Comparative Biochemistry and Physiology, Part C 236 (2020) 108815

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Comparative Biochemistry and Physiology, Part C

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Morphological, haematological and biochemical changes in African catfish T

Clarias gariepinus (Burchell 1822) juveniles exposed to clotrimazole
Temitope Dadewura Melefa, Bernard O. Mgbenka, Ifeanyi O. Aguzie, Felix A. Andong,
⁎
Uju Nwakor, Christopher D. Nwani
Department of Zoology and Environmental Biology, University of Nigeria Nsukka, Nigeria

A R T I C LE I N FO A B S T R A C T

Keywords: Clotrimazole (CLO) is an imidazole fungicide used in human and veterinary medicine for treating fungal in-
Clarias gariepinus fection. This study evaluated the changes in morphological, haematological and biochemical indices in Clarias
Morphological parameters gariepinus juveniles exposed to CLO. After the acute exposure, the 96 h LC50 value of CLO determined by probit
Haematology analysis was 38.79 mgl−1. Based on this value, fish were exposed to sublethal concentrations of 7.76, 3.89, 1.94
Biochemistry
and 0.00 mgl−1 (control) of CLO for 21 days and were allowed to recover for 7 days. The result revealed no
Clotrimazole
Toxicity
significant effect on the hepatosomatic index and condition factor of the exposed fish. There were concentration
and time-dependent significant decreases in red blood cell (RBC), haemoglobin (Hb), packed cell volume (PCV)
and mean corpuscular volume (MCV) with significant increase in the white blood cell (WBC), mean corpuscular
haemoglobin concentration (MCHC), and mean corpuscular haemoglobin (MCH) in the exposed group when
compared with the control. A mixed trend was observed in the levels of neutrophils, lymphocytes, monocytes
and eosinophils. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP) and glucose values significantly increased, while protein levels were reduced (p < 0.05)
throughout the 21-day exposure and the 7-day recovery period. The present research indicated that CLO may
have potential toxic effect on non-target organisms especially fish and, therefore, should be monitored in the
aquatic ecosystem.

1. Introduction
Clotrimazole (CLO) 1-[(2-Chlorophenyl) (diphenyl) methyl]-1H-
imidazole is an antifungal agent with a broad spectrum antimycotic
activity (Crowley and Gallagher, 2014). It belongs to the group of
imidazole fungicide used in human and veterinary medicine for treating
fungal infections (Rochette et al., 2003; Hashem et al., 2011). It is
available in various forms and has also gained interest in treating other
diseases like sickle cell diseases and some cancers (Crowley and
Gallagher, 2014). Hospital wastewater, effluents from manufacturing
plants, domestic and veterinary use of CLO may be its possible ways of
gaining entrance into the aquatic system (Frédéric and Yves, 2014).
Clotrimazole has been detected in concentration up to several ngl−1 in
river water and wastewater in Asia and Europe (Roberts and Thomas,
2006; Kahle et al., 2008; Huang et al., 2010). In Lagos Nigeria, the
concentration of CLO in surface water and ground water range
from < 1–618 ngl−1 and < 1–191 ngl−1 respectively, and was depen-
dent on whether measurement was made during rainy or dry season
(Ebele et al., 2020). It has been listed among the top ten compounds
with great potential to pose risk to the aquatic environment (OSPAR,
2013). CLO acts as an antimycotic agent by inhibiting ergosterol bio-
synthesis, thereby preventing the fungal cells from constructing a
functional cell membrane. It is also known as an inhibitor of sarco-
plasmic reticulum (Crowley and Gallagher, 2014). Toxicity studies have
shown its ability to modulate many Ca2+ homeostasis in mammals and
fish CYP450-mediated reactions (Burkina et al., 2013). Studies have
also revealed that CLO is toxic to aquatic micro algal communities
(Porsbring et al., 2008). Furthermore, it has also been reported that
CLO inhibits steroidogenesis in fish and mammals (Hinfray et al., 2011;
Wassmur et al., 2013). The continuous use and release of pharmaceu-
ticals into the aquatic environment may interfere with normal home-
ostasis of non-target organisms thereby negatively impacting their
health. Burkina et al. (2016) had studied the sublethal effects of CLO in
rainbow trout (Oncorhynchus mykiss), but to the best of our knowledge
no research has been published on the effect of CLO on morphology,
haematology and biochemical parameters on the African freshwater
catfish, Clarias gariepinus. Haematological parameters (which include
red blood cells (RBCs), haemoglobin (Hb), white blood cells (WBCs),

⁎
Corresponding author.
E-mail address: chris.nwani@unn.edu.ng (C.D. Nwani).

