Wu et al. [22], who used serum from Sparus aurata (170 U/mL) and of hybrid O. niloticus (∼70 U/mL), respectively. Regarding the ACH50 as a parameter evaluated after pollutant exposition, only two previous studies have shown a decrease of the alternative pathway activity after the Nile tilapia exposure to cadmium [22] and Acer 35 EC (lower concentration) [28], which reinforce the potential of alternative pathway activity assay as biomarker to pollutant contact. Furthermore, due to its sensitivity to environmental contaminants and its direct link with individual health status, the immune response is considered as an attractive marker for environmental biomonitoring which could help to better identify risks associated with an ecosystem contamination [29].Fig. 3 Hemolytic assay mediated by complement alternative pathway in Nile tilapia. Hemolytic activity mediated by the alternative pathway was used to evaluate the functional state of the complement system in the serum from ten Nile tilapia [male (n=5) and female (n=5)]. Data are presented as mean and standard deviation. Reference values from [21] and [22] are indicated as red and blue dot lines, respectively. ACH50 assay: functional assay that evaluates the alternative complement pathway through hemolytic activity in which the volume of serum that provides 50% lysis of rabbit red blood cells is found.

Fig 3

Biochemical parameters

Through biochemical assays it was possible to evaluate different parameters related to tissue integrity and metabolism (Table 1). The activity of liver function (ALT, AST, AP, bilirubin and albumin), renal function (urea, creatinine and uric acid), muscle function [CK (total creatine kinase), CK-MB (creatine kinase MB fraction)], global tissue injury (LDH) and metabolic parameters (glucose, total proteins and lipidemia) was evaluated in fish serum. Our proposal was to integrate this part of the serum assessment with other analyzes performed on tissues and use these results as baseline physiological parameters for further studies.

The serum biochemical parameters of Nile tilapia are presented in Table 1. No significant difference between sexes (male and female) for all the serum biochemical parameters were found, so the results are presented together. For liver function and tissue integrity parameters, we found an average of 52.3 U/mL for ALT, 81.7 U/mL for AST, 30.3 U/mL for AP and 4.3 mg/dL for albumin. Approximate values were also found for Nile tilapia control groups by other authors, such as 33 U/mL for ALT and 93 U/mL for AST [30], 39.7 U/mL for AP [31] and 4.7 g/dL for albumin [32], respectively. It is noteworthy that exposition to insecticides [33,34], herbicides [35,36,37,38], fungicides [32,39], metals [40,41,42,43], and microplastics [44,45,46] increased the serum levels of ALT, AST and AP indicating the importance of these parameters as biomarkers of pollutant contact.

Regarding renal function and tissue integrity, we found an average of 8.4 mg/dL for urea and 1.9 mg/dL for uric acid. Approximate values were found for urea and uric acid, 6.10 mg/dL and 2.96 mg/dL, respectively [47]. For creatinine there was no detection, corroborating with Chen et al. [48] that also reported measurements below the detection limit. Increase in serum urea and creatinine has been observed in fish exposed to insecticide [49], herbicides [50,51], fungicide [50], and microplastic [46]. In addition, uric acid increase in serum has been observed in fish exposed to herbicide [38,46], fungicides [32,39], and microplastic [44] .

For the muscle function and tissue integrity parameters CK-NAC and CK-MB, an average of 3654 U/L and 1329.9 U/L were found, respectively. For LDH, a parameter indicative of global tissue injury, we found an average of 260.2 U/L. The result we obtained for CK-NAC is within the normal range established by Chen et al. [48], which ranges from 246 to 20299 U/L. This parameter appears to be very promising, since Nile tilapia exposed to water polluted with different types of heavy metals had an average serum value of 21828 U/L [52]. Increased serum LDH concentration is an important indicator of cardiac, hepatic, renal and pulmonary injury. We found an average of 260.2 U/L for LDH, which is within the values found in the literature: 20,19 and 26,1 U/L [53,54], 185 to 252 U/L [55], and 450 and 550 U/L [40,56]. CK and LDH activity decreased after metal cadmium exposition [57]. In addition, LDH is reduced when fish has been exposed to herbicide [58] and fungicide [32].

Finally, intermediary metabolism parameters in the serum, such as glucose, total proteins, and lipids are frequently analyzed to examine the nutritional status of fish. We found an average of 227.2 mg/dL for glucose, 5.6 g/dL for total proteins, 252.5 mg/dL for triglycerides, 169.5 mg/dL for total cholesterol. While lower values were found by other authors for glucose 90 mg/dL [32] and 80 mg/dL [48], approximate values were found for total protein 6.88 g/dL [59], triglycerides and total cholesterol, 225 and 183 mg/dL mg/dL, respectively [32]. For cholesterol fractions, we obtained an average of 19.1 mg/dL for HDL, which had a low concentration compared to that reported in the literature, 47.67 mg/dL [60] and 76.31 mg/dL [61]. And for LDL we found an average of 12.5 mg/dL while 61.68 mg/dL was reported by [61]. These lower levels for HDL e and LDL obtained for fish serum probably were due to the high specificity for human lipoproteins, since the kits employed are for human serum. Therefore, we plan to test the methods employed by Araujo et al. [60] and Michelato et al. [61] for cholesterol fractions. While glucose, triglycerides and cholesterol increased after the glyphosate herbicide [35,36,37] total protein decreased or has no alteration [35,37,62]. On the other hand, the levels of these same metabolites can be reduced after exposure to another type of herbicide, atrazine [63]. These results of opposite behavior illustrate that the modulation of serum levels of biochemical parameters depends on the type of pollutant. This reinforces the importance of using serum and blood biomarkers for a global interpretation of what occurs in the fish's body after exposure to environmental pollutants.

Hematological profile

These fish blood cells are broadly similar in function to their mammalian counterparts and are found in all other tissues and organs throughout the body. Alterations in blood-based indicators can reflect systemic changes or disturbances in homeostasis that can alert the physiological status of an individual fish or an entire population [15]. Furthermore, several hematological parameters have been used to monitor pollution induced stress in fish.

Sometimes hematological analysis cannot be performed immediately after blood sampling and blood must be transported or stored for some time. There is some literature indicating that short-term storage of blood under refrigeration or even at room temperature does not significantly affect the results of analysis, but time of storage is limited. Furthermore, different types of anticoagulants can often influence the value of hematological parameters, which is why we chose to test three conditions: without anticoagulant - with anticoagulant (heparin or EDTA), as indicated in Table 2.Table 2 Hematological profile of Oreochromis niloticus.

