Toxopathological Studies on Some
Antimicrobial Drugs in Nile Tilapia
(Oreochromis Niloticus) and Catfish
Salah Aly M1*, Abdou Rania H2, Nashwa Abdel Atti M3, Mosleh Abomughaid M4 and Wael Nouh G4
1Department of Pathology, Faculty of Veterinary Medicine, Suez Canal University, Egypt
2 Department of Forensic Medicine & Toxicology, Faculty of Veterinary Medicine, Suez Canal University, Egypt
3Animal Health Research Institute, Ismailia Lab, Egypt
4 Department of Medical Laboratory Sciences, College of Applied Medical Sciences, University of Bisha, Saudi Arabia
Submission:April 14, 2020; Published: May 18, 2020
*Corresponding author: Salah Aly M, Department of Pathology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt
How to cite this article: Salah A M, Abdou R H, Nashwa A A M, Mosleh A M, Wael N G. Toxopathological Studies on Some Antimicrobial Drugs in Nile
Tilapia (Oreochromis Niloticus) and Catfish (Clarias Gariepinus). Open Acc J of Toxicol. 2020; 4(2):555635. DOI: 10.19080/OAJT.2020.04.555635.
Tissue distribution, residue depletion and side effect of Ciprofloxacine (CIP), Oxytetracycline (OTC) and Sulphadimethoxine (SDM) were evaluated in Nile tilapia (Oreochromis niloticus) and catfish (Clarias gariepinus). The activities of Aspartate Amino Transferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (AP), creatinine, blood urea, were estimated. Histopathological examinations were done. Fish of groups 1 were fed a basal diet and those of 2-4 fed a medicated diet containing 1g CIP/Kg, 7.5g OTC /kg and 25mg SDM/kg ration, respectively daily for five successive days. Serum, liver, kidneys and muscles samples were collected at 0, 1st, 3rd, 7th, 14th, and 21st days of treatment. CIP, OTC and SDM concentrations were estimated by ELISA. The peak concentrations of the three drugs in serum were seen at 0 day (5th day of medication); they were (1.91±0.38ug/ml) and (1.78±0.36ug/ml) for CIP, (2.15±0.41ug/ml) and (2.02±0.31ug/ml) for OTC, (3.12±0.32ug/ml) and (2.98±0.46ug/ml) for SDM in Nile tilapia and catfish; respectively. The highest kidney concentrations of CIP were (2.1±0.65ug/g) at 1st day in Nile tilapia and (1.80±0.64ug/g) at 0 day in catfish. SDM was also concentrated in kidney at 0 day post-treatment (44.2±5.1ug/g) and (31.2±4.6ug/g) in Nile tilapia and catfish; respectively. The highest residues of OTC were estimated in liver at 0 day of treatment (6.1±1.21ug/g) and (7.4±1.35ug/g) in Nile tilapia and catfish; respectively. The lowest drug residues were in muscles throughout the entire experiment. CIP didn’t detect in muscles at 14th and 21st days post-treatment. Biochemical parameters revealed significant increase in treated groups. The histopathological examination revealed variable pathological alterations in the examined organs depending on the type of antimicrobials. It could conclude the use of antimicrobials in aquaculture should be regulated, although CIP is considered as the safest one, however its use should be limited.
Fish consider one of the healthiest food as it is low in fat and rich in protein and omega 3 Fayet-Moore  & Yipel . The fish farming industry is rapidly expanding in Egypt, as well as in other countries, it has been associated with recurrent bacterial infectious diseases. Farmed Nile tilapia represents more than 58.45, while catfish production is about 3.08% of the total aquaculture harvest in Egypt Gafrd . Antimicrobial medications are used extensively in aquacultures for prophylactic or therapeutic purposes during microbial infections which may result in environmental pollution,
development of resistant bacteria and my induce toxicity to human and animals Aly  & Khalil . The availability of adequate data on the pharmacokinetics of antimicrobial agents in farmed fish is very important in order to minimize the environmental impacts of the drugs used in aquaculture. Since the excess amount of drugs can do harm to people, the European Union (EU) and the U.S. Food and Drug Administration (FDA) prescribed a Maximum Residue Limits (MRLs) for these drugs. The EU MRLs of CPX and SDM in fish were established at 100μg/kg Rezk  and 6-8μg/kg for quinolens in the edible tissues of fish Victoria .
