Acute effects of chromium on hemato-biochemical parameters and morphology of erythrocytes in striped catfish Pangasianodon hypophthalmus.

Chromium is considered the most detrimental pollutant to the aquatic organisms. The present experiment was conducted to determine the acute toxicity of chromium in view of its effects on hemato-biochemical parameters and the structure of erythrocytes in striped catfish, Pangasianodon hypophthalmus. Fish were exposed to seven different concentrations (0, 10, 20, 30, 40, 50 and 60 mg/L) of chromium, each with three replications for 96 h. After 96 h of exposure, the survived fish were sacrificed to measure hemato-biochemical parameters (hemoglobin, Hb; red blood cell, RBC; white blood cell, WBC; packed cell volume, PCV; mean corpuscular volume, MCV; the mean corpuscular hemoglobin, MCH and blood glucose). In addition, erythrocytic cellular abnormalities (ECA) and erythrocytic nuclear abnormalities (ENA) of peripheral erythrocytes were assayed. No mortality was observed up to 10 mg/L, but 90% and 100% mortality was observed at 50 mg/L and 60 mg/L, respectively after a 96 h exposure period. The 96 h LC50 value through probit analysis was 32.47 mg/L. Hb (%), RBC (×106/mm3) and PCV (%) significantly decreased at 20, 30 and 40 mg/L of chromium, whereas WBC (×103/mm3), MCV (μm3) and MCH (pg) showed the opposite scenario. Blood glucose (mg/dL) levels significantly increased at 10, 20, 30 and 40 mg/L of chromium compared to 0 mg/L. Frequencies of ECA and ENA significantly increased with increasing chromium concentrations. This study indicates that chromium is highly toxic to striped catfish.

Introduction

The striped catfish (Pangasianodon hypophthalmus) is a commercially cultured fish species in Bangladesh. It is popular for its outstanding growth performance and lucrative size, and high local and international market demand [1]. This is one of the most consumed fish species in south Asian countries. After being introduced to Bangladesh from Thailand in 1989, it has been contributing a lot (0.45 million MT in FY 2017−18) to aquaculture production, supporting livelihood of more than 0.7 millions of farmers to provide a considerable assistance in national economy and combating malnutrition of common poor people [2,3]. This fish consumes various types of food, including zooplankton, insects, algae as well as higher plants. It can exist in all types of environmental situations and show satisfactory production in high stocking density.

Heavy metal contamination severely interferes with the ecological balances of an ecosystem and produces devastating effects on the quality of the environment [4]. Heavy metals accumulate harmful chemicals in the aquatic ecosystem and consequently to humans [5]. It has been reported that heavy metal pollution reduced the production of fish [6]. Thus, it is essential to control the use of heavy metals. Moreover, heavy metals are poisonous leading the hazardous impacts on aquatic fauna that finally lead the serious human health concern [[7], [8], [9], [10], [11]].

Chromium (Cr) is one of the heavy metals which contaminates the environment through the release from various industries such as oil, paint, motor vehicle, aircraft, printing, electroplating factories, textile manufacturing facilities, steel producing factories, rinse waters, leather tanneries, dyeing, sanitary landfill leaching, the combustion of coal and oil, welding, bricks of furnaces and wood-preserving industry [12,13]. It is a ubiquitous trace element that occurs in soil, air and water. Several studies reported about the Cr concentration incidies in the environment such as 0.0376−0.0388 mg/L in Surma river [14]; 0.587 mg/L in Buriganga river [15]; 0.62–1.37 mg/L in Balu river [16]; 0.093 mg/L in ground water in Singair upazila, Manikganj [17]; 0.0326−0.0579 mg/L in Paira river [18]. Exposure of fish to chromium affects physiology and growth rate leading to deleterious health effects including allergy and even organ system-toxicity [[19], [20], [21], [22]].