Table 2	Without anticoagulant	Heparin	EDTA	
Assay	All (n=10)	Male (n=5)	Female (n=5)	All (n=10)	Male (n=5)	Female (n=5)	All (n=10)	Male (n=5)	Female (n=5)	
Total leukocytes (x103/mm3)	8.1 ± 0.8ab	8.2 ± 1.0	7.9 ± 0.7	6.2 ± 0.7	6.5 ± 0.7	5.9 ± 0.5	5.6 ± 0.6	5.3 ± 0.7	5.8 ± 0.4	
Lymphocyte (%)	91.8 ± 1.2ab	91.7 ± 1.3	91.8 ± 1.4	86.2 ± 3.6	86.0 ± 3.1	86.3 ± 4.4	80.2 ± 4.5	79.4 ± 5.1	81.0 ± 4.1	
Monocyte (%)	1.2 ± 0.5	1.1 ± 0.4	1.2 ± 0.7	0.9 ± 0.5	0.9 ± 0.2	0.8 ± 0.7	0.7 ± 0.6	0.4 ± 0.2	0.9 ± 0.8	
Neutrophils (%)	6.2 ± 1.0	6.1 ± 1.1	6.2 ± 0.9	5.7 ± 0.8	5.3 ± 0.6	6.1 ± 0.8	5.2 ± 0.7	4.7 ± 0.5	5.8 ± 0.4	
Basophils (%)	0.2 ± 0.2	0.2 ± 0.3	0.1 ± 0.2	0.2 ± 0.3	0.3 ± 0.3	0.1 ± 0.2	0.2 ± 0.2	0.0 ± 0.0	0.3 ± 0.3	
Eosinophils (%)	0.8 ± 0.3	0.9 ± 0.2	0.7 ± 0.4	0.6 ± 0.4	0.5 ± 0.4	0.6 ± 0.4	0.5 ± 0.3	0.4 ± 0.2	0.5 ± 0.4	
Thrombocytes (x104/mm3)	5.8 ± 1.0ab	5.7 ± 1.3	6.0 ± 0.9	4.5 ± 1.1	4.7 ± 1.2	4.3 ± 1.1	3.8 ± 1.2	4.3 ± 0.9	3.3 ± 1.3	
Hematocrit (%)	22 ± 1.5b	21.4 ± 1.1	22.6 ± 1.7	21.8 ± 1.4b	21.2 ± 1.1	22.4 ± 1.5	16.3 ± 1.4	16.6 ± 1.1	16 ± 1.7	
Erythrocytes (x106/mm3)	1.7 ± 0.2	1.5 ± 0.1c	1.9 ± 0.1	1.5 ± 0.2	1.5 ± 0.1c	1.8 ± 0.1	1.6 ± 0.2	1.4 ± 0.1c	1.7 ± 0.1	
Hemoglobin (g/100mL)	5.7 ± 0.5ab	5.6 ± 0.6	5.9 ± 0.4	4.6 ± 0.5	4.6 ± 0.5	4.7 ± 0.6	3.7 ± 0.6	3.7 ± 0.7	3.8 ± 0.6	
MCV (µm3)	132.7 ± 16.4b	144.6 ± 9.2	120.8 ± 12.9	135.9 ± 18.9b	145.7 ± 18.9	126 ± 14.3	104.8 ± 19.0	116.9 ± 16.1	92.8 ± 13.9	
MCH (pg/cell)	34.5 ± 5.0b	37.6 ± 4.8c	31.4 ± 3.1	28.9 ± 5.3	31.4 ± 5.4	26.4 ± 4.2	24.1 ± 5.7	26.0 ± 6.4	22.1 ± 4.9	
MCHC (g/100mL)	26.1 ± 2.4a	26.0 ± 2.8	26.1 ± 2.3	21.2 ± 1.7	21.5 ± 1.9	20.9 ± 1.5	23.0 ± 4.0	22.1 ± 3.3	23.9 ± 4.7	
Abbreviations: MCV: mean corpuscular volume, HCM: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration. Data were presented as mean and standard deviation. Indication of statistical difference when p < 0.05: a vs. heparin, b vs. EDTA, c vs. female.

The statistical analysis used considered the difference between the types of anticoagulants, the difference between males and females, and when relevant the effect of time (0, 24 h, 48 h, 7 d and 15 d). It is important to mention that all hematological parameters were evaluated at time 0 (immediately after collection) and that only total leukocytes and erythrocytes were evaluated considering the effect of time due to their packaging in specific solutions. Initially, we will compare our averages with the averages obtained by Rios [64], since that was the protocol reference used. At the end, we will briefly discuss the findings overall.

For the total leukocyte count we found a significant difference between without anticoagulant and heparin and between without anticoagulant and EDTA, with the highest average obtained for the condition without anticoagulant being 8.1 × 10³/mm³, followed by heparin 6.2 × 10³/mm³ and then EDTA 5.6 × 10³/mm³ (Table 2). There was no difference between males and females. When this material was analyzed over time (0, 24 h, 48 h, 7 d and 14 d) simulating possible periods of transport of material from the field to the laboratory, we found the same significant difference between serum and heparin and between serum and EDTA after 24 h (Fig. 4A). Evaluating individually the effect of time on each condition, we found that the mean number of total leukocytes remained stable for up to 48 hours after collection, indicating stability of the results (Fig. 4B). Our results agree with the average (13.6 × 10³/mm³) obtained by Rios [64]. The total leukocyte count is an important parameter of pollutant exposure, since it is well documented that there are alterations in its number. In most studies, an increase in this number was found after exposure to insecticides [62,65,66,67], herbicides [50,58,62,68,69], fungicide [50], microplastics [45] . On the other hand, there was also observed a decrease after exposure to metals [42,43,56] and microplastics [44,46].Fig. 4 Total leukocyte counting of Nile tilapia. Three blood collection conditions were evaluated: without anticoagulant (indicated as without), with heparin and with EDTA. In A: analysis of the number of blood leukocytes considering the different blood collection conditions within the same packaging time (0, 24 h, 48 h, 7 d and 14 d). In B: analysis of the number of blood leukocytes considering the effect of time on the same blood collection condition. Data are presented as mean and standard deviation. Indication of statistical difference when p < 0.05: a vs. heparin, b vs. EDTA, d vs. 24 h, e vs. 48 h, f vs. 7 d, and g vs. 14 d.

Fig 4

When leukocytes were evaluated differentially, a significant difference was found between without anticoagulant and heparin and between without anticoagulant and EDTA only for the number of lymphocytes (Table 2). The average percentage for the condition without anticoagulants was 91.8%, followed by heparin 86.2% and then EDTA 80.2%. Our average leukocyte percentage was lower than the average of 98% described by Rios [64]. No significant difference between males and females was found. Nevertheless, conducting a differential blood count in fish is not very common in environmental diagnostics, because reference values are missing in some species. This reinforces the objective of our manuscript in establishing a reference range, since it is known that the percentage of each type of white blood cell presents great variability depending on the class of pollutant as presented in Table 3.Table 3 Effects of environmental contaminants on serum, hematological and immunological parameters of Oreochromis niloticus.