https://doi.org/10.1016/j.cbpc.2020.108815
Received 16 March 2020; Received in revised form 16 May 2020; Accepted 29 May 2020
Available online 03 June 2020
1532-0456/ © 2020 Elsevier Inc. All rights reserved.
T.D. Melefa, et al.
packed cell volume (PCV) and differential blood indices) and bio-
chemical parameters (especially alanine aminotransferase (ALT), as-
partate aminotransferase (AST), alkaline phosphatase ALP, protein and
glucose) have proven to be useful indicators of fish health status (Nwani
et al., 2015; Burgo-Aceves et al., 2019). Despite the occurrence of CLO
in the aquatic environment, there is still scarcity of ecotoxicological
data on the effect of this pharmaceutical on tropical species of non-
target organisms. Clarias gariepinus has been selected for this work be-
cause of its wide distribution in rivers, streams, ponds, dams and lakes
in Africa especially Nigeria. The fish is also a very good model for
ecotoxicological research because of its availability throughout the
season and easy acclimatization to laboratory conditions (Adeyemi
et al., 2014). The aim of this study was to investigate the changes in
morphological, haematological and biochemical indices in C. gariepinus
exposed to CLO.
2. Materials and methods
2.1. Procurement of fish and drug
Three hundred and fifty (350) juveniles freshwater African Catfish
C. gariepinus, mean length 27.36 ± 0.23 cm and weight,
197.39 ± 2.34 g were sourced from Freedom Fisheries Ltd., Nsukka,
Enugu State and were transported to the Fisheries Wet laboratory of the
Department of Zoology and Environmental Biology, University of
Nigeria, Nsukka. Fish were acclimatized for 14 days in concrete pond
and fed daily with commercial feed (Coppens commercial feed of 4 mm,
Coppens International Helmond Netherlands). CLO stock solutions were
prepared using commercial formulations of Canesten V6 tablets man-
ufactured by Bayer Pharmaceuticals Pvt. Ltd., Village Malpur, Baddi -
173 205, Tehsil Nalagarh, District Solan, H.P., with Mfg. Lic. No. L/07/
471/MNB. (Each uncoated tablet contains CLO IP 100 mg as the active
ingredients).
2.2. Experimental Design for Acute Exposure
At total of one hundred and eighty (180) fish (198.42 ± 2.34 g and
28.46 ± 0.23 cm) were used for acute toxicity assay. They were ran-
domly distributed into six groups of thirty fish per group. Each group
consisted of three replicates of 10 fish in a glass aquarium
(60 × 30 × 30 cm) that containing 10 L of water. Fish in groups 1–5
were exposed to 35.00, 38.00, 40.00, 42.00 and 44.00 mg l−1 of CLO.
The sixth group was exposed to only dechlorinated tap water and
served as the control. The percentage mortality/survival of fish in
control and treated groups was recorded at 24, 48, 72 and 96 h duration
intervals respectively. Fishes were considered dead when their oper-
culum stops beating and were carefully removed with plastic forceps to
avoid deterioration of the test media and to prevent cannibalization.
The physicochemical properties of the test water were analyzed weekly
(American Public Health Association, 2005). The average mean of the
physicochemical properties of the test solution determined in the ex-
perimental aquarium were: dissolved oxygen 7.14 mgl−1, temperature
27.83 °C, pH 12.3 and conductivity 247.5μScm−1. The 24–96 h LC10–90
and confidence intervals of CLO were determined using SPSS 20.0
statistical software for probit analysis.
2.3. Determination of sublethal concentrations and in vivo exposure
The 96 h LC50 value of CLO in C. gariepinus following the probit
analysis was 38.79 mg l−1. Based on this value, three sub-lethal con-
centrations, which were 1/5th, 1/10th, and 1/20th of the LC50 (7.76,
3.89 and 1.94 mgl−1) respectively, were estimated by serial dilution of
the stock and used for the in vivo experiment. A total of 120 fish from
the acclimatized batch were used for the in vivo studies. The fish were
divided into four treatment groups. One group was exposed to 7.76
mgl−1 CLO, the second group was exposed to 3.89 mgl−1 CLO, the
2
Comparative Biochemistry and Physiology, Part C 236 (2020) 108815
third group was exposed to 1.94 mg l−1 and the fourth was exposed to
tap water only as the control group. All the treatment groups were in
triplicates with each treatment having 10 fish. The test solution was
renewed every alternate day to maintain the concentration of the drug.
The level of CLO in the aquaria was analyzed to ensure agreement
between nominal and actual concentrations using liquid chromato-
graphy - mass spectrometry (LC-MS) (Ajima et al., 2016). No mortality
was recorded in both the experimental and control groups. The ex-