Quinolones are effective antibacterial drugs widely used
in human and veterinary medicine because of their potential
therapeutic efficacy Plakas , Guo , Victoria  & Koc
. Ciprofloxacin is one of the most potent quinolones used
to treat infections with gram negative bacteria as Escherichia
coli, Pseudomonas aeruginosa, Salmonella spp., Shigella spp.
and Haemophilus spp., and is also effective against some grampositive
bacteria such as Staphylococcus aureus Davis  & Van
Bambeke . Oxytetracycline (OTC) is an antibacterial agent
of tetracycline family that is extensively used for treatment of
certain bacterial diseases in aquaculture all over the world Ambili
. The withdrawal time for edible tissue is differing according
to the water temperature and the type of aquatic system Jeffry
. Because of the wide spread and long-time use of OTC, many
residue studies have been recorded Rigos [15-16] & Julie .
Sulfonamides are the oldest antimicrobial agents and still
play an important role in aquaculture treatments. Sulfamethazine
(SMZ) is the most used antimicrobial drug in Veterinary field.
Sulfonamides residues have been repeatedly detected in
the aquatic environment Kolpin  & Batt . Moreover
sulphamethoxazole residues have been reported in shrimp by
Wang . Sulfamethoxazole is an effective bacteriostatic against
gram positive as well as gram negative bacteria; it affects bacteria
by inhibiting folic acid synthesis Baran . Antimicrobial drug
residues may be transferred through food-chain to human and
induce antibiotic resistance. To our knowledge, however, very few
data are available about residues of ciprofloxacin, oxytetracycline
and SDM in farmed Nile tilapia (O. niloticus) and catfish (C.
gariepinus) reared under field conditions. However, this study
aimed to investigate serum concentration peaks of ciprofloxacin,
oxytetracycline and SDM post-treatment and their residues
in liver, kidney and muscles together with serum biochemical
estimation and histopathological examinations.
Three hundred and sixty fish from each of Nile tilapia (O.
niloticus), and catfish (C.gariepinus) (weight, about 50 and 75g
for tilapia and catfish, respectively) were supplied from Central
Lab for Aquaculture Research (CLAR), Egypt and used in this
experiment that was performed in triplicates, following the
Universal Directive on the protection of animals used for scientific
purposes. Four different basal diets (control, CIP, oxytetracycline
and sulfadimethoxine) were prepared in the form of pellets to
use in the study. Basal diets were prepared by grinding the corn
to granules using 0.5mm mesh (Thomes-Willey Laboratory Mill
Model 4). Ingredients were mixed mechanically by horizontal
mixture (Hobarts model D300T) at a low speed for 30 minutes.
Oil (vegetable & cod liver) was added gradually to assure the
homogeneity of the ingredients, the mixing speed increased for
5 minutes during the addition of water (600ml water) until the
mixture began to clump. Pellets were then prepared using a pellet
machine (CPM California pellet mill Co.) with 0.5cm diameter, and
pellets were left to dry in air for 24 hrs (Table 1).
Fish with a history of no previous medication, were divided
into 4 groups (each of three replicates, 30 fish each) and held in
floating cages placed in fish farm ponds and group 1 fed a basal
diet while groups 2-4 fed a medicated diet containing 1g CIP, 7.5g
OTC and 25mg SDM/kg ration; respectively on a daily bases for
five successive days. The temperature was recorded every 12h
and adjusted to (26-30°C). The treatment was carried out once
daily at 9 a.m. for 5 successive days at a rate of 1 .0% biomass
using automatic feeders. Salinity, pH and total hardness were
adjusted to, 3±1.1%, 8.21±0.21 and 38.9±1.9mg/L; respectively.
The first sampling day was the 5th day of medication (0 day
post treatment), and on the 1st, 3rd, 7th, 14th, and 21st days after
the end of treatment with the antimicrobials. At each time of
sampling, 15 fish from each group (5fish/replicate) were netted.
Fish were anesthetized by immersion in water containing 0.1ppm
MS-222 and blood samples were collected. Serum samples
and muscle, liver and kidney specimens were collected from all
groups. Muscle samples were taken from the dorso-lateral body
area just posterior to the operculum. Each specimen was placed in
a polyethylene bag and stored at -80°C until they were analyzed.
CIP, OTC and SDM concentrations were estimated by ELISA.
The activities of Asparate Aminotransferase (AST), Alanine
Aminotransferase (ALT), Alkaline Phosphatase (AP), creatinine
and urea, were estimated using commercial diagnostic Kits
(Human Diagnostics, Germany). Methods were carried out
according to the company directions.