Table 3Pollutant	Concentration	Exposition (days)	Increase	Decrease	No change	Reference	
Insecticides	
Acer 35 EC (lambda-cyhalothrin and acetamiprid)	A-0.0017 mL/L	28	AP50			[28]	
A-0.017 mL/L	56		AP50		
Carbofuran	0.1; 0.5; 1 and 2 mg/L	2	WBC, glucose	Ht, Hb, RBC		[65]	
Chlorpyrifos	15 e 75 μg/L	3, 7 and 14	Ht, WBC, lymphocyte, neutrophil, MCV	RBC	Hb, MCH, MCHC	[66]	
Deltamethrin	5; 10 and 15 μg/L	4	Ht, Hb, RBC, WBC, lymphocyte, cholesterol, AP, AST, ALT,	Neutrophil, glucose, total protein, albumin,	Basophil, eosinophil, monocyte	[33]	
Endosulfan	12.795 μg/L	28	Neutrophil, eosinophil, monocyte, AP, ALT, AST	Hb, RBC, WBC, lymphocyte, basophil, total protein		[34]	
Fenitrothion	1.566 and 2.35 mg/L	4	ALT, creatinine, urea			[49]	
Lambda-cyhalothrin	0.48 μg/L	30	MCV	Ht, Hb, RBC, WBC, lymphocyte, neutrophil, monocyte, complement C3	Eosinophil, basophil	[72]	
Malathion	0.5 mg/L	30, 45 and 60	WBC, neutrophil	Ht, Hb, RBC, lymphocyte, monocyte, platelet, MCHC, total protein, albumin	MCV, MCH	[62]	
Sumithion	1.5; 2; 2.5 and 3 mg/L	4	WBC, glucose	Hb, RBC		[67]	
Herbicides	
Atrazine	3 and 6 mg/L	14 and 28	RBC, MCV, MCH, MCHC	Ht, Hb, glucose, total protein, triglyceride, cholesterol, urea	Albumin	[63]	
Atrazine	1.09 and 10.92 mg/L	1, 14 and 28	WBC, MCV, MCH, cholesterol, urea, creatinine, bilirubin	Ht, Hb, RBC, platelet, MCHC, total protein	AP, ALT, AST	[50]	
Atrazine	1.39 mg/L	60	Cholesterol, triglyceride	Ht, Hb, RBC, WBC, total protein, albumin	Glucose	[73]	
Glyphosate	5; 10; 20; 30 and 40 mg/L	14	Glucose, triglyceride, cholesterol, AP, LDH, ALT, AST	Ht, Hb, RBC, total protein, albumin		[35]	
Glyphosate	12.21 mg/L	30	WBC, platelet, MCV, MCH, MCHC	Ht, Hb, RBC	Lymphocyte	[68]	
Glyphosate	2 mg/L	30, 45 and 60	WBC, monocyte	Ht, Hb, RBC, lymphocyte, neutrophil, platelet, MCHC, albumin	MVC, MCH, total protein	[62]	
Glyphosate	16 mg/L	60	Triglyceride, cholesterol, LDL, ALT, AST,	HDL	Total protein, albumin	[37]	
Glyphosate	0.2, 0.8, 4, 16 mg/L	80	Cholesterol, triglyceride, ALT, AST	HDL	Total protein, LDL, albumin	[36]	
Pendimethalin	2.5 mg/L	4	WBC, MCH, glucose, total protein	Ht, MCV	RBC, albumin, AP, LDH, ALT, AST, creatinine, bilirubin	[69]	
Penoxsulam	0.9 and 1.79 mg/L	15 and 30	Ht, Hb, RBC, WBC, lymphocyte, neutrophil, monocyte, MCV, MCHC	AP, LDH, ALT, AST		[58]	
Penoxsulam	0.16, 0.31 mg/L	45	ALT, AST, urea, creatinine	Total protein, albumin		[51]	
Propanil	0.22; 0.44; 0.87 mg/L	14, 42 and 56	WBC, lymphocyte, neutrophil, glucose, total protein, triglyceride, AST, ALT	Ht, Hb, RBC, cholesterol	MCV, MCH, MCHC, basophil, eosinophil, monocyte	[80]	
Up Grade (Bentazone + MCPA)	2.91 mg/L	15	Lymphocyte, MCV, MCH, total protein, cholesterol, albumin, AP, AST, ALT, uric acid	Ht, Hb, RBC, monocyte, eosinophil, platelet	WBC, neutrophil, MCHC, glucose, creatinine	[38]	
Fungicide	
Benomyl	100; 200 and 400 μg/L	7, 21 and 35	Glucose, cholesterol, ALT, AST, creatinine, uric acid	Ht, RBC, LDH	Hb, MCV, MCH, MCHC, total protein, triglyceride, albumin, AP	[32]	
Copper sulfate	0.05 mg/L	4 and 21		Ht, Hb, RBC	WBC	[74]	
Copper sulfate	0.0069, 0.0696 and 0.6960	20 and 40		Ht, Hb, MCHC, glucose	RBC, MCV, MCH	[75]	
Copperoxychloride	32.3 mg/L	90	ALT, AST, creatinine, uric acid	Ht, Hb, WBC		[39]	
Mancozeb	0.3 and 3.03 mg/L	1, 14 and 28	WBC, cholesterol, urea, creatinine, bilirubin	Ht, Hb, RBC, platelet, total protein	MCV, MCH, MCHC, AP, ALT, AST	[50]	
Metals	
Cd	1 ppm	4	RBC		Ht	[98]	
Cd	1 mg/L	7 and 14	Glucose, total protein, albumin, ALT, AST	Cholesterol		[40]	
Cd	4.45 μM	5 and 15		AP50		[22]	
Cu	5 ppm	4			Ht, RBC	[98]	
Hg	0.02, 0.002, 0.0002 mg/L	3, 7, 10 and 14	MCV	RBC, thrombocyte	Ht, Hb, WBC, lymphocyte, neutrophil, monocyte, MCH, MCHC	[70]	
Hg	0.01 and 0.1 mg/L	7 and 14	Glucose, ALT, AST	Ht, Hb, RBC, WBC		[41]	
Pb(NO3)2	45 ppm	28		Hb, MCH, MCHC	Ht, RBC, WBC, lymphocyte, neutrophil, monocyte, MCV, total protein, albumin, ALT, AST	[81]	
SiMag	400 and 600 mg/Kg	56	ALT, AST, creatinine	Ht, Hb, WBC, total protein	RBC, triglyceride, cholesterol, HDL, LDL, albumin, urea	[42]	
Zn	5 ppm	4			Ht, RBC	[98]	
Zn	5 mg/L	7 and 14	Glucose, total protein, albumin, ALT, AST	Cholesterol		[40]	
Methyl mercury chloride	0.5, 1, 1.5, and 2 mg/kg	60	Glucose, triglyceride, cholesterol, AP, ALT, AST	Ht, Hb, RBC, WBC, lymphocyte, granulocyte, monocyte, total protein, albumin	MCV, MCHC	[43]	
Microplastics	
Microplastic	1; 10 and 100 mg/L	15	Lymphocyte, MCV, MCH, glucose, total protein, cholesterol, albumin, AP, ALT, AST, uric acid, creatinine	Ht, Hb, RBC, WBC, platelet, MCHC	Neutrophil, eosinophil, monocyte	[44]	
Microplastic	0.01; 0.1 and 1 mg/L	14	WBC, platelet, glucose, total protein, AP, AST, ALT	Ht, Hb, RBC		[45]	
Polyacrylamide microplastics	0.018; 0.03 and 0.09 g/L	28	Lymphocyte, monocyte, MCV, MCH, MCHC,total protein, AP, AST, ALT, urea, creatinine	Ht, Hb, RBC, WBC, neutrophil, eosinophil, Platelet, albumin		[46]	
Polyethylene microplastic	10 mg/L	15	Lymphocyte, MCV, MCH, cholesterol, AP, ALT	Hb, RBC, monocyte, neutrophil, eosinophil	Ht, WBC, platelet, MCHC, glucose, total protein, albumin, AST, creatinine, uric acid	[38]	
Polypropylene microplastic	100
and 500 µg/kg of b.w	30	WBC, lymphocyte, thrombocyte, cholesterol, AST, AST	Creatinine	Ht, Hb, RBC, MCV, MCHC, monocyte, neutrophil, glucose, triglyceride, total protein, albumin, AP,	[71]	
Nanoparticle	
Ag nanoparticles	4 mg/L	1, 2 and 4		Ht, Hb, RBC	MCV, MCH, MCHC	[76]	
Aluminum oxides Nanoparticles	10 mg/L	7	MCV, MCH	Hb, RBC, MCHC	Ht, MCH	[77]	
CuO nanoparticle	0.05 mg/L	4 and 21		Ht, Hb, RBC	WBC	[74]	
Iron Oxides Nanoparticles	10 mg/L	7	MCV, MCH	Ht, Hb, RBC		[77]	
Zn nanoparticle	90 mg/L	7, 14 and 28		RBC	MCV, MCH	[78]	
Zn particle	680 mg/L	7, 14 and 28		Ht, Hb, RBC, MCV	MCH	[78]	
Alpha iron oxide and gamma iron oxide	1 mg/L	30, 60 and 90	Glucose, AST, ALT, LDH	AP	Ht, Hb, RBC, WBC	[55]	
Abbreviations: Ht: hematocrit, Hb: hemoglobin, WBC: white blood cell, RBC: red blood cell, MCV: mean corpuscular volume, HCM: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, AP: alkaline phosphatase, ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactic dehydrogenase.

Fish thrombocytes are considered functionally analogous to platelets in terms of coagulation, but differ in their morphology [9]. For the thrombocytes analysis, there was a significant difference between serum and heparin and between without anticoagulant and EDTA (Table 2). As observed for total leukocytes, we found the highest average for the condition without anticoagulant 5.8 × 104/mm3, followed by heparin 4.5 × 104/mm3 and then EDTA 3.8 × 104/mm3. Our averages were similar to those presented by Rios [64], where tilapia from fee fishing presented 3.8 104/mm3, while tilapia intercropped with pigs presented an average of 4.4 104/mm3, indicating that the conditions of creation and maintenance of these fish must be considered. We did not find a significant difference between males and females for the number of thrombocytes. While a reduction in thrombocytes number was observed after metal exposition [70], an increase was reported after microplastic ingestion [71].

Hematocrit is the percentage by volume of red blood cells in the blood [9]. Regarding hematocrit, there was a significant difference between without anticoagulant and EDTA and heparin and EDTA, with the highest average for the condition without anticoagulant 22%, followed by heparin 21.8% and then EDTA with the lowest average 16.3% (Table 2). The values obtained for EDTA are below the average value of 28.6% found by Rios [64]. No significant difference between males and females was found. Exposition to different classes of pollutants, such as insecticides [62,65,72], herbicides [50,63,73], fungicides [32,50,74,75], metals [41,42,43], microplastic [44,45,46], and nanoparticles [74,76,77,78] induced a reduction in the hematocrit percentage.