Blood parameters are one of the most important determinants of physiological stress, which result from any deviation that affects the homeostasis of fish [[23], [24], [25]]. The changes might directly influence the blood parameters of fish in different water quality parameters. It has been reported that micronucleus (MN) formation in the erythrocyte is considered to examine the stress from pollution [[26], [27], [28]]. MN is a small chromatin body in the cytoplasm, formed outside the central nucleus and condensed of acentric chromosome fragments during the nuclear division [29]. Likewise, deviation from the normal structure of cells and nucleus of erythrocytes (cellular and nuclear abnormalities) is also applied to assess the stress resulted from extrinsic contaminations [[30], [31], [32], [33]]. Several experiments have reported the chromium toxicity on various fish species such as reduced the glycogen, total lipid and total protein concentration in liver, muscle and gill of rohu, Labeo rohita [34], declined the protein and carbohydrate content in gill of mrigal, Cirrhinus mrigala [35], induced the radical stress in hepatocytes of goldfish, Carassius auratus [36], caused cell death, alteration of glucose transport and endocrine disruption in fresh water fishes [37], severely disrupted the functional activites of kidney, liver and gill of grass carp, Ctenopharyngodon idella [38].

However, there is no report regarding the chromium toxicity in the striped catfish (P. hypopohthalmus), a commercially important aquaculture species. Therefore, the present experiment was designed to assess the toxicity of chromium considering its effects on hemato-biochemical parameters and morphology of peripheral erythrocytes in striped catfish.

Materials and methods

Experimental fish

Apparently healthy and active fingerlings of striped catfish (P. hypophthalmus) of weight (11.48 ± 1.67 g) and standard length (10.81 ± 0.83 cm) were procured from a private hatchery of Sadar Upazila, Mymensingh. The fingerlings were placed in a plastic bag containing 70% water and 30% oxygen and transported to the Fish Ecophysiology Laboratory, Bangladesh Agricultural University, Bangladesh. In the laboratory, fish were kept in aquariums (75 cm × 45 cm × 45 cm) containing 100 L of clean tap water and acclimatized for 21 days to the laboratory conditions. During the acclimatization period, the fish were fed twice daily with commercial grower feed (CP Bangladesh Co., Ltd., Bangladesh). Each aquarium was monitored daily to check fish mortality and dead fish were removed from the aquariums. The water quality parameters, such as temperature (30 ± 05 °C), pH (7.52 ± 0.09), dissolved oxygen (6.08 ± 0.19 mg L−1), free CO2 (6.38 ± 0.48 mg L−1) and total alkalinity (194.25 ± 9.95 mg L−1) were recorded to be in the optimum range during the rearing period. The Animal Welfare and Ethical Committee, Bangladesh Agricultural University approved the experimental procedures used in this study.

Test chemical

The analytical grade potassium dichromate (K2Cr2O7) was obtained from Merck (Darmstadt, Germany). To prepare a test solution, 2.828 g of K2Cr2O7 was dissolved with double distilled water to produce a stock solution of 1000 mg/L chromium [13]. Then a desired concentration of chromium was prepared by diluting the stock solution with double distilled water.

Acute toxicity test

Acute toxicity bioassay was followed to determine 96 h lethal concentration values (LC50) of chromium for striped catfish. Ten fingerlings were stocked in each aquarium filled with 100 L of tap water. The fish were exposed to six different concentrations (10, 20, 30, 40, 50, and 60 mg/L) of chromium and a control unit without chromium (0 mg/L) with three replications. Sufficient aeration was provided during the time of the experiment. The application of the chromium was repeated at every 24 h with a regular total exchange of water. Mortality was assessed at 24, 48, 72, and 96 h after the start of exposure, and dead fish were removed immediately. The 96 h LC50 value was determined by probit analysis.

