Sublethal effects of the herbicide thiobencarb on fecundity, histopathological and biochemical changes in the African catfish (Clarias gariepinus)
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Iranian Journal of Fisheries Sciences 19(3) 1589-1614 2020
DOI: 10.22092/ijfs.2018.119669.
Sublethal effects of the herbicide thiobencarb on fecundity,
histopathological and biochemical changes in the
African catfish (Clarias gariepinus)
Elias N.S.1; Abouelghar G.E.2*; Sobhy H.M.3;
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El Miniawy H.M.4; Elsaiedy E.G.5
Received: July 2017 Accepted: September 2018
Abstract
Fishes have been widely documented as useful bioindicator for ecotoxicological studies
because of their differential sensitivity to pollution. Thus, the present study was carried
out to determine the sub-lethal effects of commercial herbicide thiobencarb (CITRON®,
50EC) on the African catfish (Clarias gariepinus). Females fish were exposed to sub-
lethal concentration of thiobencarb (½ LC50, 0.72 ppm) for 3, 9 and 15 days. Severe
abnormality in the swimming behavior was observed in fish groups exposed for 9 and
15 days. Adverse effects on the ovary and liver weights were observed. The absolute
fecundity was significantly decreased by all thiobencarb-treatments, for which the
lowest value was observed at 15-day treatment in comparison with that of the control.
A significant decline (p0.01) in the activities of serum aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) was observed in fish as the exposure
period continued as compared to the control. Similarly, glucose and total protein levels
were also significantly declined (p0.01) with the exposure period. Histopathological
changes in the liver tissue of fish exposed to thiobencarb were characterized by
necrosis, changes in nuclear shape, formation of vacuoles and atrophy of hepatocytes.
The ovary of fish exposed to thiobencarb for 15 days showed atretic vitellogenic
oocytes and proliferation of follicular cells as well as inflammatory cells infiltration.
These results indicate that thiobencarb is toxic and has the potential to impair on the
physiological activities in African catfish. Therefore, the use of thiobencarb should be
strongly controlled and carefully monitored to minimize its negative impacts on the
aquatic ecosystems.
Keywords: Toxicity, Fecundity, Biomarkers, Biochemistry, Histopathology
1-Fish Diseases Department, Animal Health Research Institute, ARC, Dokki, Egypt
2-Department of Pesticides, Faculty of Agriculture, Menoufia University, Shebin-Elkom, Egypt
3-Institute of African Research and Studies -Department of Natural Resources, Cairo University, Egypt
4-Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt
5-General Authority for Fish Resources Development, Nasr City, Egypt
*Corresponding author's Email: gamal.abouelghar@agr.menofia.edu.eg
1590 Elias et al., Sublethal effects of the herbicide thiobencarb on…
Introduction Due to bioaccumulation of herbicides,
The use of herbicides to control weeds the concentration of herbicide in fishes
has been documented as a part of may become high enough to induce
agricultural practices throughout the toxic responses in fishes and also in
world (Nwani et al., 2010). These humans who may use these fishes for
herbicides can enter aquatic ecosystems consumption. Fishes may also be
as a result of the intensive and poorly considered good bioindicators for
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regulated usage causing harmful effects ecosystem health (Cavas, 2011). Fishes
on fish population, cellular functions, species are the most sensitive to aquatic
growth and behavior of aquatic pollutants during their early life stages
organisms (Hanazato, 2001; Zou, 2003; (Jiraungkoorksul et al., 2002). The
Relyea, 2005). Presently, there is an African catfish, Clarias gariepinus
increasing world concern over the selected in the present study seems to
indiscriminate use of the herbicides that occur nearly in all water bodies
result in environmental pollution and inhabited by fishes. It is also one of the
toxicity risk to non-target organisms most popular fishes cultured in Egypt
(Velisek et al., 2009). Thiobencarb [S- and other African countries next to the
((4-chlorophenyl) methyl) diethyl- tilapiine fishes (Tesfaye, 1998;
carbamothioate] is one of the most Nyamweya et al., 2010). This species is
commonly used herbicides against grass an aquaculture candidate that can
weeds (Johnson, 1997; Rao et al., narrow the gap between the demand
2007). This herbicide belongs to the and supply of animal protein in
thiocarbamate herbicides and is developing countries (Odo et al., 2017).
classified into group N of the Herbicide It is also considered an attractive model
Resistance Action Committee (HRAC, for toxicity studies because of its
2010). According to the HRAC, the availability throughout the year,
target site of the group N herbicides is voracious feeding habit, prolific
described as the lipid synthesis except reproduction and general hardness in
for acetyl-CoA carboxylase (ACCase). culture environments (Nwani et al.,
It is a systematic, pre-emergence 2015). It can easily acclimatize to
herbicide that acts by inhibiting shoots laboratory conditions. Thus, it is an
of emerging seedlings (USEPA, 1997). excellent model for ecotoxicological
The commercial formulation of studies.
®
thiobencarb (Citron 50%) is being Previous studies have investigated
used extensively in Egypt, at 1.5-4.5 kg the toxicological effects of several
10,000 m-2, to control weeds in herbicides in fishes (e.g. Nwani et al.,
transplanted rice before seeding and 2010; El-Sayed et al., 2013; Nwani et
early post-emergence application al., 2015; Sherif et al., 2015). However,
(Anonymous, 1993). the sub-lethal effects of thiobencarb on
Fishes, as one of the most C. gariepinus fish have not been
representative organism in freshwater, completely shown. Thiobencarb is
are often on the top of the trophic level. defined as moderate to high toxicity to
Iranian Journal of Fisheries Sciences 19(3) 2020 1591
fish by Villalobos et al. (2000), because relative liver size and relates to the
it resists hydrolysis in aquatic hepatic enzyme activity for chemical
environments and is bioaccumulated is detoxification (Fang et al., 2009). Some
in fish in vivo. However, chronic reports have demonstrated that CF
toxicity effects of thiobencarb on C. declined in fish exposed to
gariepinusare still not precisely known. environmental pollutants (Khan, 2003).