Erythrocyte counts refer to the absolute number of red blood cells in a given volume of blood [9]. For erythrocytes [also called red blood cells (RBC)], we found no significant difference between blood collection conditions (means: 1.7 × 106/mm3 for without anticoagulant, 1.6 × 106/mm3 for heparin and 1.6 × 106/mm3 for EDTA) (Table 2 and Fig. 5A), however there was a significant difference between males and females for all conditions, with males having a lower average than females. Evaluating individually the effect of time on each condition, we found that the average number of erythrocytes remained stable for up to 48 h after collection (Fig. 5B), indicating stability of the results for all conditions. Our results presented a higher average than the average of 1.1 × 106/mm3 obtained by Rios [64]. As described to hematocrit, the number of erythrocytes decrease after exposition to insecticides [62,65,66,72], herbicides [35,50,73], fungicides [32,50,74], metals [41,43,70], microplastic [44,45,46], and nanoparticles [74,76,77,78].Fig. 5 Total erythrocyte counting of Nile tilapia. Three blood collection conditions were evaluated: without anticoagulant (indicated as without), with heparin and with EDTA. In A: analysis of the number of blood leukocytes considering the different blood collection conditions within the same packaging time (0, 24 h, 48 h, 7 d and 14 d). In B: analysis of the number of erythrocytes considering the effect of time on the same blood collection condition. Data are presented as mean and standard deviation. Indication of statistical difference when p < 0.05: a vs. heparin, b vs. EDTA, d vs. 24 h, e vs. 48 h, f vs. 7 d, and g vs. 14 d.

Fig 5

Hemoglobins are iron-based oxygen-carrying molecules that make up a large proportion of red blood cells, and they are particularly important in fish as they constitute an interface between the organism and the environment [79]. For hemoglobin concentration we found a significant difference between without anticoagulant and heparin and between without anticoagulant and EDTA, the highest hemoglobin concentration for the condition without anticoagulant 5.7 g/100 mL, followed by heparin 4.6 g/100 mL and then EDTA 3.7 g/100 mL (Table 2). Our averages are relatively close to the average of 5 g/100 mL described by Rios (2012) [64]. We found no significant difference between males and females. Hemoglobin is an important parameter to be evaluated after pollutant exposure since its concentration decrease for most the pollutant classes e its subtypes: insecticides [34,62,65,67,72], herbicides [35,38,50,80], fungicide [39,50,75], metal [[41], [42], [43],81], microplastics [38,[44], [45], [46]], and nanoparticles [74,[76], [77], [78]].

Hematimetric indices are the basis for the classification of anemias, and it can be calculated if the values of hematocrit, erythrocyte count and hemoglobin are known [82]. Regarding hematimetric indices, we evaluated MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and CMCH (medium corpuscular hemoglobin concentration) (Table 2). For MCV we found a significant difference between without anticoagulant (132.7 µm3) and EDTA (104.8 µm3), and heparin (135.9 µm3) and EDTA. For MCH there was a significant difference between without anticoagulant (34.5 pg/cell) and EDTA (24.1 pg/cell). Finally, for CMCH we found a significant difference between without anticoagulant (26.1 g/100 mL) and heparin (21.2 g/100 mL). We found no significant difference between males and females for any of the hematimetric indices. In general MCV increased after exposure to insecticides [66,72], herbicides [38,50,58,68] metal [70], microplastics [38,44,45,46], nanoparticles [77]. For MCH an important increase has been observed after herbicides [38,58,63,68,69], microplastics [38,44,46] and nanoparticles [77]. Finally, for CMCH no trend was evident since both increase and decrease was found after herbicides and microplastics exposure depending on the type of the pollutant (Table 3).

Considerations on the effect of the absence and presence of anticoagulants on hematological parameters

Anticoagulants are one of several extrinsic factors that can cause species-specific reactions in the blood of animals, which can alter the organism's hematological parameters and, thus, make it difficult to determine reference values and can generate irreproducible results [83]. As the use of anticoagulants is essential in fish blood samples, due to their rapid clotting potential, especially in stressed animals, research about the interference of different anticoagulant agents is important to choose anticoagulants that cause less changes and enable more reliable results on the hematological profile of these organisms [84]. Different studies have shown that the hematological characteristics of different fish species vary according to the anticoagulant used [85]. Although EDTA is more suitable for some fish species, as it maintains hematological values more stable over time [86,87] and/or because it presents less erythrocyte hemolysis and medium corpuscular fragility [88], for most fish species EDTA seems to have a greater impact on hematological values compared to other anticoagulants, especially on red blood cells. Hattingh [89] observed that EDTA caused hemolysis in fish species and always increased cell volume. Tavares-Dias et al. [90] found that EDTA reduced Ht and Hb values when compared to heparin. According to Witeska et al. [91], EDTA induced erythrocyte swelling, causing cell membrane disruption and hemolysis followed by gradual karyolysis. In addition, some studies have shown that EDTA causes immense osmotic fragility and, consequently, hemolysis of these cells, which, in turn, ends up increasing the hemoglobin content and decreasing the red blood cell count in fish blood samples treated with this anticoagulant [92,93,94]. Heparin does not seem to cause major interference with the volume of red blood cells and, therefore, does not cause hemolysis in these cells, which makes it considered a safer anticoagulant by some authors, although in some fish species it does not appear to be effective in preventing blood clotting [84,88,95]. Sodium citrate, in turn, appears to reduce the values of most hematological elements in some fish species, such as leukocyte, lymphocyte and erythrocyte counts, compared to EDTA and heparin [86,94,96].

In summary, for total leukocytes, lymphocytes and thrombocytes we found a gradual reduction in the means when comparing the condition without anticoagulants with the anticoagulants used. Weinert et al. [97] found opposite results, where they obtained a higher average for the parameters where EDTA was used, however in this work two different anesthetics were tested (benzocaine and eugenol, control without anesthetic), different from the one we used MS-222. On the other hand, for hematocrit, hemoglobin, MCV, HCM and CHCM, we also found a gradual reduction in the means when comparing the condition without anticoagulants with the anticoagulants tested. This is in consonance with reports in the literature indicating that EDTA used as an anticoagulant causes partial hemolysis as mentioned above [[92], [93], [94]] which may explain the gradual reduction observed from the condition without anticoagulant, heparin and then to EDTA.

Furthermore, no significant difference between male and female was found for all hematological parameters, except for erythrocytes. It is a positive finding because for the establishment of biomarkers it is interesting that this type of difference does not occur. Another interesting point to consider is that the blood was collected from the same fish.

Functional changes on the alternative pathway activity, blood parameters and serum analyses induced by environmental pollutants in Nile tilapia

The use of independent reference values based on the range of natural variability of biomarkers offers the possibility to distinguish natural modulation from significant modulation linked with a toxic impact. The alternative complement pathway activity, blood parameters and serum analyses has been evaluated as an indicator of the Nile tilapia physiological state under different conditions such as toxic stress (Table 3). These findings suggest that when tilapia is exposed to insecticides, herbicides, fungicides, metals, microplastics, and nanoparticles, in general some parameters respond in a similar way, for example: hematocrit, hemoglobin, and number of erythrocytes decrease in response to these different classes of pollutants (Fig. 6). On the other hand, the levels of ALT, AST and the number of leukocytes increase when exposed to these different classes of pollutants (Fig. 6). However, for the analysis of the alternative pathway of the complement system, we found only two current studies, with an insecticide and metal, which reduce the activity of this pathway, suppressing the defense system in fish (Fig. 6). Although the studies gathered here correspond to a fraction of the whole, and to highlight the key parameters and meta-analysis should be performed, this demonstrates the need to analyze a wide variety of serum and blood biochemical parameters, specially the effect of other classes of pollutants on the fish immune system and its impact on complement system.Fig. 6 The effect of pollutants exposure on complement system, serum biochemistry analysis and hematological parameters. Environmental pollutants such as insecticides, herbicides, fungicides, metals, microplastics, and nanoparticles pose serious risks to many aquatic organisms. Fish can absorb these contaminants from water, sediments, and the food they eat. When exposed to these sources of pollution, some parameters respond in a similar way. We observed an increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST) and white blood cells (WBC). On the other hand, we observed a decrease in red blood cells (RBC), hematocrit (HT) and hemoglobin (HB). The Alternative pathway of the complement system (ACH50) seems to cause an increase after the exposure to metals and insecticides. All these parameters have been shown to change in the presence of certain contaminants and may be used as candidate indicators of environmental stress or toxicity.

