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In-feed oxolinic acid induces oxidative stress and histopathological alterations in Nile tilapia Oreochromis niloticus

Authors:

Abstract

The aquaculture industry urgently requires effective bacterial disease management strategies, necessitating better regulation of antibiotic application. This study investigated the effects of oral oxolinic acid (OA) administration on Oreochromis niloticus at the recommended dose of 12 mg (1 ×) and overdose of 36 mg (3 ×)/kg biomass/day for 7 consecutive days in terms of growth, oxidative stress, residue accretion and histopathology relative to the control. The 1 × and 3 × groups experienced dose-dependent mortalities (3.33-8.33 %). The OA residues peaked in the liver and kidney tissues with dosing and declined upon discontinuation. The residues persisted in the kidney even on day 35 post-dosing. Elevated malondialdehyde and total nitric oxide levels signified oxidative stress and correlated with the tissue level changes in various organs. Histologically, glycogen-type vacuolation and cellular hypertrophy were observed in the liver. The kidney had hydropic swelling, renal epithelium degradation, nephrocalcinosis, vacuolation, and necrosis. Splenic alterations were confined to ne-crosis and a slight increase in sinusoidal space. Intestinal tissues exhibited a depletion of absorptive vacuoles, epithelial layer degradation, mucinous degeneration, and necrosis. Gills displayed epithelial hyperplasia, thickening of secondary lamellae, and erosion. Nevertheless, the cohort administered the recommended dose exhibited recovery with OA discontinuation. However, none of the assessed parameters normalized in the overdosed group even after 35 days of dose suspension. The results indicated that O. niloticus can safely adapt to and tolerate the toxic effects of OA. As the recommended dose of OA elicited reversible bioresponses effectively in tilapia, it can be utilized in aquaculture with due caution following regulations.
In-feed oxolinic acid induces oxidative stress and histopathological
alterations in Nile tilapia Oreochromis niloticus
Thangapalam Jawahar Abraham
a,*,1
, Masud Bora
a
, Avishek Bardhan
a
, Arya Sen
a
,
Ratnapriya Das
a
, Ranjit Kumar Nadella
b
, Prasanna Kumar Patil
c
a
Department of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal 700094,
India
b
Fish Processing Division, ICAR-Central Institute of Fisheries Technology, Willington Island, Cochin, Kerala 682029, India
c
Aquatic Animal Health and Environment Division, ICAR-Central Institute of Brackishwater Aquaculture, Raja Annamalai Puram, Chennai, Tamil Nadu 600028, India
ARTICLE INFO
Handling Editor: Prof. L.H. Lash
Keywords:
Aquaculture
Antibiotic-feed
Histopathology
Oral administration
Oxidative stress
ABSTRACT
The aquaculture industry urgently requires effective bacterial disease management strategies, necessitating
better regulation of antibiotic application. This study investigated the effects of oral oxolinic acid (OA)
administration on Oreochromis niloticus at the recommended dose of 12 mg (1 ×) and overdose of 36 mg (3 ×)/kg
biomass/day for 7 consecutive days in terms of growth, oxidative stress, residue accretion and histopathology
relative to the control. The 1 ×and 3 ×groups experienced dose-dependent mortalities (3.338.33 %). The OA
residues peaked in the liver and kidney tissues with dosing and declined upon discontinuation. The residues
persisted in the kidney even on day 35 post-dosing. Elevated malondialdehyde and total nitric oxide levels
signied oxidative stress and correlated with the tissue level changes in various organs. Histologically, glycogen-
type vacuolation and cellular hypertrophy were observed in the liver. The kidney had hydropic swelling, renal
epithelium degradation, nephrocalcinosis, vacuolation, and necrosis. Splenic alterations were conned to ne-
crosis and a slight increase in sinusoidal space. Intestinal tissues exhibited a depletion of absorptive vacuoles,
epithelial layer degradation, mucinous degeneration, and necrosis. Gills displayed epithelial hyperplasia,
thickening of secondary lamellae, and erosion. Nevertheless, the cohort administered the recommended dose
exhibited recovery with OA discontinuation. However, none of the assessed parameters normalized in the
overdosed group even after 35 days of dose suspension. The results indicated that O. niloticus can safely adapt to
and tolerate the toxic effects of OA. As the recommended dose of OA elicited reversible bioresponses effectively
in tilapia, it can be utilized in aquaculture with due caution following regulations.