periment lasted for 28 days. Fish were exposed to CLO for 21 days and
allowed to recover from the effect of the drug for 7 days. During the
experiment, the fish were fed daily small quantity of food approxi-
mately 1% of total body weight about an hour before renewal of the test
solution to avoid mortality.
2.4. Blood sample collection
On each sampling day, approximately 3.0 ml of blood sample was
collected from the fish caudal vein using 2 mL heparinized syringe and
stored in small EDTA bottles. Two fish from each triplicate experiment
and control were sampled on each sampling day. Part (~1.5 ml) of the
collected blood was used for estimation of the haematological para-
meters. The remaining (~1.5 ml) blood collected was centrifuged at
10000g at 4 °C for 20 min to separate the plasma which was used for
estimation of plasma glucose, protein, alanine aminotransferase (ALT),
aspartate aminotransferase (AST) and alkaline phosphatase (ALP).
Sampled fish were not replaced in the aquarium.
2.5. Estimation of haematological parameters
The red blood cell (RBC) count was estimated using an improved
microscope Neubauer counter and Toison's solution as the blood di-
luting fluid (Mgbenka et al., 2005). The white blood cell (WBC) count
was determined also with a Neubauer microscopic counter using Turk's
solution as the blood diluting fluid. Counting of the neutrophils,
monocytes, lymphocytes, eosinophils and basophils in the blood smears
were determined as described by Anderson (2003). The cyanmethane-
moglobin method (Blaxhall and Daisley, 1973) was followed in de-
termining the haemoglobin (Hb) concentration in the blood. The
packed cell volume (PCV) was estimated by centrifugation of the blood
for 5 min at 1400g in heparinized glass capillaries using a micro hae-
matocrit centrifuge. The PCV, Hb and RBC were used in computing the
erythrocyte indices such as mean cell volume (MCV), mean cell hae-
moglobin volume (MCH) and mean cell haemoglobin concentration
(MCHC), according to Dacie and Lewis (1984):
PCV(%) × 10
MCV (fl/cell) =
RBC count in million/mm3
Hb(g/dl) × 10
MCH (pg/cell) =
RBC count in million/mm3
Hb(g/dl) × 10
MCHC (g/dl) = × 100
PCV (%)
2.6. Determination of biochemical parameters
Levels of plasma ALT, AST and ALP were determined by the
methods of Reitman and Frankel (1957) while glucose and protein were
estimated by the methods of Cooper and McDaniel (1970) and Lowry
et al. (1951) respectively.
2.7. Determination of morphometric indices
The body weight and standard length of each fish were determined
after each exposure interval. The blood was collected and the liver
dissected and weighed. The condition factor (CF) and hepatosomatic
T.D. Melefa, et al.
index (HSI) were calculated using the methods of White and Fletcher
(1985) shown below:
CF = Body weight (g)/Standard lenght (cm)3 × 100
HSI = Liver weight (g)/Body weight (g) × 100
2.8. Statistical analysis
The data obtained were analyzed statistically using SPSS 20.0 (IBM
Corporation, Armonk, New York, USA). Generalized linear model cou-
pled to univariable two-way analysis of variance (ANOVA) with mor-
phological, biochemical and haematological parameters as dependent
factors and concentration and exposure duration as fixed factors were
used to determine effects of the clotrimazole on the fish. Least square
difference (LSD) and Duncan were used as post-hoc tests. Level of sig-
nificance for the tests was set at 95% probability level (i.e. p < 0.05).
Results are summarized as mean ± standard deviation (SD).
3. Results
3.1. Morphological indices
The mean weight of the fish for the duration of the study is sum-
marized in Table 1. The weight was not different between the groups
for the duration of the study (Treatment: F = 0.984, df = 3, p = 0.410;
Duration: F = 0.624, df = 4, p = 0.648). For the duration of exposure,
the condition factor (CF) of the fish was not altered by the drug
(F = 0.512, df = 3, p = 0.676) (Supplementary Table S1). The he-
patosomatic indices (HSI) of the fish for the duration of the study are
presented in (Supplementary Table S2). The drug had no significant
effect on the HSI.
3.2. Haematological parameters
The haematological parameters of C. gariepinus generally responded
to CLO exposure (Table 2). The response was significant with respect to
treatment concentration and exposure duration. PCV (F = 38.364,
df = 3, p < 0.001), RBC (F = 10.099, df = 3, p < 0.001) and Hb
(F = 38.440, df = 3, p < 0.001) decreased significantly in fish ex-
posed to the drug. The effect on PCV and Hb decreased with the
duration of exposure. WBC was not significantly affected by the treat-