Fig 6

Final considerations

To limit the risk of false positive and false negative results, many authors suggest comparing biomonitoring results to reference values [17,99,100]. But unfortunately, these values are scarce and often difficult to interpret, as they are not emphasized in ecotoxicological studies. Our study provides important information about baseline levels on O. niloticus, as model species in ecotoxicological bioassays involving biomarkers of early effect. Moreover, it currently appears difficult to find reference sites free from any anthropogenic pressure for field studies. This supports the trend towards defining generic reference values.

Conclusion

In conclusion, our study represents the first step to determine useful baseline biochemical values for O. niloticus. This study was conducted in laboratory-controlled conditions, using fish raised from the same spawning period. Therefore, unwanted sources of variation such as the individual age, pathogen presence, food availability, or potential environmental pollution are one factor that may influence the physiological response of this species. In this way, future studies should combine this reference value approaches with active biomonitoring to facilitate obtaining data in multiple conditions.

CRediT authorship contribution statement

Lorena Bavia: Writing – original draft, Methodology, Formal analysis, Conceptualization. Ana Paula da Silva: Writing – original draft, Methodology, Data curation. Milena Carvalho Carneiro: Writing – original draft, Visualization, Software. Melyssa Kmecick: Writing – original draft, Methodology, Formal analysis. Roberta Pozzan: Writing – original draft, Methodology, Formal analysis. Juan Esquivel-Muelbert: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Data curation. Lourdes Isaac: Writing – review & editing, Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Maritana Mela Prodocimo: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Data curation, Conceptualization.

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.

Data availability

Data will be made available on request.

Acknowledgments

This work was funded by the National Council for Scientific and Technological Development - CNPq (Universal project number 406258/2021-0 ). We would especially like to thank Quibasa-Bioclin for providing all biochemical kits.
==== Refs
References