To evaluate toxic stress of The hepatosomatic index (HSI) reflects
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environmental contaminants, the relative liver size and is linked to
biochemical parameters have been the hepatic enzymes activities for
widely used. These parameters are detoxification of compounds, indicating
including the levels of plasma proteins, exposure to pollutants (Yeom et al.,
glucose, and other enzymes, like 2007; Fang et al., 2009). Toxic
alanine aminotransferase (ALT) and substances in the water may affect the
aspartate aminotransferase (AST) fish growth by changing metabolism
(Burtis and Ashwood, 1996; El-Sayed directly and increasing the energy
et al., 2007; Kumari et al., 2011; Nwani required to maintain homeostasis, or
et al., 2015). Therefore, biochemical they can indirectly influences growth
analysis of blood plasma are useful in by reducing food availability
monitoring the physiological status of (Sadauskas-Henrique et al., 2011).
fish, health in the aquatic environment, Previous studies related to the effects
and considered as an important of pesticides on fish reproduction are
biological factors in aquaculture rare and do not comprise the diverse
(Hochachka and Mommsen, 1995). range of events involved in
Biochemical markers such as stress reproduction such as oocyte maturation,
proteins and metabolic enzymes spawning, fecundity and fertilization
generally reflect stress conditions in (Mir et al., 2011). Pesticide-induced
fish induced by a broad range of reproductive failure or dysfunction is
environmental factors (Schwaiger et al., evident from the available reports on
1997; Marchand et al., 2008; Velisek et Indian fishes (Singh and Singh, 1982;
al., 2013). Singh et al., 1997). Mir et al. (2011)
Morphological indices, especially investigated the effects of sub-lethal
condition factor (CF) and HSI, have concentrations of the organophosphate
been proposed as an “exposure index” pesticide dimethoate on the
to environmental contaminants gonadosomatic index (GSI) of the fish,
(Kopecka and Pempkowiak, 2008; Cyprinus carpio communis. The
Nwani et al., 2015). The CF, a somatic maximum reduction in GSI values was
biomarker, is indicative of health and obtained at the highest concentrations
reflects feeding conditions as well as of dimethoatein of the treated fish
energy consumption and metabolism ovaries showed histomorphological
(Schulz and Martins-Junior, 2001; disorders. Furthermore, the reduced
Alberto et al., 2005). The GSI was found directly proportional to
hepatosomatic index (HIS) refers to the the pesticide concentration and the
1592 Elias et al., Sublethal effects of the herbicide thiobencarb on…
duration of the exposure (Mir et al., both adaptive processes and detrimental
2011). The sub-lethal effects of another effects in fish induced by organic
insecticide, fipronil, on the reproduction pollutants (Cengiz and Unlu, 2006).
dynamics of the zebrafish (Danio rerio) However, scarce information is
were also reported (Boaru et al., 2013). available on histopathological changes
The effects on reproductive parameters in fish ovaries due to herbicides'
were evident even at the lowest toxicity and particularly thiobencarb.
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concentration of fipronil when applied, Singh (2015) showed that exposure of
while exposure to the highest dose common carp (C. caprio) to sub-lethal
decreased the spawning, fecundation concentrations of dimethoate, for 96 h
and hatching percentages in comparison and 36 days, caused considerable
with the control group (Boaru et al., structural damage to ovaries, which
2013). included breaking of ovigerous
Histopathology provides a rapid lamellae, lifting and fragmentation of
method to detect effects of irritants, follicular lining and zona radiata of
especially chronic ones, in various mature oocytes.
organs (Johnson et al., 1993; Bernet et Thus, this study investigated the
al., 1999). Greenfield et al. (2008) impact of sub-chronic toxic effects of
explained that the histological the herbicide thiobencarb on biometric
alterations in selected target organs are indices, fecundity, serum biochemical
sensitive biomarkers for xenobiotic analysis, and histopathology in the
effects, they occurred earlier and African catfish (C. gariepinus), an
provided a better evaluation of the economically important freshwater fish
effects of aquatic pollution than any worldwide.
single biochemical parameter. The
histopathological pictures of the organs Materials and methods
can corroborate with the biochemical Experimental fish and chemical
changes accounting for the functional The adult African catfish, Clarias
disruptions in the activity of the organs gariepinus, with 205.0±4.03 g mean
due to cellular damage (Kalaiyarasi et weight and total length 34.2±0.16 cm
al., 2017). The exposure of fish to were purchased from a private
chemical contaminants likely induces a commercial catfish farm and used as
number of lesions in different organs laboratory models. They were
(Bucke et al., 1996). Since fish liver is transported and placed in large plastic
regarded as a major site of storage, containers to the Biology Unit, Fish
biotransformation and excretion of Diseases Department at Animal Health
pesticides, histological changes in the Research Institute, Dokki, where they
liver were chosen as criteria for the sub- were acclimatized for 3 weeks in plastic
lethal action of thiobencarb. tanks (capacity of 300L). Water was
Histological investigations of fish liver renewed daily to help C. gariepinus
repeatedly proved to be an acclimatize to the new environment.