Hemato-biochemical study

The blood sample was collected from the survived fish after 96 h of exposure. Six fish (n = 6) from each aquarium were sacrificed for the hemato-biochemical assessment. The counting of red blood cell (RBC) and white blood cell (WBC) was done with the help of Neubauer hemocytometer under a light microscope. The hemoglobin (Hb, %) was measured directly by a digital EasyMate® GHb (Model: ET 232, Hb/Glu double monitoring system, Bioptic technology Inc. Taiwan 35057) with hemoglobin strips. Similar to Hb, blood glucose (mg/dL) was measured immediately following blood sample collection by glucose strips using the digital EasyMate® GHb. The packed cell volume (PCV, %), mean corpuscular volume (MCV, μm3) and the mean corpuscular hemoglobin (MCH, pg) were calculated using the following formulas [39]:PCV (%) = % Hb × 3MCV (μmMCH (pg) = (% Hb/RBC in millions) × 10

Analysis of cellular and nuclear abnormalities of blood cells

Erythrocytic cellular abnormalities (ECA) and erythrocytic nuclear abnormalities (ENA) were analyzed after exposure to different concentrations of chromium. A drop of blood was taken on the clean microscopic slide and smeared smoothly directly after collection. After air-drying for 10 min, the smeared slide was fixed by methanol and finally stained by 5% Giemsa solution. Then, the stained slide was washed with tap water and kept overnight for air drying at room temperature. For preservation, the slide was mounted using DPX solution. Then the ECA and ENA were observed under an electronic microscope (MCX100, Micros Austria) with magnification of ×40. Three different slides were prepared from each fish blood and 2000 cells were counted from each one.

An oval-shaped erythrocyte cell with a condensed nucleus is considered as the regular erythrocyte cell and ECA was identified by comparing desperation of the regular cell. Among ECA, echinocytic cells are irregular shape and having serrated edges structure entire the surface of the cell membrane; elongated cells are slightly egg-shaped, slender structures which have more length compared to width; fusions are more than two cells, which are joining each other and form heavier clotting; twins are two cells joined together by cellular membrane; teardrop cells when deformed a nipple at the top point and tapering the endpoints of erythrocyte.

ENA were classified [40,41] briefly such as, binucleated is the two nuclei that are equal in size but completely separated from each other in a cell. Micronucleus is the circular chromatin bodies formed in the same staining shape out of the central nucleus. Blebbed is that condition when small evaginations of the euchromatin bearing with the nuclear membrane. A notched nucleus is resulting from osmosis in a hypertonic solution of the nucleus, which did not contain any nuclear materials owing to the shrinkage. Nuclear bud is the evagination generated like a bud in the nucleus. The nuclear bridge had thin strands connected between two individual nuclei of the cell.

Statistical analysis

Values were expressed as mean ± standard deviation (SD). The LC50 value of chromium was determined through probit analysis. The data were analyzed by using one-way ANOVA, followed by post hoc test to find out the statistically significant differences among treated values with control. Statistical analysis was executed by using SPSS version 14.0 for Windows (SPSS Inc., Chicago, IL, USA) at level of significance p < 0.05.

Results

Median lethal concentration of chromium for striped catfish

After 96 h of exposure, there was no mortality up to 10 mg/L, but 86.67 ± 5.77% and 100 ± 0.00% mortality were recorded at 50 mg/L and 60 mg/L concentrations, respectively (Table 1). The 96 h LC50 value was 32.47 mg/L with upper limit and lower limit 38.63 mg/L and 27.81 mg/L, respectively, where the linear regression equation was y = 133.1x − 0.144.81, R2 = 0.9305 (Fig. 1).

Table 1

Mortality percentages of fish exposed to different concentrations of chromium.

Concentrations of chromium (mg/L)	Percent (%) dead fish at different time intervals (mean ± SD)
	24 h	48 h	72 h	96 h
0	–	–	–	–
10	–	–	–	–
20	–	–	–	13.33 ± 5.77
30	–	–	23.33 ± 5.77	43.33 ± 5.77
40	–	10.00 ± 0.00	30.00 ± 0.00	70.00 ± 0.00
50	–	33.33 ± 5.77	46.67 ± 5.77	86.67 ± 5.77
60	10 ± 0.00	40 ± 0.00	60 ± 0.00	100.00 ± 0.00

Fig. 1

Graph showing linear transformation and the relationship of probit of log concentration of chromium used to determine LC50 of P. hypophthalmus.