Tissues specimens from the muscles, liver and kidneys were
collected at 5th day post-treatment and processed routinely
according to Drury and Wallington (1980). Sections were stained with hematoxylin and eosin (H&E) and examined by light
Statistical analysis was performed using the one way analysis
of variance (ANOVA) followed by Duncan’s multiple range test
to determine the differences among the six fish groups (mean
at significance level of P<0.05). All analyses were run on the
computer using SAS program Chris Hemedinger .
Mean concentrations of the drugs (mean ± SE) vs. time in the
serum, liver, kidney and musculature were recorded in (Table
1-3). The peak concentrations of the three drugs in serum were
at 0 day. The lowest drug residues were seen in the muscles
throughout the entire experiment.
Ciprofloxacine: Results obtained after oral dose of 1 g CIP/kg
ration for 5 successive days were shown in (Table 2). The highest
recorded concentrations of CIP in sera of Nile tilapia and Catfish
were (1.91±0.38ug/ml) and (1.78±0.36ug/ml), respectively at 0
day. CIP concentrations were identified all over the experiment in
kidneys with the highest concentrations (2.1±0.65ug/g) at 1st day
in Nile tilapia and (1.80±0.64ug/g) at 0 day in kidneys in catfish.
CIP neither detected in muscles of Nile tilapia nor of Catfish at 14th
and 21st days post-treatment while, were not detect in livers of
both kinds of fish at 21st days post-treatment.
Oxytetracycline: (Table 3) shows the serum, liver, kidney
and muscle concentrations of OTC versus time in Tilapia and
Catfish after oral administration of 75mg OTC/kg ration for 5
successive days. Peaks of OTC in serum were (2.15±0.41ug/
ml) and (2.02±0.31ug/ml) at 0 day in Nile tilapia and Catfish;
respectively while, it was not detect in sera of both fish species
after 14th and 21st days but detected only in one Catfish (0.03μg /
ml) at 7th day post treatment. The highest tissue residues of OTC
were (6.1±1.21ug/g) and (7.4±1.35ug/g) in liver of Nile tilapia
and Catfish; respectively at 0 day of the treatment. In Nile tilapia
and Catfish the OCT concentrations in kidneys were 0.08±0.04
and 0.05±0.02 (μg /g); respectively at 21st day post treatment.
The lowest drug residues were in muscles throughout the entire
experiment. OCT concentrations were detected in muscles of
Nile tilapia and Catfish at (0.10±0.03ug/g) and (0.14±0.02ug/g);
respectively after 21 days post treatment
Sulphadimethoxine: (Table 4) showed the mean
concentrations of SDM in Nile tilapia and Catfish sera and tissues
versus time profile after oral administration of 25mg SDM/kg
ration for 5 successive days. The highest serum concentrations of
SDM were (3.12±0.32ug/ml) and (2.98±0.46ug/ml) at 0 day in Nile
tilapia and Catfish; respectively while it was detected in only one
Tilapia fish (0.04μg /ml) at 7th day of treatment and not thereafter
was detected. SDM was detected in kidneys of both Tilapia and
catfish all over the experiment. SDM highest concentrations
in kidney were at 0 day post-treatment (44.2±5.1ug/g) and
(31.2±4.6ug/g) in Nile tilapia and Catfish; respectively. At 21st day
of treatment; SDM was not detected in muscles and liver of Catfish
but detected only in one Tilapia fish (0.11ug/g and 0.03ug/g in
liver and muscles; respectively).
(Figure 1,2) represented the biochemical results at 5th day
of oral administration of CIP, OTC and SDM in both Nile tilapia
and Catfish. ALT was significantly increased in both fish species
after 5 days of oral administration of the three drugs compared
with control. In Tilapia fish AST was significantly increased after
administration of the three tested drugs while, in Catfish AST was
significantly increased after administration of OTC and SDM in
comparison with control. Creatinine was significantly increased
in Nile tilapia with all three drugs but in Catfish it was significantly
increased with OTC and SDM whereas not increased with CIP. Urea
was significantly increased in Tilapia fish after administration
of all drugs except OTC while, in Catfish urea was significantly
increased in both OTC and SDM but not significantly changed in
case of CIP compared with control.