extraordinarily sensitive tool to reveal The fish were fed daily with a
Iranian Journal of Fisheries Sciences 19(3) 2020 1593
commercial pellet diet (35% crude toxicity test, the mortality and survival
protein) at 3% of their body weight rates of the fish were recorded daily
twice a day. The water used was under each test concentration. Fish are
analyzed weekly by standard methods considered dead if there is no visible
(Eaton, 2005) for temperature, movement (e.g. gill movements) and if
dissolved oxygen, hardness and pH, touching of the caudal peduncle making
which were recorded as 23.5±1.5°C, no reaction. Dead fish are removed to
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7.5±0.4 mg L-1, 230.5±4.5 mg L-1 as avoid pollution of the water. The LC50-
CaCO3 and 7.8±0.5, respectively. 96 h value of thiobencarb was
During the acclimatization period, determined using the PC-Probit
experimental fish were monitored for software based on Finney's Probit
disease conditions and mortality analysis procedure (Finney, 1971).
(OECD, 1992). In the present study, a
commercial formulation of thiobencarb Sub-lethal toxicity tests
herbicide (CITRON® 50 EC) Based on the 1.44 mg L-1 (LC50-96 h-
manufactured by Rhône-Poulenc Ag acute toxicity) obtained value, a sub-
company, was used as stock solution. lethal concentration of 0.72 mg L-1
corresponding to ½ LC50 was used for
96 hour-acute toxicity test this study. A total 120 acclimatized-
To determine the LC50-96 h value of fish females of mean weight 215.0±3.5
thiobencrab for the African catfish, g were used in this experiment. The fish
acute toxicity, bioassays were were divided into four groups at which
conducted in 40 L glass aquaria a set of 30 fish were introduced in
(60×30×30 cm size) in a semi-static triplicate treatments (10 fish/ replicate)
laboratory system. The study was for each group in a 50 liter- capacity
conducted according to the aquarium. Fish in Group-1 served as
Organization for Economic control which was held in tap water
Cooperation and Development (OECD, with no pesticide exposure. Fish in
1992), guideline No. 203. A set of 10 treated groups were exposed to the
fish specimens were randomly exposed selected concentration of thiobencarb
to different concentrations (0, 0.35, for different periods as follows: 3 days
0.70, 1.40, 2.80 and 5.60 mg L-1) (Group-2), 9 days (Group-3) and 15
obtained by serial dilution of the stock days (Group-4). Semi-static system was
solution. The experiment was set in used in this experiment at which the test
triplicate to obtain the LC50-96 h value solution was renewed every two days
of thiobencarb exposure for African (Nwani et al., 2015). During the
catfish. Feeding was stopped 24 h prior experiment, fish were fed with
to and during the 96-h exposure period commercial pellet feed at 3% of body
in order to prevent interference with mass once a day and mortality was
stomach contents and wastes in the fish recorded. To prevent oxygen depletion,
culture water (Smith et al., 2007; experimental tanks were continuously
Olufayo, 2009). During the acute oxygenated by using an air pump. Dead
1594 Elias et al., Sublethal effects of the herbicide thiobencarb on…
fish were immediately removed to hepatopancreas were dissected and
avoid possible deterioration of the weighed in order to determine the GSI
water quality. During the test period, and HSI for each fish according to the
fish behavior was observed daily for following formulae:
hyperactivity, equilibrium status, GSI (%)=(Gonad weight/BW×100) and
swimming rate, fins movement, jerky HIS (%)=(Liver weight/BW×100)
movements, air gulping and skin
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discoloration. The water temperature, Estimation of fecundity
pH, dissolved oxygen concentration and Fecundity has been considered as the
salinity were measured on each renewal number of ripening eggs in the
day. female prior to spawning (Bagenal
and Braum, 1978). Gravimetric method
Biochemical analysis was used for estimation of the fish
At the end of the duration of each fecundity (Bithy et al., 2012).Three
exposure, 4-6 fish from each replicate matured female samples were randomly
of each group were randomly selected selected from each group. Then these
for blood sampling. Blood was matured females were dissected and the
collected by puncturing the caudal vein whole gonads from each specimen were
with a 2 mL- plastic heparinized removed out intact. The matured
syringe needle. The blood was emptied ovaries were weighed nearest gm by a
into 5 mL- heparinized blood bottles sensitive scale. These ovaries were
without any anticoagulant and then split longitudinally and kept in
centrifuged at 1,000 xg for 10 minutes Petri-dish. Then, weights of right and
to obtain the serum for analysis. The left gonad were measured. Three
enzymatic aspartate AST and ALT samples, each was taken from the
activities were analyzed according to anterior, middle and posterior regions
the method of Reitman and Frankel of each ovaries and kept in Petri-dish.