1 Chen H. Luo D. Application of haematology parameters for health management in fish farms Rev. Aquac. 15 2023 704 737 10.1111/raq.12753
2 Bavia L. Santiesteban-Lores L.E. Carneiro M.C. Prodocimo M.M. Advances in the complement system of a teleost fish, Oreochromis niloticus Fish Shellfish Immunol. 123 2022 61 74 10.1016/J.FSI.2022.02.013 35227880
3 Segner H. Wenger M. Möller A.M. Köllner B. Casanova-Nakayama A. Immunotoxic effects of environmental toxicants in fish - how to assess them? Environ. Sci. Pollut. Res. 19 2012 2465 2476 10.1007/s11356-012-0978-x
4 Chen M. Ding M. Li Y. Zhong X. Liu S. Guo Z. Yin X. Fu S. Ye J. The complement component 1 q (C1q) in Nile tilapia (Oreochromis niloticus): functional characterization in host defense against bacterial infection and effect on cytokine response in macrophages Dev. Comp. Immunol. 87 2018 98 108 10.1016/j.dci.2018.05.023 29890197
5 Baudou F.G. Eissa B.L. Ossana N.A. Mastrángelo M.M. Ferro J.P. Campos L.B. Ferrari L. First baseline for bioenergetic biomarkers in Cnesterodon decemmaculatus as test organism in ecotoxicological studies Ecotoxicol. Environ. Saf. 208 2021 10.1016/j.ecoenv.2020.111639
6 Kobayashi A. Suzuki Y. Sugai S. Specificity of transaminase activities in the prediction of drug-induced hepatotoxicity J. Toxicol. Sci. 45 2020 515 537 32879252
7 Lallès J.P. Biology, environmental and nutritional modulation of skin mucus alkaline phosphatase in fish: a review Fish Shellfish Immunol. 89 2019 179 186 10.1016/j.fsi.2019.03.053 30928666
8 Farhana A. Lappin S.L. Biochemistry, Lactate Dehydrogenase 2022 StatPearls https://www.ncbi.nlm.nih.gov/books/NBK557536/ (accessed November 12, 2023)
9 Shahjahan M. Islam M.J. Hossain M.T. Mishu M.A. Hasan J. Brown C. Blood biomarkers as diagnostic tools: An overview of climate-driven stress responses in fish Sci. Total Environ. 843 2022 10.1016/j.scitotenv.2022.156910
10 Rojas V. Morales-Lange B. Avendaño-Herrera R. Poblete-Morales M. Tapia-Cammas D. Guzmán F. Marshall S.H. Mercado L. Detection of muscle-specific creatine kinase expression as physiological indicator for Atlantic salmon (Salmo salar L) skeletal muscle damage Aquaculture 496 2018 66 72 10.1016/j.aquaculture.2018.07.006
11 Atli G. Ariyurek S.Y. Kanak E.G. Canli M. Alterations in the serum biomarkers belonging to different metabolic systems of fish (Oreochromis niloticus) after Cd and Pb exposures Environ. Toxicol. Pharmacol. 40 2015 508 515 10.1016/j.etap.2015.08.001 26310509
12 Javed M. Ahmad M.I. Usmani N. Ahmad M. Multiple biomarker responses (serum biochemistry, oxidative stress, genotoxicity and histopathology) in Channa punctatus exposed to heavy metal loaded waste water Sci. Rep. 7 2017 10.1038/s41598-017-01749-6
13 Sunshine H. Iruela-Arispe M.L. Membrane lipids and cell signaling Curr. Opin. Lipidol. 28 2017 408 413 10.1097/MOL.0000000000000443 28692598
14 Craig M. Yarrarapu S.N.S. Dimri M. Biochemistry, Cholesterol 2023 StatPearls
15 Kroon F. Streten C. Harries S. A protocol for identifying suitable biomarkers to assess fish health: a systematic review PLoS One 2017 12 10.1371/journal.pone.0174762
16 Fazio F. Fish hematology analysis as an important tool of aquaculture: a review Aquaculture 500 2019 237 242 10.1016/J.AQUACULTURE.2018.10.030
17 Barrick A. Marion J.M. Perrein-Ettajani H. Châtel A. Mouneyrac C. Baseline levels of biochemical biomarkers in the endobenthic ragworm Hediste diversicolor as useful tools in biological monitoring of estuaries under anthropogenic pressure Mar. Pollut. Bull. 129 2018 81 85 10.1016/j.marpolbul.2018.02.006 29680571
18 Kroon F.J. Kuhnert P.M. Henderson B.L. Wilkinson S.N. Kinsey-Henderson A. Abbott B. Brodie J.E. Turner R.D.R. River loads of suspended solids, nitrogen, phosphorus and herbicides delivered to the Great Barrier Reef lagoon Mar. Pollut. Bull. 65 2012 167 181 10.1016/j.marpolbul.2011.10.018 22154273
19 El-Sayed A.-F.M. Tilapia Culture 2019
20 R. Kour, S. Bhatia, K.K. Sharma, Nile Tilapia(Oreochromis niloticus) as a successful biological invader in Jammu (J&K) and its impacts on native ecosystem, 2014. http://www.ijims.com.
21 Sunyer J.O. Tort L. Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway Vet. Immunol. Immunopathol. 45 1995 333 345 7676614
22 Wu S.M. Shih M.J. Ho Y.C. Toxicological stress response and cadmium distribution in hybrid tilapia (Oreochromis sp.) upon cadmium exposure Comp. Biochem. Physiol. C Toxicol. Pharmacol. 145 2007 218 226 10.1016/j.cbpc.2006.12.003 17251063
23 Moreno-Indias I. Dodds A.W. Argüello A. Castro N. Sim R.B. The complement system of the goat: Haemolytic assays and isolation of major proteins BMC Vet. Res. 8 2012 10.1186/1746-6148-8-91
24 L. Bavia, Í.A. de Castro, S.M.G. Massironi, L. Isaac, Basal physiological parameters of two congenic mice strains: C5 deficient C57BL/6 and C5 sufficient A/J, Immunol. Lett. 159 (2014) 47–54. 10.1016/J.IMLET.2014.02.010.
25 Da Silva R.D. Rocha L.O. Fortes B.D.A. Vieira D. Fioravanti M.C.S. Parâmetros hematológicos e bioquímicos da tilápia-do-Nilo (Oreochromis niloticus L.) sob estresse por exposição ao ar Pesquisa Veterinária Brasileira 32 2012 99 107
26 Mgbenka B.O. Oluah N.S. Umeike I. Effect of Gammalin 20 (lindane) on differential white blood cell counts of the African catfish, Clarias albopunctatus Bull. Environ. Contam. Toxicol. 71 2003 248 254 10.1007/s00128-003-0157-3 14560374
27 M.J.T. Ranzani-Paiva, S.B. de Pádua, M. Tavares-Dias, M.I. Egami, Métodos para análise hematológica em peixes, EDUEM, 2013. 10.7476/9788576286530.
28 Guedegba N.L. Imorou Toko I. Ben Ammar I. François L. Oreins N. Palluel O. Mandiki S.N.M. Jauniaux T. Porcher J.M. Scippo M.L. Kestemont P. Chronic effects of a binary insecticide Acer 35 EC on Nile tilapia Oreochromis niloticus through a multi-biomarker approach Chemosphere 273 2021 128530 10.1016/J.CHEMOSPHERE.2020.128530
29 Bado-Nilles A. Jolly S. Porcher J.M. Palluel O. Geffard A. Gagnaire B. Betoulle S. Sanchez W. Applications in environmental risk assessment of leucocyte apoptosis, necrosis and respiratory burst analysis on the European bullhead, Cottus sp Environ. Pollut. 184 2014 9 17 10.1016/j.envpol.2013.07.049 24012786
30 Sutthi N. Thaimuangphol W. Rodmongkoldee M. Leelapatra W. Panase P. Growth performances, survival rate, and biochemical parameters of Nile tilapia (Oreochromis niloticus) reared in water treated with probiotic Comp. Clin. Path. 27 2018 597 603 10.1007/s00580-017-2633-x
31 Mohammady E.Y. Soaudy M.R. Mohamed A.E. EL-Erian M.M.A. Farag A. Badr A.M.M. Bassuony N.I. Ragaza J.A. El-Haroun E.R. Hassaan M.S. Can dietary phytogenic mixture improve performance for growth, digestive enzyme activity, blood parameters, and antioxidant and related gene expressions of Nile tilapia, Oreochromis niloticus? Anim. Feed Sci. Technol. 290 2022 10.1016/j.anifeedsci.2022.115369
32 Min E.Y. Kang J.C. Effect of waterborne benomyl on the hematological and antioxidant parameters of the Nile tilapia, Oreochromis niloticus Pestic. Biochem. Physiol. 92 2008 138 143 10.1016/j.pestbp.2008.07.007
33 El-Sayed Y.S. Saad T.T. El-Bahr S.M. Acute intoxication of deltamethrin in monosex Nile tilapia, Oreochromis niloticus with special reference to the clinical, biochemical and haematological effects Environ. Toxicol. Pharmacol. 24 2007 212 217 10.1016/j.etap.2007.05.006 21783813
34 Hussein M.M.A. Elsadaawy H.A. El-Murr A. Ahmed M.M. Bedawy A.M. Tukur H.A. Swelum A.A.A. Saadeldin I.M. Endosulfan toxicity in Nile tilapia (Oreochromis niloticus) and the use of lycopene as an ameliorative agent Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 224 2019 10.1016/j.cbpc.2019.108573
35 Acar Ü. İnanan B.E. Navruz F.Z. Yılmaz S. Alterations in blood parameters, DNA damage, oxidative stress and antioxidant enzymes and immune-related genes expression in Nile tilapia (Oreochromis niloticus) exposed to glyphosate-based herbicide Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 249 2021 10.1016/j.cbpc.2021.109147
36 Zheng T. Jia R. Cao L. Du J. Gu Z. He Q. Xu P. Yin G. Effects of chronic glyphosate exposure on antioxdative status, metabolism and immune response in tilapia (GIFT, Oreochromis niloticus) Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 239 2021 10.1016/j.cbpc.2020.108878