1. Introduction
Global aquaculture is projected to continue its robust growth, driven
by increasing demand for seafood and advancements in farming tech-
nologies. Tilapia production, particularly of Nile tilapia Oreochromis
niloticus, is expected to play a signicant role, contributing approxi-
mately 8 % of global aquaculture output, with genetically improved
strains enhancing growth rates and disease resistance [1]. Adopting
genetically enhanced strains, such as Genetically Improved Farmed
Tilapia (GIFT), has signicantly improved growth rates and production
efciency, helping to meet the rising domestic and international demand
for tilapia [2]. Nanotechnology and eco-friendly compounds are
revolutionizing the aquaculture industry by improving disease man-
agement and feed efciency. Nanoparticles, encapsulated vaccines,
natural biopolymers, and plant-derived compounds enhance growth and
health [3]. In addition, sensor technologies like wearable sensors and
electronic noses monitor water quality and volatile organic compounds,
ensuring optimal conditions for aquatic life. These technologies have
potential in healthcare and environmental monitoring, enhancing
human and animal life quality through innovative solutions [4]. With
advancements in technology and a growing market for aquaculture
products, the future of tilapia farming appears bright [2]. However,
disease outbreaks and environmental sustainability challenges necessi-
tate continuous research and innovation to ensure the industrys
* Corresponding author.
E-mail address: abrahamtj1@gmail.com (T.J. Abraham).
1
00000003-05811307
Contents lists available at ScienceDirect
Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep
https://doi.org/10.1016/j.toxrep.2025.102020
Received 1 December 2024; Received in revised form 1 April 2025; Accepted 2 April 2025
Toxicology Reports 14 (2025) 102020
Available online 4 April 2025
2214-7500/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
long-term success [5]. Disease management remains a signicant chal-
lenge in tilapia farming, as various diseases can impact productivity and
sustainability. The misuse of antibiotics in aquaculture has contributed
to resistance development, complicating disease treatment and raising
public health concerns due to the potential transfer of resistant bacteria
through the food chain [68]. Pathogens such as Aeromonas hydrophila,
Streptococcus agalactiae, Edwardsiella ictaluri and others have shown
signicant antibiotic resistance [911]. Additionally, antibiotic residues
in tilapia can be a public health concern, worsening resistance, and food
quality issues [12]. Addressing these challenges is critical, and adopting
improved management practices, including prudent antibiotic use, and
exploring alternatives will be essential for sustainable tilapia farming
[13].
Oxolinic acid (OA), a rst-generation uoroquinolone antibiotic, has
proven effective in managing bacterial diseases in tilapia aquaculture,
particularly those caused by pathogens like A. hydrophila and E. tarda
[14,15]. Its mechanism of action involves inhibiting bacterial DNA
gyrase, an enzyme critical for DNA replication, thereby limiting bacte-
rial growth in infected sh [16]. A perusal of the literature indicated that
OA is widely used in major aquaculture-producing nations [1719], and
several sh bacterial pathogens have developed resistance to potential
aquaculture antibiotics [20,21]. As per the World Organization of Ani-
mal Health, uoroquinolones including OA should not be used as a
rst-line treatment unless justied, when used as a second-line treat-
ment, it should ideally be based on the results of bacteriological tests,
and extra-label/off-label use should be limited and reserved for in-
stances where no alternatives are available [22]. However, its use is
currently prohibited by Indian regulatory authorities in shrimp aqua-
culture [23]. There appears to be a lack of knowledge on the effects of
OA on cultured sh in the Indian and Southeast Asian context. Hence, to
ensure its responsible and regulated use in nsh aquaculture, further
research is required on its biosafety, pharmacokinetics, and withdrawal
period across various farmed aquatic species. Such studies would pro-
vide the scientic basis for developing guidelines to ensure the safe and
effective use of the drug while minimising its environmental impact.