(1957). Serum protein and glucose All the ovaries were kept in Petri-dish
levels were analyzed according to the with few drops of Gilson’s fluid for 10
methods of Young (1990) and Schmidt minutes with periodical shaking. The
and Schmidt (1963), respectively. All number of ripen eggs (F1) for the sub-
chemical reagent-kits were purchased sample was estimated by using the
from BioMed Diagnostics, Giza, Egypt. following equation (Yelden and Avsar,
2000):
Determination of biometric indices Fecundity (F1)=[(No. of eggs in sub-
Immediately after blood sampling, body sample×gonad weight)/Weight of sub-
weight (BW) and body length (BL) sample]
were recorded. The CF of each fish was Later, by taking the mean number of
calculated according to the formula: three sub-samples (F1, F2, F3), the total
CF=BW (g)/ BL (cm)3 100. After (absolute) fecundity for each female
measuring the fish biometric fish was estimated F= (F1+F2+F3)/3.
characteristics, both ovaries and In addition, the relative fecundity
Iranian Journal of Fisheries Sciences 19(3) 2020 1595
parameters were also estimated for each Results
fish as absolute fecundity (F) divided Toxicity and behavioral changes of fish
by: body length (FBL), body weight The 96 h-acute toxicity test of
(FBW) and gonad weight (FGW). thiobencarb herbicide to C. gariepinus
adult females showed that LC50-96 h
Tissue histopathological analysis value was found to be 1.44 mg L-1. The
At the end of each exposure period, fish sub-lethal concentration (½ LC50) of
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were randomly selected for thiobencarb affected behavioral
histopathological examinations using characteristics of C. gariepinus. The
the routine standard histological control specimens were not hyperactive
technique (Bancroft and Cook, 1994). and showed normal swimming patterns,
Liver and ovaries tissues were isolated color and fins movements throughout
from the control - and thiobencarb- the exposure period. Several distinct
treated groups. Physiological saline unusual swimming behavior and
solution (0.75% NaCl) was used to increased deformities in fish exposed to
rinse and clean up the tissues. They sub-lethal concentration of thiobencarb
were fixed in neutral buffered formalin were observed during the exposure
(10% NBF) for 48 hours. The samples period including: lack of balance,
fixed in formaldehyde solution were agitated or jerky swimming, air
processed through graded series of gulping, sudden quick movement,
alcohols, cleared in xylene and excessive secretion of mucus.
embedded in paraffin wax. The tissues Moreover, the color of fish skin was
were sectioned into thin sections (5-7 changed from normal darkly
µm), dehydrated and stained with pigmentation in the dorsal and lateral
Haematoxylin and Eosin (H&E) stain parts to very light pigmentation in the
(Bancroft and Cook, 1994). dorsal and lateral part, as well as
Histopathological lesions were peeling of the skin was observed.
examined using light microscope and Severe abnormality in swimming
photographed. behavior was observed in fish groups
exposed for 9 and 15 days where faster
Statistical analysis opercular movement, surfacing and
Data obtained were subjected to swallowing of air were observed. With
statistical analysis using one-way increase in duration of the exposure,
analysis of variance (ANOVA) and swimming and body movements were
means separated by Fishers Least retarded and copious mucus was
Significant difference test at 95% secreted and deposited in the buccal
probability. Statistical analyses were cavity and on the gills.
performed using a computer program
SPSS, version 14 for Windows. Biometric parameters
There was a significant reduction
(p0.01) in the mean body weight of C.
gariepinus from groups exposed to
1596 Elias et al., Sublethal effects of the herbicide thiobencarb on…
selected concentration of thiobencarb exposure periods in comparison with
for periods of 9 and 15 days in that of the control. The most significant
comparison with that of control group decrease (p0.01) in the CF (0.4) was
(Table 1). Ovarian weight revealed in the fish exposed for 15 days in
highly significant (p0.01) decrease in comparison with that of the control
C. gariepinus females exposed to (0.8). Also, the results presented in
thibencarb in all three different groups Table 1 showed that mean GSI values
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in comparison with the control group. were significantly (p0.01) decreased
Mean ovarian weights of fish from in fish females from all three groups
group exposed to thiobencarb for 3 and exposed to thiobencarb as compared to
15 days were highly decreased by 87.1 that of the control. The GSI values were
and 89.5% , respectively, than that of 2.8, 3.5 and 2.8 in fish exposed to
the control. Mean hepatic weight was thiobencarb for periods of 3, 9, and 15
also significantly decreased (p0.01) in days, respectively, as compared to that
fish from groups exposed to herbicide of the control (17.0). However, there
for 9 and 15 days as compared to that of were no significant differences (p0.05)
the control. Likewise, there was a in the mean body length, HSI and CF
significant reduction (p0.01) in the between thiobencarb-treatments and the
condition coefficient (CF) in the fish control group.
exposed to thiobencarb for different
Table 1: Growth parameters, condition coefficient (CF), gonadosomatic index (GSI) and
hepatosomatic index (HSI) in catfish females (Clarias gariepinus) after exposure to
thiobencarb herbicide for different periods (mean±SE).
Body Ovarian Hepatic
Exposure Body weight
length weight weight GSI HSI CF
period (g)
(cm) (g) (g)
Control 230.1 ± 30.0 33.0 ± 1.2 42.0 ± 1.1 3.3 ± 0.03 17.0 ± 3.3 1.1 ± 0.0 0.8 ± 0.0
3-days 229.3 ± 3.1 35.0 ± 3.3 5.4 ± 1.2* 2.5 ± 0.6 2.8 ± 1.1* 1.2 ± 0.1 0.5 ± 0.1*
9-days 156.5 ± 5.2* 32.0 ± 2.2 13.0 ± 5.2* 1.6 ± 0.4* 3.5 ± 1.0* 1.1 ± 0.1 0.5 ± 0.1*
15-days 169.0 ± 4.4* 34.0 ± 3.0 4.4 ± 1.2* 1.7 ± 0.6* 2.8 ± 0.9* 1.0 ± 0.2 0.4 ± 0.03*
*Significant differences compared with control value, p
Iranian Journal of Fisheries Sciences 19(3) 2020 1597
Table 2: Relative and absolute fecundity of catfish females (Clarias gariepinus) after exposure to
thiobencarb herbicide for different periods (mean ±SE).