ment at the concentrations used (F = 2.730, p = 0.114). There was
interaction effects of treatment concentrations and duration of exposure
on PCV (F = 8.297, df = 12, p < 0.001), Hb (F = 16.910, df = 12,
p < 0.001), and WBC (F = 10.853, df = 12, p < 0.001).
A mixed trend was observed in the levels of the white blood cell
differentials (neutrophils, lymphocytes, monocytes and eosinophils).
Neutrophil count was increased by the treatment (F = 9.831, df = 3,
p < 0.001), while lymphocytes count decreased (F = 8.378, df = 3,
p < 0.001). However, monocyte and eosinophil showed no response to
the drug (Table 3). The effect on the neutrophil, lymphocytes, mono-
cytes and eosinophil depended on interaction between treatment and
Table 1
Changes in the body weight of Clarias gariepinus juvenile exposed to clotrimazole.
Conc. (mgl−1) Duration (day)
1 7
Control 86.70 ± 55.51a1 84.30 ± 61.19a1
1.94 97.93 ± 42.77a2 83.63 ± 12.04a12
3.89 93.27 ± 39.07a1 99.77 ± 45.50a1
7.76 81.47 ± 28.44a1 79.80 ± 28.35a1
Values as mean ± SD. Values with different alphabet superscript along a column were significantly different between concentrations; and values with different
number superscript across a row were significantly different (p < 0.05).
3
Comparative Biochemistry and Physiology, Part C 236 (2020) 108815
duration (p < 0.05). Basophil was not detected in all the group for the
duration of the study.
The haematological indices MCV, MCH and MCHC were affected
significantly by the drug (F = 28.938, 37.206, 37.206, df = 12,
p < 0.001 respectively), and the effects depended on exposure dura-
tion (F = 18.247, 11.224, 63.385, df = 12, p < 0.001 respectively).
MCH and MCHC increased as the drug concentration increased while
MCV decreased (Table 4). The effect on MCV (F = 6.044, df = 12,
p < 0.001) and MCHC (F = 12.175, df = 12, p < 0.001) also in-
creased with duration of exposure.
3.3. Biochemical parameters
The liver enzymes ALT, AST and ALP were affected by the clo-
trimazole exposure (Table 5). Their activities increased as the con-
centration of the drug, and duration of exposure increased. The effect of
the drug on all three enzymes was significant, and depended on dura-
tion of exposure which were both positively related.
Total protein responded negatively to CLO exposure (Fig. 1). It
decreased as concentration of the drug increased (F = 1096.587,
df = 3, p < 0.001); the effect was much obvious as the duration of
exposure increased (F = 336.077, df = 4, p < 0.001), and interacted
positively with it (F = 44.734, df = 12, p < 0.001). Plasma glucose
concentration was affected on exposure of C. gariepinus to CLO (Fig. 2).
Glucose concentration was increased by the drug, in a pattern related to
the duration of exposure (F = 16.335, df = 12, p < 0.001). The effect
was more obvious at the lower drug concentration.
4. Discussion
4.1. Morphometric parameters
In the present study, we evaluated the effect of different treatments
of CLO on the morphometric changes in Clarias gariepinus. Condition
factor is used to assess the general health status of fish (Kumolu-
Johnson and Ndimele, 2010), while the hepatosomatic index is an in-
dicator of energy reserve by the liver (Moslemi-aqdam et al., 2014).
Fish usually have smaller liver in poor environment, an indication of
poor energy reserve (Moslemi-aqdam et al., 2014). There was no sig-
nificant change in fish weight for the duration of the exposure. The
condition factor (CF) of the fish was not altered throughout the dura-
tion of the exposure. This agrees with the findings of Burkina et al.
(2016) when rainbow trout was exposed to CLO at environmentally
relevant concentrations of 0.01, 1.0 and 10 μgl−1. They similarly re-
ported that CLO exposure had no significant effect on the condition
factor of the fish, Oncorhynchus mykiss. However, the CLO concentration
and species of fish they used were different from what we used in the
present study, and their exposure duration was 42 days. In our study,
there was also no significant effect of CLO on hepatosomatic indices
(HSI) in C. gariepinus. Li et al. (2011a) obtained similar results when O.
mykiss was exposed to the anticonvulsant drug carbamazepine. Simi-
larly, no significant effect on CF and HSI has been reported when C.
gariepinus was exposed to praziquantel (Nwani et al., 2014a) and
14 21 7-Day recovery
62.10 ± 57.45a1 70.83 ± 41.32a1 93.60 ± 55.44a1
61.43 ± 16.42a12 58.58 ± 24.50a12 50.30 ± 2.94a1
92.27 ± 22.12a1 108.83 ± 54.49a1 68.40 ± 25.81a1
74.50 ± 31.21a1 72.97 ± 19.92a1 68.77 ± 21.91a1
T.D. Melefa, et al. Comparative Biochemistry and Physiology, Part C 236 (2020) 108815