This study, thus, focused on evaluating the effects of in-feed OA on the
residue accretion, lipid peroxidation and histopathological alterations in
the vital organs of O. niloticus juveniles to provide insights into its safety
and potential application in aquaculture systems as a second-line
treatment when primary treatment fails.
2. Materials and methods
2.1. Experimental setup and design
Oreochromis niloticus juveniles (21.26 ±0.04 g; 12.75 ±0.15 cm)
were procured from Sonarpur sh farm, West Bengal, India. Upon
arrival, the experimental sh underwent decontamination by immersion
in 2-ppm potassium permanganate solution for 5 min before introduc-
tion into the circular tanks containing 300-L chlorine-free aerated bor-
ewell water and acclimatized for 15 days at 50 sh/tank with
continuous aeration. The sh were given commercially available
oating pellet feed twice daily, equivalent to 2 % body weight (BW).
Any uneaten feed, faeces, or other wastes were removed daily by
siphoning. About 50 % of the water exchange was done in three-day
intervals. Juveniles without infections were randomly chosen from the
stock tanks and placed 40 each in nine tanks. The sh were assigned into
three groups, viz., Group 1 as control (0 ×); Group 2, as the recom-
mended dose group that received 12 mg OA/kg biomass/day (1 ×) [24]
and Group 3, as the overdosed group that received 36 mg OA/kg bio-
mass/day (3 ×), in triplicates. Throughout the experimental period, the
ranges of water quality parameters such as pH (7.507.80), dissolved
oxygen (5.395.47 mg/L), water temperature (24.0032.00C), nitrite
(0.170.20 mg/L), and nitrate (0.280.33 mg/L) were sustained
optimally.
2.2. Oxolinic acid (OA) feed preparation and administration
The OA (Sigma-Aldrich, India, CAS-No: 14698294) dose was
determined to provide an estimated inclusion rate of 0, 12, and 36 mg/
kg biomass/day. The OA feeds were prepared by feed top-dressing as
described in Abraham et al. [25]. The required amount of OA was mixed
in 5 mL soybean oil and mixed thoroughly with 1 kg basal pellet feed in
an airtight container. The control feed was prepared as above but
without OA. After proper mixing, the OA and control feeds were spread
separately, dried overnight under the fan, and stored in airtight con-
tainers at room temperature. The sh groups were fed the respective
feeds at 2 % BW thrice daily in equal rations. From day 17 (pre-dosing),
all groups were fed with the control feed. Group 1 was fed with the
control feed throughout the experiment. During the 7 days of OA-dosing
(OD) period (Days 814), 1 ×and 3 ×feeds were fed to groups 2 and 3,
respectively. All groups were fed the control feed during the 35 days
(Days 1549) post-OA-dosing (POD) period. The unconsumed feed, if
any, in each tank after an hour of each feeding was removed, air-dried,
pooled day-wise and weighed carefully. Observations on mortality,
behaviour, pigmentation, and dermal lesions on the skin were recorded
daily. The biomass was determined weekly to adjust the feed quantity
and to assess the growth rate by estimating the biomass difference.
2.3. Collection of blood and plasma
Sampling was done with minimum handling stress to the experi-
mental sh on days 0 and 7 of OD and days 14 and 35 POD. Two sh/
tank were promptly anaesthetized by placing them in clove oil (40
μ
L/L)
mixed with water [25]. The blood was drawn using 2 mL sterile syringes
via a caudal puncture and transferred to 1.5 mL Eppendorf tubes rinsed
with 5 % ethylenediamine tetraacetic acid (EDTA) to prevent coagula-
tion [26]. The blood was then centrifuged at 4500 rpm for 15 min at
30C. The resultant plasma was shifted to Eppendorf tubes and
well-preserved at 20C.