Effects of chromium on hemato-biochemical parameters

The values of RBC (×106/mm3), Hb (%) and PCV (%) ranged from 1.09 ± 0.02 to 1.65 ± 0.43, 8.7 ± 0.3 to 10.1 ± 0.9 and 25.86 ± 4.2 to 30.3 ± 2.7, respectively in the fish blood exposed to different concentrations of Cr. The total number of RBC, Hb and PCV significantly (p < 0.05) declined at 20, 30 and 40 mg/L compared to the control (0 mg/L) after the exposure of period (Table 2). Conversely, total number of WBC (×103/mm3), MCV (μm3) and MCH (pg) significantly increased at 10, 20, 30 and 40 mg/L compared to the control (0 mg/L) of chromium (Table 2). The blood glucose (mg/dL) level increased significantly at 20 (172.0 ± 18.6) mg/L, 30 (170.5 ± 34.5) mg/L and 40 (189.0 ± 16.6) mg/L compared to control (117.2 ± 14.4) (Table 2).

Table 2

Hemato-biochemical parameters after 96 h exposure to different concentrations of chromium.

Parameters	Concentration of chromium (mg/L)
	0	10	20	30	40
RBC (×106/mm3)	1.65 ± 0.43a	1.40 ± 0.30ab	1.26 ± 0.13b	1.29 ± 0.16b	1.09 ± 0.02b
WBC (×103/mm3)	10.85 ± 2.29a	16.29 ± 3.19b	16.45 ± 3.96b	17.27 ± 3.02b	16.61 ± 1.31b
Hb (%)	10.1 ± 0.9a	9.8 ± 1.2ab	8.62 ± 1.4b	8.7 ± 0.3b	8.7 ± 0.4b
PCV (%)	30.3 ± 2.7a	29.4 ± 3.6a	25.86 ± 4.2b	26.1 ± 0.9b	26.1 ± 1.2b
MCV (μm3)	183.64 ± 9.25a	210 ± 10.52b	205.23 ± 12.02b	202.33 ± 11.21b	239.45 ± 13.25b
MCH (pg)	61.21 ± 4.21a	70.00 ± 5.41b	68.41 ± 3.26 b	67.44 ± 4.56 b	79.82 ± 5.62 b
Blood glucose (mg/dL)	117.2 ± 14.4a	138.0 ± 9.5ab	172.0 ± 18.6b	170.5 ± 34.5b	189.0 ± 16.6b

Values of in a row with different alphabetical superscripts are significantly (p < 0.05) different. All values are expressed as mean ± SD.

Effects of chromium on cellular and nuclear anomalies of blood cells

The blood smear of striped catfish exhibited different erythrocytic cellular abnormalities (ECA) after exposure to different chromium concentrations, such as elongated (Fig. 2b), twin (Fig. 2c), fusion (Fig. 2d), teardrop (Fig. 2e) and echinocytic (Fig. 2f). The frequencies of ECA showed significant (p < 0.05) increases in the blood of fish exposed to 10, 20, 30 and 40 mg/L of chromium concentrations compared to 0 mg/L (Table 3). The present experiment also showed different ENA in the blood of fish, such as micronucleus (Fig. 3b), notched nuclei (Fig. 3c), binuclei (Fig. 3d), blebbed (Fig. 3e) and nuclear bud (Fig. 3f) in different concentrations of chromium. The frequencies of ENA of fish treated in different chromium concentrations are shown in Table 4. ENA has been significantly (p < 0.05) increased after the exposure period at 10, 20, 30 and 40 mg/L compared to 0 mg/L.

Fig. 2

Various erythrocytic cellular abnormalities (ECA) in Giemsa stained blood smears of P. hypophthalmus treated with different concentrations of chromium; (a) Normal, (b) elongated, (c) twin, (d) fusion, (e) teardrop, (f) echinocytic. The different ECA were observed under an electronic microscope with magnification of ×40.

Table 3

Frequencies of erythrocytic cellular abnormalities (ECA) after 96 h exposure to different concentrations of chromium.