Table 2
Changes in blood parameters of Clarias gariepinus exposed to clotrimazole.
Conc. (mgl−1) Duration (day)

1 7 14 21 7-Day recovery

a2 a12 a12 a1
PCV (%) Control 51.50 ± 3.50 50.00 ± 2.00 47.50 ± 1.50 48.50 ± 5.50 44.00 ± 4.02b12
1.94 53.00 ± 1.02a5 42.50 ± 0.05b3 40.50 ± 0.50b2 37.50 ± 0.50b1 49.50 ± 1.50a4
3.89 45.50 ± 1.50b5 38.50 ± 0.50c3 35.50 ± 0.50c2 35.50 ± 0.50bc1 43.00 ± 1.00b4
7.76 37.00 ± 1.00c34 36.00 ± 1.00d3 33.50 ± 0.50d2 29.00 ± 1.00c1 38.50 ± 0.50c4
RBC (×106) Control 9.70 ± 0.10a1 9.50 ± 0.10a1 9.60 ± 0.20a1 9.45 ± 0.15a1 9.70 ± 0.10a1
1.94 9.35 ± 0.50ab1 8.95 ± 0.15b2 8.90 ± 0.30b2 8.10 ± 0.10b1 8.65 ± 0.45b2
3.89 8.90 ± 0.30bc2 8.75 ± 0.35b2 8.55 ± 0.45bc2 7.70 ± 0.10c1 8.65 ± 0.45b2
7.76 8.35 ± 0.55c2 8.00 ± 0.40c2 8.05 ± 0.35c2 7.30 ± 0.10d1 8.00 ± 0.10c1
HB (%) Control 10.67 ± 0.15a12 10.43 ± 0.20b1 10.73 ± 0.13a2 10.69 ± 0.17a2 10.65 ± 0.05a12
1.94 10.58 ± 0.20a4 10.62 ± 0.20a4 10.21 ± 0.20b2 96.60 ± 0.02b2 10.27 ± 0.04b3
3.89 10.24 ± 0.05b4 10.20 ± 0.35c34 10.04 ± 0.35c2 98.45 ± 0.02bc1 10.14 ± 0.02c3
7.76 10.05 ± 0.04c2 10.03 ± 0.06d2 9.81 ± 0.04d1 9.76 ± 0.04c1 10.05 ± 0.04d2
WBC (×103) Control 10,110 ± 300a1 9950 ± 250a1 9900 ± 500a1 9900 ± 100a1 9953 ± 200a1
1.94 9800 ± 400ab3 9500 ± 100c23 8200 ± 200b1 8400 ± 200b1 9000 ± 400b2
3.89 9550 ± 150ab3 9100 ± 300b3 7800 ± 400bc2 7250 ± 150c1 8100 ± 300c2
7.76 9250 ± 550c4 8650 ± 500d3 7500 ± 100c2 6100 ± 100d1 8400 ± 200c3

Values as mean ± SD. Values with different alphabet superscript along a column were significantly different between concentrations; and values with different
number superscript across a row were significantly different (p < 0.05).

ivermectin (Odo et al., 2020). The no significant effect obtained for CF
and HSI in the present study may indicate that CLO does not have major
effect on the length-weight relationship and on the liver weight relative
to the fish body weight respectively.
4.2. Haematological parameters
Haematological parameters have proven to be very useful indicators
of pollution and physiological dysfunction in environmental and
aquaculture studies (Burgo-Aceves et al., 2019). Observable variations
in these parameters provide information on fish health status after ex-
posure to pharmaceuticals (Nwani et al., 2014a,b; Ogueji et al., 2017).
The present study revealed a significant decrease in the values of red
blood cell (RBC) and haemoglobin (Hb) in the CLO-exposed fish when
compared with the control. Red blood cells (also known as ery-
throcytes) carry haemoglobin to supply oxygen to all tissues and or-
gans. The studies of various vertebrates including fish have shown that
these organisms possess a very unique erythropoietic response to en-
vironmental stress. The significant reductions in RBC and Hb in the
exposed fish are indications of impairment in the generation of ery-
throcytes. This may have altered the proper functioning of the red
blood cells leading to anaemic diseases in the fish. Burkina et al. (2016),
have also reported that CLO significantly lowered the RBC counts of
juvenile rainbow trout (O. mykiss) exposed to very low concentrations
(0.01, 1.0 and 10 μgl−1) of CLO. They noted this to be the main effect of
CLO on haematological parameters as the drug could not have pro-
nounced effect on the other blood parameters probably because of the
very low concentrations of CLO that the fish were exposed to. The
significant reduction in the haemoglobin levels in the present study
may be as a result of the inhibition of the haemoglobin synthesis. This
may lead to decreased oxygen supply to the tissues and organs of the
fish. Other authors have also reported similar effects of pharmaceuticals
on RBC and Hb in fish (Li et al., 2011b; Ogueji et al., 2019). The ob-
served significant reduction in the values of packed cell volume (PCV)
and mean corpuscular volume (MCV) in this study may be as a result of
anaemia and impaired osmoregulation across the gill epithelium due to
accumulation of CLO in the gill region of the exposed fish (Nwani et al.,
2015). On the other hand, significant increases were observed in mean
corpuscular haemoglobin concentration (MCHC), and mean corpus-
cular haemoglobin (MCH). Red cell indices (MCV, MCH and MCHC) are
important anaemic diagnostic tools in fish (Coles, 1986; Ogueji et al.,
2017). Variations in these blood indices signify different types of