2.4. Assessment of lipid peroxidation and total nitric acid
The lipid peroxidation, represented by plasma malondialdehyde
(MDA), was evaluated by the thiobarbituric acid reactive substances
(TBARS) assay by assessing the coloured MDA-thiobarbituric acid (TBA)
adduct (1:2), produced from the reaction of MDA and TBA [27]. The
MDA was quantied using a microplate reader (Dynamica, Australia) at
535 nm following the kit guidelines (HiMedia, India). The nitric oxide
estimation kit (HiMedia, India) was used for the total nitric oxide (TNO)
assay, which relied on nitrate reduction (NO
3
) by a reducing agent at
37C. The resultant nitrite and the endogenous nitrite were converted to
a blue-coloured azo compound with Griess reagent [28]. The plasma
TNO was determined spectrophotometrically at 540 nm, and computed
following the kit instructions.
2.5. Quantication of oxolinic acid residues
The sh, after blood collection, were euthanized by increasing the
clove oil dose to 100
μ
L/L, carefully removed the kidney and liver tis-
sues, washed gently in owing water to remove the blood traces and
homogenized. The acidied acetonitrile (ACN) was utilized for OA
extraction, involving a solvent mixture comprising 5 mL n-hexane,
10 mL 0.05 M EDTA, and 10 mL 0.1 % formic acid in ACN. The process
included vortexing for a minute, incubating in a shaker at 650 rpm for
30 min, and centrifuging at 4000 rpm for 10 min at 4C. Aliquots were
puried using an Oasis hydrophilic-lipophilic balance (HLB) solid-phase
extraction (SPE) cartridge for the OA analyte. The elution from the SPE
method was analyzed by reverse-phase liquid chromatography on a C18
LC column. The analytical procedure relied on QTRAP LC-MS/MS. As
detailed in our earlier research [25], the detection was done through
electrospray ionization and tandem mass spectrometry on an ion trap
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
2
mass spectrometer, and separation via a Raptor C18 2.7 µm;
100 ×2.1 mm (Restek). The in-house validation concept led to valida-
tion using diverse matrices as per the Commission Decision
2002/657/EC [19]. The method was validated at the National Referral
Laboratory, ICAR-Central Institute of Fisheries Technology, Kochi,
India. Validation parameters were determined based on data from mass
spectrometry analysis utilizing Analyst 1.6.3 version. The response was
measured in terms of peak area, exhibiting satisfactory linearity of
analytes with an r
2
value >0.99. All the analytes showed minimal
carryover (<0.5 %) in matrix-based calibration.
2.6. Histopathology and qualitative assessment
For histopathology, the liver, kidney, spleen, intestine, and gills were
collected on OD-days 0 and 7, and POD-days 14 and 35, and xed in
Bouins solution for 48 h. Standardized procedures were followed for
tissue processing, embedding, cutting into 5
μ
m sections, and perform-
ing double staining with hematoxylin and eosin, following the guide-
lines of Roberts [26]. Photomicrography was captured using an
Olympus microscope (BX51) set with a 16 MP camera (SCO-LUX). Image
acquisition and processing were carried out using ToupView software
(ToupTek-x64, 4.11). The major histopathological changes in different
organs were identied and qualitatively assessed on a six-point ordinal
scale, according to the damage in the respective tissues from their
normal architecture [29].
2.7. Statistical analyses
Data on MDA, TNO, and OA residues in mean ±standard deviation
were analyzed by one-way ANOVA. The signicance of differences
among treatments and dosing periods was ascertained using the Tukey
post-hoc test for mean comparisons. The non-parametric KruskalWallis
test assessed the qualitative scores of histopathological changes with
pair-wise comparisons at p <0.05 using IBM-Statistical Package for
Social Sciences, Version 22.0.
3. Results
3.1. Feed intake, abnormalities, mortalities, and biomass
The control group exhibited vigorous and assertive feeding behav-
iour. During the OD tenure, feed intake was reduced by 13 % in the
1 ×and 3 ×groups. With dose termination, it steadily increased and
almost reached 100 % in both groups. The sh displayed no aberrant
behaviour, except for the increased production of skin and gill mucus in
the 3 ×group. During the OD phase, mortalities recorded in the 1 ×and
3 ×groups were 3.33 ±1.44 and 8.33 ±1.44 %, respectively, which
differed signicantly. On day 35 POD, insignicantly higher mortalities
were noted in both groups. There were signicant variations in the
biomass among groups, with the 3 ×group showing the lowest growth
rate.