ECA	Concentration of chromium (mg/L)
	0	10	20	30	40
Elongated shape	0.31 ± 0.09a	1.51 ± 0.13b	1.53 ± 0.15b	1.57 ± 0.18b	1.75 ± 0.18b
Twin	0.41 ± 0.11a	1.63 ± 0.15b	1.66 ± 0.18b	1.59 ± 0.19b	1.95 ± 0.19b
Fusion	0.68 ± 0.07a	1.66 ± 0.13b	1.67 ± 0.15b	1.63 ± 0.18b	1.83 ± 0.18b
Tear-drop	0.50 ± 0.07a	1.53 ± 0.11b	1.56 ± 0.11b	1.57 ± 0.15b	1.77 ± 0.15b
Echinocytic	0.45 ± 0.07a	1.53 ± 0.13b	1.66 ± 0.15b	1.48 ± 0.18b	1.88 ± 0.18b

Values in a row with different alphabetical superscripts are significantly (p < 0.05) different. All values are expressed as mean ± SD. Three slides were prepared from each fish and 2000 cells were scored from each slide and three fish were analyzed from each group.

Fig. 3

Various erythrocytic nuclear abnormalities (ENA) in Giemsa stained blood smears of P. hypophthalmus treated with different concentrations of chromium; (a) Normal, (b) micronucleus, (c) notched, (d) binuclei, (e) blebbed, (f) nuclear bud. The different ENA were observed under an electronic microscope with magnification of ×40.

Table 4

Frequencies of erythrocytic nuclear abnormalities (ENA) after 96 h exposure to different concentrations of chromium.

ENA	Concentration of chromium (mg/L)
	0	10	20	30	40
Micronucleus	0.15 ± 0.03a	0.48 ± 0.07b	1.21 ± 0.09c	1.56 ± 0.11c	1.72 ± 0.11c
Notched	0.16 ± 0.03a	0.85 ± 0.09b	1.10 ± 0.13b	1.60 ± 0.15c	1.69 ± 0.15c
Binuclei	0.18 ± 0.05a	0.83 ± 0.09b	0.95 ± 0.13b	1.46 ± 0.18c	1.61 ± 0.18c
Blebbed	0.53 ± 0.09a	1.66 ± 0.15b	1.63 ± 0.15b	1.68 ± 0.18b	1.80 ± 0.18b
Nuclear bud	0.67 ± 0.09a	1.60 ± 0.13b	1.66 ± 0.15b	1.61 ± 0.19b	1.83 ± 0.19b

Values in a row with different alphabetical superscripts are significantly (p < 0.05) different. All values are expressed as mean ± SD. Three slides were prepared from each fish and 2000 cells were scored from each slide and three fish were analyzed from each group.

Discussion

Chromium is one of the most harmful contaminants to aquatic organisms. Hence we aimed to test the acute toxicity of chromium considering its effects on hemato-biochemical parameters and structure of peripheral erythrocytes in striped catfish. Different changes were observed in hemato-biochemical parameters and morphology of erythrocytes at different concentrations of chromium, which indicates its stressful impacts on physiology in the striped catfish.

Several studies reported the assessment of the acute toxicity of chromium on different fish species. In the present study, 96 h LC50 value of striped catfish was 32.47 mg/L, which is less than the values of 50.00 mg/L for Channa punctatus [42], 155.00 mg/L for Cyprinus carpio [43], 39.40 mg/L for Labeo rohita [34] 60 mg/L for Colisa fasciatus [36], 100.00 mg/L for Catla catla [37] and 119.52 mg/L for Oreochromis niliticus [38]. More or less similar 96 h LC50 values were recorded as 34.00 mg/L for Cirrhinus mrigala [27,30], 35.72 mg/L for Heteropneustes fossilis [44] and 36.65 mg/L for Clarius batrachus [45]. In contrast, lower 96 h LC50 values were recorded as 7.33 mg/L for Labeo bata and 10.37 mg/L for Puntius sarana [46]. The toxicity level of any chemical depends on the physiological situations of the fish exposed, their habitat and also the purity of the chemicals [47,48].