Exposure
FBW FBL FGW Absolute fecundity (F)
period
Control 1969.9 ± 5.3 3814.4 ± 3.5 9899.8 ± 1.9 33135 ± 2.0
3-days 1506.6 ± 2.2* 3541.1 ± 4.4* 1489.8 ± 4.4* 532.5 ± 2.2*
9-days 1263.3 ± 2.3* 3451.7 ± 4.4* 1767.7 ± 6.6* 1090.9 ± 4.0*
* * *
15-days 1125.5 ± 2.5 3474.7 ± 6.6 2560.6 ± 2.5 181.8 ± 6.6*
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*Significant differences compared with control value, p1598 Elias et al., Sublethal effects of the herbicide thiobencarb on…
showed normal typical paranchymatous treatment, the liver demonstrated
appearance. The liver was made up of dissociated hepatocytes and few
hepatocytes that were polygonal cells melanomacrophage cells infiltration in
with a central spherical nucleus and the portal area (Fig. 1-C). After 15 days
densely stained nucleolus. The liver of of treatment, there was karyopyknosis
fish exposed to thiobencarb for 3 days of hepatocytes with increased number
showed vacuolar degeneration and of melanomacrophages and
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necrobiotic changes in hepatocytes and mononuclear cells infiltration in portal
few mononuclear cells infiltration in the areas of the liver (Fig. 1-D).
portal area (Fig. 1-B). After 9 days of
A B
C D
Figure 1: (A) Liver tissue of control fish showing normal histological structure of the portal vein with
hepatocytes in the hepatic parenchyma, a quite homogeneous cytoplasm and sinusoids. H&E ×100.
(B) Liver tissue of Clarias gariepinus exposed to 0.72 mg L-1 thiobencarb for 3 days shows vacuolar
degeneration, necrosis of the hepatic cells rupture of sinusoids, and few mononuclear cells
infiltration in the portal area. H&E, 100. (C) Liver tissue of C. gariepinus exposed to 0.72 mgL-
1
thiobencarb for 9 days shows dissociation of hepatocytes, few melanomacrophages and
mononuclear cells infiltration in the portal area (H and E 400). (D) Liver tissue of C. gariepinus
exposed to 0.72 mg L-1 thiobencarb for 15 days shows karyopyknosis of hepatocytes,
melanomacrophages and mononuclear cells infiltration in the portal area (H and E 200).
The histological structure of ovaries in vitellogenic and oocyte stages is shown
the C. gariepinus from thiobencarb- in Fig. 2-A. The ovaries of C.
exposed and control groups are shown gariepinus treated with thiobencarb for
in Fig. 2 (A-E). Normal histological 3 days showed many oocytes in the
structure of the ovaries in the control previtellogenic stage and atretic
group with pre-vitellogenic, vitellogenic oocytes with an increasedIranian Journal of Fisheries Sciences 19(3) 2020 1599
size of follicular cells as well as vitellogenic oocytes with an increase in
liquefaction of yolk globules (Fig. 2-B). size of the follicular cells and
After 9 days of treatment, an increase in liquefaction of yolk globules were
atretic ovarian follicles was shown in observed (Fig. 2-D). In addition,
addition to presence of mononuclear inflammatory cells infiltration were
cells and melanomacrophages in observed in the interstitium of the
interstituim of the ovary (Fig. 2-C). ovaries (Fig. 2-E).
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After 15 days of treatment, atretic
A
B C
*
*
D E
*
*
Figure 2: (A) Histological section of ovary in the control group shows vitellogenic oocytes with moderate numbers of
vitelline granules, few perinucleolar oocytes, and cortical alveoli oocytes (H and E 200). (B) Ovarian tissue of C.
gariepinus exposed to 0.72 mg L-1 thiobencarb for 3 days shows chromatin-nucleolus and, perinucleolar oocytes
(previtellogenic stage), two atretic vitellogenic oocytes with an increased size of follicular cells and liquefaction of
yolk globules (asterisk) (H and E 200). (C) Ovarian tissue of C. gariepinus exposed to 0.72 mg L-1 thiobencarb for
9 days shows chromatin-nucleolus and, perinucleolar oocytes, atresia vitellogenic oocytes with follicular cells
entering the oocyte to phagocytose degenerating material (asterisk), melano-macrophages, interstitial mononuclear
cells (H and E 400). (D) Ovarian tissue of C. gariepinus exposed to 0.72 mg L-1 thiobencarb for 15 days shows
atresia vitellogenic oocytes, an increased ovarian follicle cells, liquefaction of yolk globules and corrugation of
vitelline envelope (asterisk) (H and E 200). (E) Ovarian tissue of C. gariepinus exposed to 0.72 mg L-1 thiobencarb
for 15 days shows inflammatory cell infiltration in the interstitium (H and E 400).1600 Elias et al., Sublethal effects of the herbicide thiobencarb on…
Discussion to that reported by USEPA (2008) on
Thiobencarb is a systemic pre- different fish species: e.g. bluegill
emergence herbicide that is used to sunfish (0.56-2.6 ppm), channel catfish
control broadleaved weeds, annual (2.28 ppm), rainbow trout (1.05-1.5
grasses. It is one of the widely used ppm), depending on percentage of
herbicidesin Egypt to control weeds in technical grade of active ingredient.