Table 3
Changes in white blood cell differentials of Clarias gariepinus exposed to clotrimazole.
Parameter Conc. (mgl−1) Duration (day)

1 7 14 21 7-Day recovery

a1 b12 a1 b12
N (%) Control 61.00 ± 1.00 59.00 ± 1.00 58.50 ± 1.50 59.00 ± 1.00 58.50 ± 1.50b1
1.94 58.50 ± 0.50b1 61.00 ± 1.00ab2 61.00 ± 1.00a2 62.00 ± 0.00a2 61.00 ± 1.00a2
3.89 62.00 ± 2.00a1 61.00 ± 2.00ab1 60.50 ± 3.50a1 61.50 ± 1.00a1 62.50 ± 0.50a1
7.76 57.50 ± 0.50b1 63.00 ± 1.00a2 59.00 ± 1.00a1 58.00 ± 0.00a1 59.00 ± 1.00b1
L (%) Control 35.50 ± 1.50ab1 38.50 ± 1.50a1 38.50 ± 0.50a1 37.00 ± 0.00a1 38.00 ± 0.00b1
1.94 37.00 ± 0.00a2 34.50 ± 2.50ab1 35.50 ± 0.00a12 34.00 ± 1.00b1 34.50 ± 0.50b1
3.89 34.00 ± 2.00b1 33.00 ± 2.00ab1 34.50 ± 3.50a1 34.00 ± 1.00b1 34.00 ± 1.00a1
7.76 37.50 ± 0.50a4 32.00 ± 1.00b1 35.50 ± 0.50a2 37.00 ± 1.00a34 38.00 ± 0.00a23
M (%) Control 2.00 ± 0.00ab1 2.50 ± 0.50b1 2.50 ± 0.50a1 2.50 ± 0.50a1 2.50 ± 0.50a1
1.94 3.00 ± 1.00b1 1.50 ± 0.50c1 2.00 ± 0.00a1 2.50 ± 1.50a1 2.50 ± 0.50a1
3.89 1.50 ± 0.50a1 3.50 ± 0.50b3 1.50 ± 0.50a1 2.50 ± 0.50a2 2.00 ± 0.00a12
7.76 3.00 ± 0.00a1 2.50 ± 0.50a1 3.00 ± 1.00a1 2.50 ± 0.50a1 2.50 ± 0.50a1
E (%) Control 1.50 ± 0.50b1 2.00 ± 0.00a12 2.50 ± 0.50a12 1.50 ± 0.50a1 3.00 ± 1.00a2
1.94 1.50 ± 0.50b1 3.00 ± 1.00a2 2.00 ± 1.00a12 1.50 ± 0.50a1 2.00 ± 0.00a12
3.89 2.50 ± 0.50a12 2.00 ± 0.00a12 3.00 ± 1.00a2 2.50 ± 0.50a12 1.50 ± 0.50a1
7.76 2.00 ± 0.00ab1 2.50 ± 0.50a1 2.50 ± 0.50a1 2.50 ± 0.50a1 2.00 ± 1.00a1

Values as mean ± SD. Values with different alphabet superscript along a column were significantly different between concentrations; and values with different
number superscript across a row were significantly different (p < 0.05). N = neutrophil, L = lymphocytes, M = monocytes, E = eosinophil.

4
T.D. Melefa, et al. Comparative Biochemistry and Physiology, Part C 236 (2020) 108815

Table 4
Changes in red blood cell indices of Clarias gariepinus exposed to clotrimazole.
Conc. (mgl−1) Duration (day)