In the present study, significant changes were observed in the different parameters of the blood. It has been reported that different physiological parameters in fish are changed as a result of stress [49]. Erythrocytes count is considered as a quite stable index and fish species always maintain this index within the standard limit by compensating different physiological techniques in stressful conditions. In this study, the Hb level and the number of RBC decreased in different chromium concentrations. Previous studies also reported that the volume of Hb and the number of RBC decreased significantly in freshwater fish when exposed to different heavy metals like as zinc, cadmium and nickel causing disorder such as anemia, erythropenia, leucopoiesis, etc [[50], [51], [52]]. A similar decreased value of Hb was also reported in common carp exposed to organophosphorus pesticide sumithion [25,53] and malathion [54]. The significant reduction of Hb level and the number of RBC in the present study might be due to iron deficiency and structural alterations of heme leading to decline hemoglobin synthesis. On the other hand, significant increases in WBC counts might be due to the increase of antibody production [55], which helped the organism in survival and healing during exposure to toxic pollutants [56,57]. This increase of WBC also might be due to leukocytosis under chemical strees caused by chromium, resulted direct stimulation of immunological resistance [58]. Moreover, chromium exposure could lead to activate radical oxidation processes and to nutrients imbalance, which can ultimately lead to the development of the observed changes in immunological and biochemical factors [59].

Blood glucose plays an important role in producing energy for the central nervous system, which is controlled by the hormone. Therefore, blood glucose levels are commonly used as a stress bio-indicator in fish [60]. In the present study, blood glucose levels increased in the fish reared in different chromium concentrations, which might be due to the transformation of glucose from glycogen that met the increasing energy demands at high chromium concentration. Hyperglycemia produced in animals due to the secretion of catecholamine and glucocorticoids from the adrenal tissue of the fish and stress acts as a stimulant to secrete these hormones rapidly [61,62]. This type of raise may result because of boosted gluconeogenesis through which stressed fish try to fulfill their high energy demand [63].

Erythrocytes are commonly used as an important diagnosing tool to know the functional and structural situation of fish exposed to any toxicants. Erythrocytes are capable to respond to environmental obsesses and alterations of erythrocyte represent the most common reflection toward toxicants present in water bodies [64]. In the present study, different morphological (ECA and ENA) changes of erythrocytes were observed due to exposure in different chromium concentrations. Morphological changes of erythrocytes may also occur due to the increase of lipid peroxide compound of the cell membrane [30,65]. Erythrocyte structures are changed due to the damage of the cell membrane, ion-permeability, and absorption of cells by the toxic substances [26]. It has been reported that chromium exerts a genotoxic effect through DNA damage [66,67] and altered the nucleus structure of blood cells [68,69]. Due to the failure of tubulin polymerization, differential nuclear abnormalities of erythrocytes, such as binucleated, nuclear bud, nuclear bridge, karyopyknosis, notched nuclei, etc. might be formed under stress triggered by chromium [30,70,71]. Moreover, the formation of binucleated cells and notched nuclei are also possible because of mitotic fuses by aneugenic actions of toxicants [72]. Furthermore, it can be assumed that these types of abnormalities in erythrocyte may cause morphological changes in the plasma membrane that affects deformation in the surface as well as increase susceptibility of erythrocyte to brust during crossing small capillaries.

In summary, chromium is considered highly toxic to striped catfish that altered hemato-biochemical parameters, cellular and nuclear structure of erythrocytes. Also, it reduces the fish survival rate even at low concentrations. However, further studies related to enzymatic changes and oxidative stress of fish are needed to enlarge scope of knowledge and make possible the development of an efficient biomarker for chromium.

Author contributions

S M Majharul Islam and Md. Fazle Rohani conceived, designed and performed the experiments, analyzed data and drafted the manuscript. Seyed Akib Zabed, Md. Tarikul Islam and Rayeda Jannat were assisted in data collection. Yeasmin Akter assisted in data analysis and edited the manuscript. Md. Shahjahan assisted in the experimental design, analyzed data and edited the manuscript. All authors reviewed and approved the final manuscript.

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.