rice fields. The evaluation of Morphological and behavioral
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thiobencarb toxicity to the African changes are generally indicative of
catfish, C. gariepinus, is important irreversible damage. The results of the
because this species is widely present study showed several abnormal
distributed in freshwater bodies in swimming behavior and increased
Egypt and they are sensitive to deformities in fish exposed to sub-lethal
pollutants. The majority of the results concentration of thiobencarb (½ LC50)
indicate that thiobencarb is moderately during the exposure period. The
toxic to fish on an acute basis. Fish are observed behavioral changes in C.
often used as sentinel organisms for gariepinus exposed to thiobencarb in
ecotoxicological studies because they the present study, which indicated
play number of roles in the trophic web, internal effects on body physiology,
accumulate toxic substances and may be attributed to a neurotoxic effect
respond to low concentration of of thiobencarb. Several abnormal
Mutagens (Cavas and Ergene- behaviors such as incessant jumping
Gözükara, 2005). Therefore, the use of and gulping of air, losing their balance,
fish biomarkers as indices of the effects consciousness, rolling movement,
of pollution are increasing importance surface to bottom movement, sudden
and can permit early detection of quick movement, remaining in vertical
aquatic environmental problems position for a few minutes with anterior
(Lopez-Barea, 1996; Van Der Oost et side or terminal mouth up near the
al., 2003). Biochemical and surface of the water, trying to gulp air
physiological biomarkers are frequently and tail in a downward position, were
used for detecting or diagnosing sub- similar to the observations of Nwani et
lethal effects in fish exposed to al. (2010). Such behavioral changes in
different toxic substances (De la Torre C. gariepinus were also observed after
et al., 1999). In ecotoxicology, the exposure to paraquat herbicide which
LC50-96 h is one of the most valuable indicated internal effects on body
parameters for assessing the toxic physiology, might be attributed to a
effects of pollutants. Herein, the LC50- neurotoxic effect of paraquat (Nwani et
96 h value (i.e. 1.44 mg L-1) was al., 2015; Ladipo et al., 2011).
obtained from C. gariepinus exposed to Similarly, respiratory stress, erratic
thiobencarb, which indicated that this swimming and death of fish were
herbicide is highly toxic for fish. The observed in juvenile African catfish, C.
LC50 value of thiobencarb obtained for gariepinus, exposed to glyphosate
C. gariepinus in this study is very close herbicide for 4 days which varies withIranian Journal of Fisheries Sciences 19(3) 2020 1601
the toxicant concentration (Ayoola, indicators of overall health in fish and
2008). Pandey et al. (2009) indicated have been used in toxicological studies
that the introduction of toxicant into an as indicators of stress (Nwani et al.,
aquatic system might decrease the 2015). HSI is defined as the ratio of
dissolved oxygen concentration which liver weight to body weight. It provides
will impair respiration leading to an indication on status of energy
asphyxiation. Therefore, death could reserve in animals. In a poor
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occur either by direct poisoning or environmental condition, fish usually
indirectly by making the medium have a smaller liver (with less energy
unconducive for the fish or even by reserved in the liver). HSI has been
both (Nwani et al., 2010). reported the decrease in fish exposed to
The CF and HSI are indicators of high concentrations of cadmium and
overall health in fish and have been zinc (Alkahemal-Balawi et al., 2011).
used in toxicological studies as In the present study, there was no
indicators of stress (Lohner et al., significant difference in HSI between
2001). The condition factor shows the the exposed and the control fish on
degree of wellbeing of the fish in their different exposure periods. Previous
habitat which it is an index expression studies showed that normal HSI values
for the interaction between biotic and were also observed in different fish
abiotic factors in the physiological species: e.g. Carassius auratus exposed
condition of fishes (Olurin and to herbicide alachlor (Yi et al., 2007),
Aderibigbe, 2006). In the present C. carpio exposed to alachlor (Mikula
study, there was a significant reduction et al., 2009; Ensibi et al., 2013) and
(p0.01) in CF on day 15, following Oncorhynchus mykiss exposed to
exposure to 0.72 mg L-1. A similar carbamazepine (Li et al., 2011). Nwani
decrease in CF was reported by Nwani et al. (2015) indicated that normal CF
et al. (2015) in C. gariepinus fish and HSI in fish exposed to paraquat
exposed to paraquat herbicide at 1.37 may indicate that herbicide did not
mg L-1 by day 15 and to 2.75 mg L-1 by affect the liver at the beginning of the
day 5 in comparison with the control. exposure, however, with the
Similar results were reported by Khan progression of the experiment, the liver
(2003) in four species of flatfish was affected and this has indicated in a
inhabiting two areas in Placentia Bay, decline in CF and HSI. Therefore, the
Newfoundland, contaminated either reduction in CF and HSI may indicate a
with polycyclic aromatic hydrocarbons decrease in the overall condition of the
(PAHs) or polychlorinated biphenyls fish, which may be due to the effects of
(PCBs). Also, Roussel et al. (2007) paraquat (Nwani et al., 2015). GSI is a
found that condition factor in three- metric that represents the relative
spined stickleback (Gasterosteus weight of the gonads to the fish weight
aculeatus) was affected at medium and and considered generally a good
high copper concentrations. The indicative of reproductive success
condition factor, HSI and GSI are (Lowerre-Barbieri et al., 2011). It has1602 Elias et al., Sublethal effects of the herbicide thiobencarb on…
been widely used to evaluate used (as biomarkers) to identify
reproduction timing because it is possible environmental contaminations
inexpensive and easy to compute. before the health of aquatic organisms
Gonadosomatic index of fish increases is seriously affected (Jiminez and
with maturation being maximum during Stegeman, 1990; Barnhoorn, 1996). In
peak period of maturity and abruptly toxicological studies of acute exposure,
declines after spawning (Islam et al., changes in concentrations and activities
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2012). In the present study, GSI values of some enzymes may reflect cell
were significantly reduced in fish damage in specific organs (Casillas et
exposed to thiobencarb for different al., 1983; Heath, 1996). Serum
exposure periods as compared to that of aspartate AST and ALT enzyme
the control group. Recently, Silveyra et activities in fish have been used
al. (2017) showed that a lower GSI frequently as bioindicators of toxicant
could be observed in the estuarine crab and contamination of marine
Neohelice granulate fish exposed to ecosystems (Philip and Rajasree, 1996;
different concentrations of atrazine with Kim et al., 2008). ALT and AST are
concentration increase. Concerning the liver specific enzymes and they are
absolute fecundity of C. gariepinus more sensitive measure of
exposed to thiobencarb, the current hepatotoxicity and histopathologic
results revealed a highly significant changes and can be assessed within a
reduction in fecundity rates in all shorter time (Balint et al., 1997). The
treatments. This effect implies a delay changes in the enzyme kinetics of the
in ovarian maturation, caused by organs and blood in fish exposed to
several possible factors. Since highly various pollutants or stressors have
significant decreases in the GSI values been reported by several researchers.