1 7 14 21 7-Day recovery

a2 a2 a12 a12
MCV (fl) Control 53.07 ± 3.06 52.65 ± 2.86 49.47 ± 0.53 51.26 ± 5.00 45.33 ± 3.66b1
1.94 58.68 ± 0.76a2 47.49 ± 0.23b1 45.55 ± 2.10b1 46.30 ± 0.04b1 55.70 ± 3.56a2
3.89 51.12 ± 0.03b3 44.03 ± 1.19b2 41.58 ± 1.61c1 43.50 ± 0.08b12 49.76 ± 1.43b3
7.76 44.49 ± 4.13c23 45.11 ± 3.51b23 41.65 ± 1.19c12 39.72 ± 0.82c1 48.12 ± 0.02b3
MCH (pg) Control 11.00 ± 0.27b1 10.98 ± 0.09c1 11.17 ± 0.09b12 11.31 ± 0.00d12 10.98 ± 0.05ab2
1.94 11.31 ± 0.03ab1 11.89 ± 0.18ab23 11.48 ± 0.40ab12 12.29 ± 0.13c2 11.54 ± 0.35c4
3.89 11.51 ± 0.33ab1 11.66 ± 0.42bc1 11.79 ± 0.58ab12 12.79 ± 0.14b5 11.74 ± 0.59b2
7.76 12.04 ± 0.83a1 12.55 ± 0.70a12 12.20 ± 0.51a1 13.38 ± 0.13a4 12.56 ± 0.11a1
MCHC (g/dl) Control 20.80 ± 1.70bc1 20.83 ± 0.87d1 22.59 ± 0.44d12 22.20 ± 2.18d12 24.34 ± 2.09ab2
1.94 19.96 ± 0.33c1 24.99 ± 0.24c3 25.21 ± 0.26c3 26.54 ± 0.31c2 20.75 ± 0.69c4
3.89 22.51 ± 0.63b1 26.48 ± 0.25b3 28.29 ± 0.30b4 29.39 ± 0.39b5 23.58 ± 0.50b2
7.76 27.16 ± 0.63a12 27.85 ± 0.62a2 29.28 ± 0.37a3 33.66 ± 1.04a4 26.09 ± 0.24a1

Values as mean ± SD. Values with different alphabet superscript along a column were significantly different between concentrations; and values with different
number superscript across a row were significantly different (p < 0.05).

Table 5
Response of liver enzymes of Clarias gariepinus exposed to clotrimazole.
Conc. (mgl−1) Duration (day)

1 7 14 21 7-Day recovery

c1 bc23 d2 d3
ALT (U/L) Control 11.24 ± 0.04 11.45 ± 0.05 11.42 ± 0.50 11.55 ± 0.20 11.94 ± 0.35b4
1.94 11.22 ± 0.50c1 11.42 ± 0.50c2 11.55 ± 0.20c3 12.15 ± 0.20c5 11.72 ± 0.15c4
3.89 11.32 ± 0.15b1 11.57 ± 0.35b2 11.77 ± 0.35b3 12.25 ± 0.20b5 11.92 ± 0.15b4
7.76 11.42 ± 0.15a1 11.76 ± 0.12a2 11.89 ± 0.02a23 12.35 ± 0.02a4 12.04 ± 0.35a3
AST (U/L) Control 11.18 ± 0.75c1 11.63 ± 0.13b2 11.63 ± 0.25d2 11.83 ± 0.25d3 11.90 ± 0.50a3
1.94 11.10 ± 0.50bc1 11.77 ± 0.15b2 11.85 ± 0.50c2 12.02 ± 0.25c3 11.83 ± 0.50b2
3.89 11.43 ± 0.25b1 12.25 ± 0.25b2 12.18 ± 0.25b2 12.38 ± 0.25b3 11.95 ± 0.51a2
7.76 11.43 ± 0.25a1 72.25 ± 0.25a3 12.18 ± 0.25a3 12.38 ± 0.25a3 11.95 ± 0.50a2
ALP (U/L) Control 72.87 ± 0.38c1 75.49 ± 0.75d2 76.21 ± 0.38d23 76.05 ± 0.19b23 76.97 ± 1.50b3
1.94 73.06 ± 0.19c1 76.42 ± 0.19c1 77.11 ± 0.13c1 91.47 ± 0.50a2 77.17 ± 0.58b1
3.89 73.62 ± 0.00b1 77.35 ± 0.00b2 79.04 ± 0.19b3 85.76 ± 0.56a4 78.87 ± 4.10ab23
7.76 74.56 ± 0.19a1 79.04 ± 0.19a2 79.98 ± 0.00a4 88.00 ± 0.56a4 80.16 ± 0.19a3

Values as mean ± SD. Values with different alphabet superscript along a column were significantly different between concentrations; and values with different
number superscript across a row were significantly different (p < 0.05).

Control 1.94mgl-1 3.89mgl-1 7.76mgl-1

Control 1.94mgl-1 3.89mgl-1 7.76mgl-1
140 a5 b5
7 a4
b4 b4 ab5
a1 a2 a2 a3 a3 a2 b2
b3 a2 a3 120 b23 c3
Total protein (mgdl-1)

6 b2 c3 b3 c23 c2
c3 b4 d2
Glucose (mgdl-1)

b2 c4 a1
d3 d3 c2 a2 a1 100 a1
5 d2 c1 d1 b1
c1
4 80
3 60
2 40
1 20
0 0
1 7 14 21 7-day 1 7 14 21 7-day
recovery recovery
(Day) Duraon (Day)

Fig. 2. Response of Clarias gariepinus glucose to clotrimazole exposure.Values as