3.2. Lipid peroxidation, total nitric acid production and oxolinic acid
residues
The plasma MDA showed a dose-dependent signicant increase with
the highest on day 7 of OD (p <0.05). The recommended dose group
accomplished normalcy on day 14 POD, while the 3 ×group returned to
normal on day 35 POD. The plasma TNO increased signicantly and
peaked on day 7 (p <0.05), with the 3 ×group displaying the highest
level. With dose suspension and time, the TNO reduced signicantly
(p <0.05). The 1 ×group was normalized, and the 3 ×group failed to
recover on day 35 POD (Table 1). The OA residues in the liver and
kidney had a signicant accumulation that reached its highest on day 7
of OD. The OA residues depleted with dose suspension and were below
the detectable level on day 14 of POD in the 1 ×groups liver. In the
kidney, the residues remained detectable even on day 35 POD in both
groups (Table 1).
3.3. Histopathology
The normal histoarchitecture of the liver, kidney, spleen, intestine
and gill tissues is presented in Fig. 1A-E. The liver tissues and hepato-
cytes underwent considerable changes compared to the control (Fig. 2A-
F). On day 7, the 1 ×group revealed signicant glycogen-type vacuo-
lation. The POD period recorded a decrease in the degree of glycogen-
type vacuolation, and the liver tissue organization improved signi-
cantly (Fig. 2B-C). However, the 3 ×group liver tissues displayed an
increased intensity of changes, including cytoplasmic degeneration,
glycogen-type vacuolation, and cellular hypertrophy, along with minor
alterations like necrosis and karyolytic nuclear abnormalities (Fig. 2D).
Though the liver tissues exhibited signs of recovery from day 14 POD
(Fig. 2E), glycogen-type vacuolation and cellular hypertrophy were
prominent on day 35 POD (Fig. 2F). Compared to the control, the OA-fed
O. niloticus had considerable renal tissue damage (Fig. 3A-F). The
1 ×group exhibited necrotic areas, vacuolation, renal epithelium
impairment, hydropic swelling, and nephrocalcinosis on day 7 of OD
(Fig. 3A). On day 14 POD, the renal tissues started showing recovery and
the abnormalities decreased, although mild degenerative renal epithe-
lium, nephrocalcinosis and vacuolation persisted (Fig. 3B). On day 35
POD, the intensity of nephrocalcinosis and hydropic swelling reduced
(Fig. 3C). The 3 ×group had a signicantly higher degree of damage on
day 7 (Fig. 3D). From day 14 POD, the kidney tissues showed signs of
recovery and with reduced damages (Fig. 3E, F).
The spleen tissues of OA-dosed O. niloticus demonstrated changes like
increased sinusoidal space, splenic necrosis, and hemosiderin deposits in
a dose-dependent fashion. The 1 ×groups spleen on day 7 demon-
strated splenic necrosis and mildly increased sinusoidal space (Fig. 4A)
In the 3 ×group, these alterations were signicantly (p <0.05) severe
(Fig. 4D). On days 14 and 35 of POD, both the groups spleen tissue
showed signicant (p <0.05) recovery despite the persistence of
increased sinusoidal space and splenic necrosis (Fig. 4B, C), although the
recovery was less in 3 ×group compared to the 1 ×group (Fig. 4E, F).
Table 1
Plasma thiobarbituric acid reactive substances (TBARS), total nitric oxide (TNO), and liver and kidney tissue oxolinic acid (OA) residues in OA-dosed Oreochromis
niloticus.