were observed by effect of thiobencarb, e.g. cypermethrin (Velisek et al., 2006),
it seems to be a lower energetic nuracron (Gabriel et al., 2010),
investment in fish reproduction was nonylphenol and octylphenol (Kumaran
happened as a result of the exposure to et al., 2011) and prometryn (Velisek et
this herbicide (Álvarez et al., 2015).The al., 2013). Goel et al. (1982) reported
significant reduction in fish fecundity that the results of reduced activities of
could be also interpreted as a AST, ALT, alkaline phosphatase (ALP)
consequence of the possible hormonal and lactate dehydrogenase (LDH) in
disruption exerted by the herbicide, various organs of fish (Pontius
leading to an arrested oocyte growth conchonius) exposed to Malathion in
(Álvarez et al., 2015). Additionally, various organs implies destruction in
some kind of interference of this the tissues of the animals. Similar
herbicide with the endocrine system results were observed by Hedayati et al.
controlling ovarian growth in fish may (2010) who reported that exposure of
become a more plausible hypothesis. yellowfin sea bream (Acanthopagrus
Biochemical and physiological latus) to sublethal concentrations of
indicators such as enzymes, could be mercury for 3 weeks decreased theIranian Journal of Fisheries Sciences 19(3) 2020 1603
activities of both ALT and AST. ALT significantly increased in C.
Gabriel et al. (2012) demonstrated also gariepinus after exposure for 15 days to
that exposure of C. gariepinus to sub- two sub-lethal concentrations (1/20-
lethal levels of cypermethrin caused an and 1/10- LC50) of paraquat. The
inhibition in the metabolic enzymes, elevated activities of serum AST and
AST, ALT, ALP and LDH in the ALT indicate liver damage or enhanced
organs (gill, kidney, liver) and tissues transamination. Increased
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(muscles plasma). These findings were transamination during pesticide
in agreement with our present study challenge has been attributed to the
where the activity levels of serum AST need to meet higher energy demanded
and ALT in fish exposed to by fish (Philip et al., 1995).Proteins are
thiobencarb, at different periods were involved in major physiological events,
significantly decreased than those so the evaluation of the protein content
reported in the control fish. This was can be considered as a diagnostic tool
correlated by the vacuolar degeneration to determine the physiological indices
and necrobiotic changes with of biota. Blood glucose level in fish is
karyopyknosis found in hepatocytes. known to be very useful as a criterion
Such changes in the enzyme activities for diagnosis of liver and muscle tissue
disrupt physiological and biochemical functions (Shakoori et al., 1996). In the
processes (De la Torre et al., 2005). In present study, both protein and glucose
the present study, the lower values of levels were significantly decreased after
serum ALT and AST in fish exposed to exposure to thiobencarb at different
thiobencarb as compared to the control periods as compared to those in the
suggest a disruption of the transfer of control fish. The reduction in glucose
the α-amino acid group of alanine to α- levels after exposure to thiobencarb
ketoglutarate which results in the might be due to rapid utilization of
formation of pyruvic acid. This implies blood glucose during hyper excitability
that there was a disruption of the in treated fish, as well as the nervous
feeding of amino-acids into energy manifestation which was a
cycle through alanine-pyruvate pathway characteristic behavior of herbicide
representing anaerobic tendency of the toxicity. However, Nwani et al. (2015)
tissues (Ghorpade et al., 2002). In found that glucose was significantly
contrast, increases in AST and ALT increased when C. gariepinus exposed
activities were reported in freshwater to two sub-lethal concentrations of
teleost (Mystus vittatus) after chronic paraquat (1.37 and 2.75 mg L-1) for 15
exposure for 30 days to sub-lethal days, while protein levels declined. The
concentrations of Metasystox (4 ppm) increase of glucose levels may be a
and Sevin (7 ppm) (John, 2007), in physiological response to meet the high
juvenile rainbow trout (O. mykiss) metabolic demands caused by
exposed to 96 h-LC50 of carbamazepine continued exposure to paraquat (Nwani
(Li et al., 2011). Similarly, Nwani et al. et al., 2015). On the contrary, the
(2015) reported that the levels AST and reduction in the protein level may be1604 Elias et al., Sublethal effects of the herbicide thiobencarb on…
associated with liver and kidney structure destruction of liver in
damages caused by toxicant stress and thiobencarb- treated fish clearly shows
the consequent utilization of available the effect of pesticide in destroying the
protein for metabolic activities (Nwani cellular membrane and so necrosis of
et al., 2015). The highly significant liver cells. Similar histopathogical
hypoproteinemia found in fish exposed symptoms were reported by Banaee et
to thiobencarb was in agreement with al. (2013) in gourami fish (Trichogaster
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Abbas et al. (2007) who indicated that trichopterus) when exposed to sub-
the existence of thiobencarb residues in lethal concentrations of parquet. Due to
liver tissue due to its lipophilic nature disturbance in cellular and osmotic
causing its dysfunction. regulation power of cellular and