Fig. 1. Response of Clarias gariepinus total protein to clotrimazole exposure.
mean ± SD. Bars with different alphabet label per day were significantly dif-
Values as mean ± SD. Bars with different alphabet label per day were sig-
ferent between concentrations; and values with different number labels per
nificantly different between concentrations; and values with different number
experimental group were significantly different (p < 0.05).
labels per experimental group were significantly different (p < 0.05).

substances, production, transportation and distribution of antibodies in

anaemia (macrocytic or microcytic). The increased MCH and MCHC in
immune response (Ajima et al., 2016; Burgo-Aceves et al., 2019).
the exposed group observed in this study may indicate macrocytic
Changes in white blood cell affect the maintenance of functional im-
anaemia. A similar trend was reported by Ogueji et al. (2017), when C.
mune system in fish (Liu et al., 2017). The significant increase in WBC
gariepinus was exposed to sublethal concentrations of diazepam. In this
in the exposed group may indicate an immune system response to the
study, a significant increase in the white blood cells was observed
toxic effects of CLO. Similar findings have been reported by Nwani et al.
among the exposed groups. The white blood cells have many functions
(2014b) in C. gariepinus exposed to chloramphenicol; Ajima et al., 2016
which include defense against infections and invasions by foreign
in Oreochromis niloticus exposed to verapamil and Ogueji et al. (2018) in

5
T.D. Melefa, et al.
C. gariepinus exposed to diazepam. Changes in WBC differential counts
are indications of exposure to environmental stress in living organisms
(Dogan and Can, 2011). The observed mixed trend in the values of
neutrophil, lymphocyte, monocyte and eosinophil in the CLO-exposed
fish may be due to physiological response to the stress imposed by the
exposure to the pharmaceutical (Nwani et al., 2016).
4.3. Biochemical parameters
The liver plays a major role in metabolism and detoxification of
xenobiotics. Exposure to toxicants can lead to hepatotoxicity, conse-
quently leading to the release of liver enzymes into the bloodstream. In
this study, the concentration of liver enzymes vis: alanine amino-
transferase (ALT), aspartate aminotransferase (AST) and alkaline
phosphatase (ALP) increased significantly in the exposed group. The
increase was more obvious at the highest concentration of 7.76 mgl−1.
The concentration remained high even till the 7-day post withdrawal in
all the exposed groups. The observed increase in the activities of these
enzymes may be an indication of the hepatotoxic effects of CLO on the
exposed fish. The results from this study agree with the findings of
Ajima et al. (2015) who reported an increase in the levels of AST and
ALT in fish exposed to chronic levels of diclofenac. Nwani et al. (2015)
also reported an elevated level of plasma ALT and AST in carbendazim-
exposed fish. A significant decrease in protein levels was observed in
this study. This may be as a result of the toxic effects of CLO on the liver
of the exposed fish leading to the inhibition of protein synthesis
(Lavanya et al., 2011; Nwani et al., 2015). The decrease may also be
due to metabolic utilization of the ketoacids for synthesizing glucose in
order to compensate for the high energy requirements imposed by the
drug (Dogan and Can, 2011; Ogueji et al., 2017). This observation
agrees with the findings of Nwani et al. (2013), Ajima et al. (2016) and
Ogueji et al. (2017). A significant increase (p < 0.05) in the blood
glucose level was observed on days 14 and 21 respectively in this study.
The significant difference was retained even on withdrawal of the drug
for 7 days. This may be as a result of the disruption in normal glycolysis.
The increase is an indication of hyperglycaemia (Tsatsakis et al., 2009;
Nwani et al., 2015). Similar trends have been observed by other authors
in catfish exposed to different pharmaceuticals (Ajima et al., 2015;
Ogueji et al., 2020; Odo et al., 2020).
5. Conclusion
The results of the present study demonstrated that exposure of C.
gariepinus to sublethal concentrations of the pharmaceutical drug, CLO,
elicited changes in haematological and biochemical parameters of the
fish but has no significant effect the morphometric parameters. The
studied parameters may help to improve risk assessment strategies in
evaluating the toxicological effects of CLO on non-target species. The
occurrence and distribution of environmental contaminants like CLO
should be closely monitored especially in developing countries where
most of the wastes from industries, hospitals and homes are discharged
into the environment without treatment. Further studies using other
biomarkers in the different fish tissues could improve our under-
standing on the toxicodynamics of the drug.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cbpc.2020.108815.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
6
Comparative Biochemistry and Physiology, Part C 236 (2020) 108815
Acknowledgements
The authors will like to appreciate the Head of Zoology and
Environmental Biology Department, University of Nigeria, for permis-
sion to have access to laboratory facilities.
Disclosure statements
All experiments were carried out in accordance with the principles
guiding the use and handling of experimental animals as approved by
the University of Nigeria ethical committee.
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