The oral administration of 1g CIP/kg ration for 5 successive
days in Nile tilapia and Catfish at 5th days post-treatment,
revealed minimal histopatholoigical alterations in comparison
with the other treated groups. The musculature exhibited hyaline
degeneration in few muscle bundles (Figure 3), the liver displayed
nuclear pyknosis of some hepatocytes with mild parenchymal edema (Figure 4) while the kidneys showed proliferation of
melanomachrophage cells and mild tubular nephrosis in the renal
epithelium (Figure 5). The oral administration of 75mg OTC/kg
ration, for 5 successive days in Tilapia and Catfish at 5th day posttreatment,
revealed edema and focal hyaline degeneration in the
musculature (Figure 6). Focal proliferation of melanomoacrophage
cells was observed in the liver and kidney parenchyma. Wide
spread vacuolar degeneration in the hepatocytes (Figure 7) and
tubular nephroses in the renal tubular epithelium (Figure 8) were
The oral administration of 25mg SDM/kg ration, for 5
successive days in both Nile tilapia and Catfish at 5th days posttreatment,
revealed edema and hyaline degeneration as well
as focal Zenker’s necrosis in the musculature with focal of
mononuclear leukocytic infiltration (Figure 9). The liver exhibited
wide spread vacuolar degeneration as well as coagulative necrosis
in the hepatocytes with some mononuclear cells infiltration and
melanomacrophages (Figure 10). The kidney showed tubular
nephrosis mainly vacuolar degeneration with few cells exhibited
coagulative necrosis, hyaline casts and few mononuclear cells
infiltrations were evident (Figure 11).
Using of antimicrobial drugs in aquaculture production is one
of the main sources of environmental pollution Pruden ; Rico
& Van den Brink . During the past years there was increase in
the occurrence of antibiotic resistant bacteria and this is of critical
implications on public health Gouvêa  & Rezk . Quesada
 & Guidi  mentioned that tetracycline, oxytetracycline
(tetracyclines), enrofloxacin (quinolones), and sulfadimethoxine
(sulfonamide) are most commonly used antibiotics in aquaculture
worldwide and the presence of their residues in food could resulted
in health hazards and toxic effects. Therefore, understanding the depletion of drugs from different tissues of fish is of extreme
importance and the drug residues must be assessed in order to
determine the time needed before the antimicrobials disappear
from the tissues and to judge when the treated fish can be safely
consumed. There are limited data about the occurrence of drugresidues
in intensive culture of freshwater fishes in Egypt, hence
the goal of this study was to estimate tissue distribution and
residue depletion after oral administration of CIP, OTC and SDM in
Nile tilapia (O. niloticus) and catfish (C. gariepinus).
The elimination and residues of antimicrobials depend
upon dose, duration, fish species, and aquaculture conditions
He . Nile tilapia and catfish are kinds of tropical fish and the
appropriate temperature for survival is ranging between 24–
32°C. The water temperature in this study was 26-30°C and the
research was conducted on healthy fish in conditions those are
quite close to actual aquaculture. In this study the withdrawal
time of CIP from serum in both O. niloticus and C. gariepinus was
almost 7days. Guo  concluded that CIP in eels eliminated from
plasma for about 298h, after oral gavage of a single dose (10μg /
kg). Wu  reported that, elimination half life of enrofloxacine
and its metabolite ciprofloxacin were 15.61, 16.83, and 17.19h
in muscle, liver, and plasma of Tilapia; respectively. Ciprofloxacin
concentration was 0.3 and 0.1μg/g in liver and muscle of Chinese
mitten-handed crab after single intramuscular injection of 5.0mg
enrofloxacin/kg body weight Guanghong . The maximum
enrofloxacin concentrations in the muscle, liver and plasma of O.
niloticus were 3.61μg/g, 5.96μg/g and 1.25μg/ml; respectively
after oral dose of enrofloxacine (50mg/kg) for 7 days and the
predicted withdrawal time was 22 days Weihai . Withdrawal
time of CIP from muscle and liver under our experimental
conditions was 14 days in both O. niloticus and C. gariepinus.
Enrofloxacin metabolized into ciprofloxacin therefore, extended
withdrawal time for enrofloxacin is recommended. Renal CIP
concentrations in both O. niloticus and C. gariepinus were
0.12μg/g and 0.10μg/g; respectively at 21 days post-treatment.
The main target organ for CIP metabolism is kidney Ole . Our
results showed that, serum OTC concentrations at 0 day posttreatment
(5th day of medication) in Nile tilapia and catfish were
2.15 and 2.02μg/mL; respectively. Food and Drug Administration
(FDA) regulations specify OTC treatment in finfish culture at 55
to 83mg/kg fish per day for 10 days with a 21-day withdrawal
prior introducing for food. After 21 days, OTC concentrations
must be below the tolerance of 2ppm (μg/g). The mean peak
concentrations of OTC at 0 day post-treatment in fish muscle of O.
niloticus and C. gariepinus were 0.94 and 0.99μg/g; respectively.