The literature on histopathological biological membranes, the volume of
effects of thiobencarb on C. gariepinus the nuclei and nucleoli increase and it
fish is somewhat rare. Ayoola (2008) leads to necrosis of liver cells (Ahmad
reported that the liver of C. gariepinus et al., 2000). Necrosis of some portions
exposed to glyphosate concentration of the liver tissue that were observed
showed an infiltration of leukocytes, probably resulted from the excessive
increasing hepatocyte size with work required by the fish to get rid of
pykrotic nuclei, fatty infiltration, the toxicant from its body during the
congested central vein, severe necrotic, process of detoxification by the liver
hemorrhage and vacuolization . In the (Banaee et al., 2013). The inability of
study of done by Risbourg and Bastide fish to regenerate new liver cells may
(1995), the exposure of fish to atrazine also have led to necrosis of hepatic cells
herbicide increased in the size of lipid of sinusoids (Mostakim et al., 2015).
droplets, vacuolization in the liver. The Histological sections of ovaries of
most frequent encountered types of females C. gariepinus exposed to sub-
degenerative changes are those of lethal concentration of thiobencarb
hydropic degeneration, cloudy swelling, showed different levels of alterations
vacuolization and focal necrosis. The with exposure period increase including
liver of the exposed fish had slightly an increase of oocytes in the
vacuolated cells showing evidence of previtellogenic stage with an increased
fatty degeneration. In the present study, size of follicular cells and liquefaction
the results of histopathological of yolk globules and inflammatory cells
responses of liver and ovary in C. infiltration in the interstitium of ovary.
gariepinus exposed to sub-lethal In the study of Avigliano et al. (2014),
concentration of thiobencarb showed the exposure of estuarine crab,
different levels of damage with Neohelice granulate, females to pure
increasing of exposure period. The liver glyphosate at 2.5 mg L-1 stimulated
histology showed important alterations ovarian maturation over control levels,
including necrosis, changes in nuclear mainly in terms of a higher GSI and a
shape, formation of vacuoles and the higher percentage of vitellogenic
atrophy of hepatocyte cells. The oocytes, suggesting that exposure toIranian Journal of Fisheries Sciences 19(3) 2020 1605
glyphosate disrupts the hormonal effects of pollutants in water bodies.
system controlling reproduction. More detailed laboratory studies with
Moreover, the lower degree of validated assays are needed before they
maturation shown by control ovaries in can be established as specific
comparison with ovaries belonging to biomarkers. There are several endpoint
females exposed to glyphosate (at 2.5 measures that can be used to assess sub-
mg L-1) was in close correlation with lethal effects (Mensah et al., 2014). For
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the augmented GSI and HIS, suggesting example, at the ‘physical’ level,
a possible effect of glyphosate as an measures of growth, morphological
endocrine disruptor (Avigliano et al., changes, and behavioral changes
2014). The results of another recent exposed fish are used as endpoint
study by Álvarez et al. (2015) showed indicators. Also, measures of
that exposure of N. granulate females reproductive performance that are often
to atrazine (2.5, 5 and 15 mg L-1) for used to assess sub-lethal response
32-days produced a significant include sexual maturity, fecundity,
reabsorption of previtellogenic oocytes gonad histopathology, and alterations in
in all experimental groups. Since in reproductive characteristics.
those groups most of oocytes were Additionally, biochemical measures
primary, the augmented reabsorption used as possible endpoints to assess
was only observed in this type of exposed fish include metabolic
oocyte. This result could be interpreted disruption and lipid peroxidation
as a consequence of the possible ((Mensah et al., 2014). Moreover,
hormonal disruption exerted by organosomatic indices (e.g, CF, HSI
atrazine, leading to an arrested oocyte and GSI) are common approaches for
growth, and/or as a non-specific assessing fish health and may provide
response to the stress induced by the information for evaluating
chronic exposure to atrazine, as environmental stress (Adams et al.,
reported by several previous studies 1993). Thus, further studies on the
concerning multiple stress factors chronic effects of thiobencarb, and the
(Power, 2002). parameters examined herein, are still
This study has proved convincingly needed for a greater insight into the
the sub-lethal effects of commercial mechanism of thiobencarb toxicity.
formulation of thiobencarb (CITRON®)
herbicide to the African catfish, C. Acknowledgement
gariepinus. Our results provide a The authors are thankful to the Head,
description of serum biochemical and Department of Fish Disease at Animal
histopathological alterations in the liver Health Research Institute, Dokki, for
and ovaries of the African catfish, C. providing support, materials, and
gariepinus, that can be used as baseline equipment for the experiments.
information for further studies and
suggest that C. gariepinus is a useful
bioindicator animal for monitoring the1606 Elias et al., Sublethal effects of the herbicide thiobencarb on…
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