37 Zheng T. Jia R. Cao L. Du J. Gu Z. He Q. Xu P. Yin G. Alleviative effects of Ginkgo biloba extract on oxidative stress, inflammatory response and immune suppression induced by long-term glyphosate exposure in tilapia (Oreochromis niloticus) Aquaculture 546 2022 10.1016/j.aquaculture.2021.737325
38 Mohamed I.A. Soliman H.A.M. Hana M. Lee J.S. Sayed A.E.D.H. Toxicity of mixture of polyethylene microplastics and Up Grade® pesticide on Oreochromis niloticus juvenile: I. Hemato-biochemical and histopathological alterations Environ. Toxicol. Pharmacol. 101 2023 104213 10.1016/J.ETAP.2023.104213
39 Hassaan M.S. Goda A.M.A.S. Mahmoud S.A. Tayel S.I. Protective effect of dietary vitamin E against fungicide copperoxychloride stress on Nile tilapia, Oreochromis niloticus (L.) fingerlings Int. Aquat. Res. 6 2014 10.1007/s40071-014-0058-6
40 Firat Ö. Kargin F. Individual and combined effects of heavy metals on serum biochemistry of nile tilapia Oreochromis niloticus Arch. Environ. Contam. Toxicol. 58 2010 151 157 10.1007/s00244-009-9344-5 19488801
41 Cogun H.Y. Firat Ö. Firat Ö. Yüzereroǧlu T.A. Gök G. Kargin F. Kötemen Y. Protective effect of selenium against mercury-induced toxicity on hematological and biochemical parameters of Oreochromis niloticus J. Biochem. Mol. Toxicol. 26 2012 117 122 10.1002/jbt.20417 22162128
42 M.N. Alandiyjany, A.T.Y. Kishawy, A. Abdelfattah-Hassan, H. Eldoumani, S.T. Elazab, S.A.M. El-Mandrawy, A.A. Saleh, N.A. ElSawy, Y.A. Attia, A.H. Arisha, D. Ibrahim, Nano-silica and magnetized-silica mitigated lead toxicity: their efficacy on bioaccumulation risk, performance, and apoptotic targeted genes in Nile tilapia (Oreochromis niloticus), Aquatic Toxicol. 242 (2022). 10.1016/j.aquatox.2021.106054.
43 Alam R.T.M. Zeid E.H.A. Khalifa B.A. Hamed Arisha A. Reda R.M. Dietary exposure to methyl mercury chloride induces alterations in hematology, biochemical parameters, and mRNA expression of antioxidant enzymes and metallothionein in Nile tilapia Environ. Sci. Pollut. Res. 28 2021 31391 31402 10.1007/s11356-021-13014-5/Published
44 Hamed M. Soliman H.A.M. Osman A.G.M. Sayed A.E.D.H. Assessment the effect of exposure to microplastics in Nile tilapia (Oreochromis niloticus) early juvenile: I. Blood biomarkers Chemosphere 228 2019 345 350 10.1016/j.chemosphere.2019.04.153 31039541
45 Das B.C. Ramanan P A. Gorakh S.S. Pillai D. Vattiringal Jayadradhan R.K. Sub-chronic exposure of Oreochromis niloticus to environmentally relevant concentrations of smaller microplastics: accumulation and toxico-physiological responses J. Hazard. Mater. 458 2023 10.1016/j.jhazmat.2023.131916
46 Raza T. Rasool B. Asrar M. Manzoor M. Javed Z. Jabeen F. Younis T. Exploration of polyacrylamide microplastics and evaluation of their toxicity on multiple parameters of Oreochromis niloticus Saudi J. Biol. Sci. 30 2023 10.1016/j.sjbs.2022.103518
47 El-Hawarry W.N. Biochemical and non-specific immune parameters of healthy Nile tilapia (Oreochromis niloticus), Blue tilapia (Oreochromis aureus) and their interspecific hybrid (♂ O. aureus X ♀ O. niloticus) maintained in semi-intensive culture system Online J. Anim. Feed Res. 2 2012 84 88
48 Chen C.-Y. Wooster G.A. Getchell R.G. Bowser P.R. Timmons M.B. Blood chemistry of healthy, nephrocalcinosis-affected and ozone-treated tilapia in a recirculation system, with application of discriminant analysis Aquaculture 218 2003 89 102 www.elsevier.com/locate/aqua-online
49 Abu Zeid E.H. Khalil A.L.S.A. Effects of acute fenitrothion insecticide exposure on DNA damage and oxidative stress biomarkers and health of Nile tilapia fingerlings, Oreochromis niloticus L World J. Fish Mar. Sci. 6 2014 361 370 10.5829/idosi.wjfms.2014.06.04.85138
50 Kanu K.C. Okoboshi A.C. Otitoloju A.A. Haematological and biochemical toxicity in freshwater fish Clarias gariepinus and Oreochromis niloticus following pulse exposure to atrazine, mancozeb, chlorpyrifos, lambda-cyhalothrin, and their combination Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 270 2023 109643 10.1016/J.CBPC.2023.109643
51 Saber T.M. ElHady M. Ali H.A. Effect of dietary vitamin E on biochemical, oxidative stress and immunological parameters in Nile tilapia exposed to penoxsulam Afr. J. Aquat. Sci. 44 2019 237 245 10.2989/16085914.2019.1628699
52 Adham K.G. Ibrahim H.M. Hamed S.S. Saleh R.A. Blood chemistry of the Nile tilapia, Oreochromis niloticus (Linnaeus, 1757) under the impact of water pollution Aquat. Ecol. 36 2002 549 557 10.1023/A:1021137122046
53 Hamed H.S. Amen R.M. Elelemi A.H. Mahboub H.H. Elabd H. Abdelfattah A.M. Moniem H.A. El-Beltagy M.A. Alkafafy M. Yassin E.M.M. Ismail A.K. Effect of Dietary Moringa oleifera Leaves nanoparticles on growth performance, physiological, immunological responses, and liver antioxidant biomarkers in Nile tilapia (Oreochromis niloticus) against zinc oxide nanoparticles toxicity Fishes 7 2022 360 10.3390/FISHES7060360
54 Hamed H.S. El-Sayed Y.S. Antioxidant activities of Moringa oleifera leaf extract against pendimethalin-induced oxidative stress and genotoxicity in Nile tilapia, Oreochromis niloticus (L.) Fish Physiol. Biochem. 45 2019 71 82 10.1007/S10695-018-0535-8/FIGURES/2 29982916
55 Ates M. Demir V. Arslan Z. Kaya H. Yilmaz S. Camas M. Chronic exposure of tilapia (Oreochromis niloticus) to iron oxide nanoparticles: effects of particle morphology on accumulation, elimination, hematology and immune responses Aquatic Toxicol. 177 2016 22 32 10.1016/J.AQUATOX.2016.05.005
56 Fırat Ö. Cogun H.Y. Yüzereroğlu T.A. Gök G. Firat Ö. Kargin F. Kötemen Y. A comparative study on the effects of a pesticide (cypermethrin) and two metals (copper, lead) to serum biochemistry of Nile tilapia, Oreochromis niloticus Fish Physiol. Biochem. 37 2011 657 666 10.1007/s10695-011-9466-3 21229307
57 Almeida J.A. Diniz Y.S. Marques S.F.G. Faine L.A. Ribas B.O. Burneiko R.C. Novelli E.L.B. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination Environ. Int. 27 2002 673 679 10.1016/S0160-4120(01)00127-1 11934117
58 Galal A.A.A. Reda R.M. Abdel-Rahman Mohamed A. Influences of Chlorella vulgaris dietary supplementation on growth performance, hematology, immune response and disease resistance in Oreochromis niloticus exposed to sub-lethal concentrations of penoxsulam herbicide Fish Shellfish Immunol. 77 2018 445 456 10.1016/j.fsi.2018.04.011 29626668
59 Abdel-Tawwab M. Hamed H.S. Effect of bisphenol A toxicity on growth performance, biochemical variables, and oxidative stress biomarkers of Nile tilapia, Oreochromis niloticus (L.) J. Appl. Ichthyol. 34 2018 1117 1125 10.1111/jai.13763
60 de M. Araujo D. Pezzato A.C. Barros M.M. Pezzato L.E. Nakagome F.K. Hematologia de tilápias-do-nilo alimentadas com dietas com óleos vegetais e estimuladas pelo frio Hematology of Nile tilapia fed diets with vegetable oils and stimulated by cold Pesqui Agropecu Bras 46 2011 294 302
61 Michelato M. Furuya W.M. Graciano T.S. Vítor L. Vidal O. Xavier T.O. Batista De Moura L. Rossetto V. Furuya B. Digestible methionine + cystine requirement for Nile tilapia from 550 to 700 g Revista Brasileira de Zootecnia 42 2013 7 12 www.sbz.org.br
62 Hassan M.A. Hozien S.T. Abdel Wahab M.M. Hassan A.M. Ameliorative effect of selenium yeast supplementation on the physio-pathological impacts ofchronic exposure to glyphosate and or malathion in Oreochromis niloticus BMC Vet. Res. 18 2022 10.1186/s12917-022-03261-0
63 S.Y. Hussein, M.A. El-Nasser, S.M. Ahmed, Comparative Studies on the Effects of Herbicide Atrazine on Freshwater Fish Oreochromis niloticus and Chrysichthyes auratus at Assiut, Egypt, (1996).
64 Rios F.S. Hematologia de peixes Ribeiro C.A.de O dos Reis Filho H.S. Grötzner S.R. Técnicas e Métodos Para a Utilização Prática de Microscopia 1 ed. 2012 Guanabara-Koogan Rio de Janeiro 341 360
65 Barbieri E. Ruiz-Hidalgo K. Rezende K.F.O. Leonardo A.F.G. Sabino F.P. Effect of carbofuran pesticide in juvenil Oreochromis niloticus on toxicity, routine metabolism and hematological parameters Boletim Do Instituto de Pesca 43 2017 513 526 10.20950/1678-2305.2017v43n4p513
66 Zahran E. Risha E. Awadin W. Palić D. Acute exposure to chlorpyrifos induces reversible changes in health parameters of Nile tilapia (Oreochromis niloticus) Aquatic Toxicol. 197 2018 47 59 10.1016/j.aquatox.2018.02.001
67 Sharmin S. Islam M.T. Sadat M.A. Jannat R. Alam M.R. Shahjahan M. Sumithion induced structural erythrocyte alteration and damage to the liver and kidney of Nile tilapia Environ. Sci. Pollut. Res. 28 2021 36695 36706 10.1007/s11356-021-13263-4