Comparable to other studies carried out in farmed fish; Bjorklund
& Bylund  found peak OTC concentrations of 0.6-1.5ug/g in
farmed rainbow trout and salmon. Our study showed that, OCT
concentration in muscle was 0.10μg/g and 0.14μg/g in O. niloticus
and C. gariepinus at 21 day post-treatment. Rigos  recorded
plasma and muscle concentrations of OCT were 0.9±0.2μg/ml and
3.0 ±1.1 μg/g in seabream 150 hours post single intravascular
injection (40mg/kg) while, at 24h post-oral dosing (75mg/kg)
muscle and liver concentrations of OCT were 0.008 and 6.2±1.8
(μg/g); respectively. Julie  observed that OCT concentrations
in muscles of adult rainbow trout were below 2ug/g by 21 days
after withdrawal of OTC medicated feed for 10 days. Bjorklund
& Bylund  reported OCT concentration in muscle of rainbow
trout (Salmo gairdneri ) to be below 1ug/g by 14 days after drug
withdrawal. Josè  concluded OTC concentrations in sea bream
muscle were lower than in all the other tissues and declined
under 0.1ug/g 20 days after treatment ceased. Meanwhile, Rigos
 concluded poor intestinal absorption of OCT and that oral
administration was unsuccessful in sharp snout sea bream. Reda
 found that, the OTC residues in O. niloticus muscles were
0.05ug/g after a withdrawal period of 15 days when supplemented
in diet at 100mg/kg diet for 12 weeks, this level was lower than
the MRLs of OTC (0.1ug/g) that established by commission
regulations, EU . The differences between these species are
likely the result of physiological differences between species and/
or differences in experimental design. Hepatic accumulation of
OCT in our work was observed in both O. niloticus & C.s gariepinus
(0.51 and 0.98μg/g) 21 day post-treatment, respectively. Hepatic
metabolism is the major route for OCT metabolism in different
fish species. Rigos  and Bjorklund & Bylund  recorded
OTC hepatic accumulation. Ole  recorded the highest average
concentrations of SDM in plasma and muscles of Atlantic salmon
(14.30μg/ml and 17.72μg/g, respectively) after oral administration
in feed for 5 consecutive days as well as the withdrawal time was
288, 300 and 350 hrs in muscle, liver and kidney; respectively. The
elimination half-life of SDM from blood of rainbow trout was 24.5
hours after a single oral administration (200mg/kg), at a water
temperature of 15°C Kauzauki . Our work showed that, the
highest average concentration of SDM in liver, kidney and muscle
were 8.95, 44.2 and 2.15μg/g; respectively in Nile tilapia at 0 day
post-treatment. The corresponding values in catfish were 6.14,
31.2 and 2.02μg/g; respectively. SDM was not detectible at the 21th
day post-treatment in muscle of C. gariepinus and detected only in
one O. niloticus.
The antimicrobial drugs based on dose and type may negatively
impact the liver and kidney functions with significant changes
in enzymatic parameters and histopathological picture [48-55].
Also, the three tested medications had residues in the liver, kidney and muscles of Nile tilapia and catfish, the lowest drug residues
were in muscles. CIP is considered as the safest one with the least
residues. For the control of fish bacterial diseases, preventive
measures should be applied and during urgent need, the selection
of correct antimicrobial agent is very important through frequent
antimicrobial sensitivity testing. An antimicrobial with minimal
residue limit should be selected to protect animal and human
health from potential hazards caused by contaminated fish.
However, further studies are needed to estimate the toxicity of
therapies in the aquatic creatures and environment.
The authors declare that they have no significant competing
financial, professional or personal interests that might have
influenced the performance or presentation of the work described
in this manuscript.
Wu WH, Lin LM, Zhu XB, Wang XT, Huang XP, et al. (2004) Residues of enrofloxacin and its metabolite in Jifu Tilapia and Penaeus chinensis. Fish Sci 23: 5-8.
Guanghong Wu, Yong Meng, Xiaohua Zhu, Cheng Huang (2006) Pharmacokinetics and tissue distribution of enroXoxacin and its metabolite ciproXoxacin in the Chinese mitten-handed crab Eriocheir sinensis. Analytical Biochemistry 358: 25-30.
Commission regulation EU (2010) On pharmacologically active substances and their classification regarding maximum residue limits in food stuffs of animal origin. Official journal of the European Union.
Ole Bent Samuelsen, Lisa Purse, Peter Smith, Arne Ervik (1997) Multiple-dose pharmacokinetic study of Romet”’ in Atlantic salmon (Sdmo sahr) and in vitro antibacterial activity against Aeromonas salmonicida. Aquaculture 152: 13-24.