68 Samanta P. Pal S. Mukherjee A.K. Senapati T. Jung J. Ghosh A.R. Assessment of adverse impacts of glyphosate-based herbicide, Excel Mera 71 by integrating multi-level biomarker responses in fishes Int. J. Environ. Sci. Technol. 16 2019 6291 6300 10.1007/s13762-018-2013-3
69 El-Sayed Y.S. Samak D.H. Abou-Ghanema I.Y. Soliman M.K. Physiological and oxidative stress biomarkers in the freshwater monosex Nile tilapia, Oreochromis niloticus L., exposed to pendimethalin-based herbicide Environ. Toxicol. 30 2015 430 438 10.1002/tox.21919 24293324
70 Ishikawa N.M. Ranzani-Paiva M.J.T. Lombardi J.V. Ferreira C.M. Hematological parameters in Nile Tilápia, Oreochromis niloticus exposed to sub-letal concentrations of mercury Braz. Arch. Biol. Technol. 50 2007 619 626 10.1590/S1516-89132007000400007
71 do Nascimento L.S. de Oliveira S.L. da Costa C.C. Aracati M.F. Rodrigues L.F. Charlie-Silva I. Conde G. Mansano C.F.M. Andreani D.I.K. de Andrade Belo M.A. Deleterious effects of polypropylene microplastic ingestion in Nile tilapia (Oreochromis niloticus) Bull. Environ. Contam. Toxicol. 111 2023 10.1007/S00128-023-03772-Y
72 Khalil S.R. Elhakim Y.A. Abd El-fattah A.H. Ragab Farag M. Abd El-Hameed N.E. EL-Murr A.E. Dual immunological and oxidative responses in Oreochromis niloticus fish exposed to lambda cyhalothrin and concurrently fed with Thyme powder (Thymus vulgaris L.): Stress and immune encoding gene expression Fish Shellfish Immunol. 100 2020 208 218 10.1016/j.fsi.2020.03.009 32165248
73 Ahmed M.M. Mohammed A.T. Farag M.R. Hassan M.A. Mawed S.A. Alagawany M. Zizzadoro C. Di Cerbo A. Abdel-Latif H.M.R. Dietary supplementation of Nile tilapia (Oreochromis niloticus) with Panax ginseng essential oil: positive impact on animal health and productive performance, and mitigating effects on atrazine- induced toxicity Front. Mar. Sci. 9 2022 10.3389/fmars.2022.920057
74 Firat Ö. Erol R. Firat Ö. Effects of individual and Co-exposure of copper oxide nanoparticles and copper sulphate on Nile tilapia Oreochromis niloticus: nanoparticles enhance pesticide biochemical toxicity Acta Chim. Slov. 69 2022 81 90 10.17344/acsi.2021.6995 35298018
75 Vasconcelos R.T. Copatti C.E. Albinati A.C. Campeche D.F.B. Bonfá H. Melo J. Waterborne copper sulfate toxicity in Nile tilapia (Oreochromis niloticus) juveniles affect survival, growth, and physiology J. Appl. Aquac. 2022 10.1080/10454438.2022.2128964
76 Abdel-Khalek A.A. Hamed A. Hasheesh W.S.F. The potential use of orange and banana peels to minimize the toxicological effects of silver nanoparticles in Oreochromis niloticus Bull. Environ. Contam. Toxicol. 108 2022 985 994 10.1007/s00128-022-03488-5 35275237
77 Abdel-Khalek A.A. Badran S.R. Marie M.A.S. The efficient role of rice husk in reducing the toxicity of iron and aluminum oxides nanoparticles in Oreochromis niloticus: hematological, bioaccumulation, and histological endpoints Water Air Soil Pollut. 231 2020 10.1007/s11270-020-4424-2
78 Abdel-Khalek A.A. Hamed A. Marie M.A. The accumulation potency of bulk and nano zinc metal and their impacts on the hematological and histological perturbations of Oreochromis niloticus Water Air Soil Pollut. 227 2016 10.1007/s11270-016-2908-x
79 Brittain T. Root effect hemoglobins J. Inorg. Biochem. 99 2005 120 129 10.1016/j.jinorgbio.2004.09.025 15598496
80 Yaji A. Iheanacho S. Ogueji E. Sublethal exposure and toxicity effect of Propanil on hematology and serum biochemistry in Oreochromis niloticus in a Static Bioassay Gazi Univ. J. Sci. 31 2018 1048 1062 http://dergipark.gov.tr/gujs
81 Palipoch S. Jiraungkoorskul W. Tansatit T. Preyavichyapugdee N. Jaikua W. Kosai P. Protective efficiency of Thunbergia laurifolia leaf extract against lead (II) nitrate-induced toxicity in Oreochromis niloticus J. Med. Plants Res. 5 2011 719 728 http://www.academicjournals.org/JMPR
82 Sarma P.R. Red cell indices Clinical Methods: The History, Physical, and Laboratory Examinations 1990 Butterworths https://www.ncbi.nlm.nih.gov/books/NBK260/ (accessed 17 March 2024)
83 Witeska M. Biardzka J. Kniaz J. The effects of heparin concentration, storage time, and temperature on the values of hematological parameters in Cyprinus carpio Turk .J. Vet. Anim. Sci. 41 2017 351 356 10.3906/vet-1611-35
84 Ciepliński M. Kasprzak M. Grandtke M. Steliga A. Kamiński P. Jerzak L. The effect of dipotassium EDTA and lithium heparin on hematologic values of farmed brown trout Salmo trutta (L.) spawners Aquac. Int. 27 2019 79 87 10.1007/s10499-018-0308-5
85 Witeska M. Kondera E. Ługowska K. Bojarski B. Hematological methods in fish – Not only for beginners Aquaculture 547 2022 737498 10.1016/J.AQUACULTURE.2021.737498
86 Faggio C. Arfuso F. Piccione G. Zumbo A. Fazio F. Effect of three different anticoagulants and storage time on haematological parameters of Mugil cephalus (Linneaus, 1758) Turk. J. Fish. Aquat. Sci. 14 2014 615 621 10.4194/1303-2712-v14_3_03
87 Gonzales-Flores A.P. Mejia Perez F.I. Huanuiri Quinteros K.A. Sanchez Callejas I.Y. Vasquez Rojas J.L. Fernandez-Mendez C. Effect of heparin and EDTA as anticoagulants on hematological values in farmed juvenile of Arapaima gigas Aquac. Int. 30 2022 263 271 10.1007/s10499-021-00796-1
88 Lulijwa R. Alfaro A.C. Young T. Venter L. Decker P. Merien F. Meyer J. Effect of anticoagulants on farmed giant kokopu, Galaxias argenteus (Gmelin 1789) haematological parameters and erythrocyte fragility J. Fish. Biol. 99 2021 684 689 10.1111/jfb.14746 33811326
89 Hattingh J. Heparin and ethylenediamine tetra-acetate as anticoagulants for fish blood Pflugers Arch. 355 1975 347 352 10.1007/BF00579855/METRICS 813183
90 Tavares-Dias M. Sandrim E.F.S. Influence of anticoagulants and blood storage on hematological values in tambaqui, values in tambaqui, values in tambaqui, values in tambaqui, Colossoma macropomum Acta Sci. 20 1998 151 155
91 Witeska M. Wargocka W. Disodium EDTA used as anticoagulant causes hemolysis in common carp blood Turk .J. Vet. Anim. Sci. 35 2011 99 104 10.3906/vet-0908-51
92 Walencik J. Witeska M. The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.) Comp. Biochem. Physiol. C Toxicol. Pharmacol. 146 2007 331 335 10.1016/j.cbpc.2007.04.004 17509941
93 Maqbool A. Ahmed I. Sheikh Z.A. Museum Z. Sheikh A. Effects of two commonly used anticoagulants on haematology and erythrocyte morphology of rainbow trout (Oncorhynchus mykiss), ∼ 239 ∼ Int. J. Fish. Aquat. Stud. 2 2014 239 243 www.fisheriesjournal.com
94 Jan K. Ahmed I. Sheikh Z.A. Fazio F. Impact of three anticoagulants and their storage time on hematological parameters of snow trout, Schizothorax labiatus, habiting in river Sindh of Indian Himalayan region Comp. Clin. Path. 31 2022 747 755 10.1007/s00580-022-03375-9
95 Sheikh Z.A. Ahmed I. Comparative evaluation of two anticoagulants used for the analysis of haematological, biochemical parameters and blood cell morphology of himalayan snow trout, Schizopyge plagiostomus Tissue Cell 67 2020 10.1016/j.tice.2020.101398
96 Sousa A.G. Pacheco A.H.B. Siqueira-Pinto G.A. dos Reis G.T.J. Fugimura M.M.S. Vaz L.J. Marcusso P.F. Ramos-Espinoza F.C. da Silva Claudiano G. Comparative study of hematological parameters of Colossoma macropomum anesthetized with benzocaine and eugenol by using different anticoagulants Aquac. Int. 29 2021 977 988 10.1007/s10499-021-00668-8
97 Weinert N.C. Volpato J. Costa Á. Antunes R.R. De Oliveira A.C. Mattoso C.R.S. Saito M.E. Hematology of Nile tilapia (Oreochromis niloticus) subjected to anesthesia and anticoagulation protocols Semina: Ciencias Agrarias 36 2015 4237 4250 10.5433/1679-0359.2015v36n6Supl2p4237
98 Duran S. Erdem C. Effects of sublethal concentrations of copper, zinc and cadmium, appliedsingly and in mixture, on some hematological parameters of Oreochromis niloticus (L., 1758) Fresenius Environ. Bull. 27 2023 2977 2980
99 Barrick A. Châtel A. Marion J.M. Perrein-Ettajani H. Bruneau M. Mouneyrac C. A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor Mar. Pollut. Bull. 108 2016 275 280 10.1016/j.marpolbul.2016.04.056 27184131
100 Burgeot T. Gagné F. Forget-Leray J. Bocquené G. Acethylcholinesterase: methodology development of a biomarker and challenges of its application for biomonitoring Aquat. Living Resour. 17 2010 309 316 www.Bequalm.org