Plasma oxidative stress markers OA residues
Treatment day TBARS (µM) TNO (µM) Liver (µg/kg) Kidney (µg/kg)
1 ×3 ×1 ×3 ×1 ×3 ×1 ×3 ×
Day 0 1.16 ±0.05 1.16 ±0.05 61.15 ±0.58 61.15 ±0.58 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00
Day 7 OD 1.41 ±0.05* 1.54 ±0.05
*@
138.75 ±1.00* 172.50 ±1.15
*@
35.60 ±1.00* 94.20 ±0.10
*@
94.23 ±0.95* 270.30 ±0.61
*@
Day 14 POD 1.22 ±0.05 1.27 ±0.05* 97.50 ±1.00* 132.50 ±1.10
*@
BDL 1.33 ±0.01 2.17 ±0.07 4.37 ±0.02
Day 35 POD 1.16 ±0.05 1.17 ±0.05 62.50 ±0.58 82.50 ±0.72
*@
BDL BDL 1.76 ±0.02 2.60 ±0.10
OD: Oxolinic acid dosing; POD: Post-oxolinic acid-dosing; TBARS: Thiobarbituric acid reactive substances as malondialdehyde (MDA); TNO: Total nitric oxide; BDL:
Below detectable limit; 1 ×: 12 mg/kg biomass/day; 3 ×: 36 mg/kg biomass/day. *: Signicant increase compared to day 0 (p <0.05); @: Signicant increase
compared to the 1 ×group (p <0.05).
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
3
Oral OD for 7 consecutive days caused considerable alterations in the
intestine of O. niloticus. The 1 ×group documented minor changes like
loss of absorptive vacuoles and degenerated epithelial layer on day 7 of
OD compared to the control (Fig. 5A). On day 14 of POD, the 1 ×group
showed trivial recovery (Fig. 5B). However, on day 35 POD, recovery of
tissue histoarchitecture was considerable in the 1 ×group with signi-
cant (p <0.05) reduction in the aberrations to an almost normal level,
except for the swollen lamina propria (Fig. 5C). In the 3 ×group, sig-
nicant (p <0.05) alterations such as loss of absorptive vacuoles,
mucinous degeneration and degenerated epithelial layer were observed
on day 7 (Fig. 5D). On day 14 POD, signicant (p <0.05) recovery was
noted, which continued till day 35 POD, although the swollen lamina
propria and mucinous degeneration were persistent (Fig. 5E, F). The
gills of the 1 ×group recorded epithelial hyperplasia, thinning of sec-
ondary lamellae, curling of secondary lamellae, and thickening of sec-
ondary lamellae on day 7 of OD compared to the control (Fig. 6A). On
day 14 POD, a signicant reduction (p <0.05) and recovery in the gill
tissues were observed (Fig. 6B). On day 35 POD, signicant reductions
(p <0.05) and recovery in the gill tissues was observed with the
persistent minor alterations like curling and thickening of secondary
lamellae (Fig. 6C). The 3 ×group showed a signicant hike in
alterations compared to the 1 ×group (Fig. 6D). On day 14 and 35 POD,
signicant reduction in alterations (p <0.05) and recovery of the gill
tissues were observed with substantial persistence of swollen tips of the
secondary lamella, erosion of secondary lamellae, epithelial hyperpla-
sia, and chloride cells (Fig. 6E, F). The qualitative scores of the major
histopathological changes in the vital organs of OA-dosed O. niloticus
juveniles are presented in Table 2.
4. Discussion
Oreochromis niloticus administered OA orally showed a signicant
and dose-dependent reduction in feed intake, survival, and growth. The
sh were able to mount biological responses post-dosing, and feed
intake recovered. Mortalities were found in both dosing groups, indi-
cating OA-induced stress and intoxication. These observations corrob-
orated the works of Abraham et al. [25], who asserted dose and
time-dependent OA intoxication in O. niloticus and supported the ob-
servations of Hentschel et al. [30] on drug dose and toxicity. The study
found signicant changes in sh biomass during and after OD, indi-
cating a negative effect of OA on sh growth. The control group showed
a 1.25-fold increase in biomass, while the OD groups showed a
Fig. 1. Normal histoarchitecture of the control feed-fed Oreochromis niloticus showing [A] liver, [B] kidney, [C] spleen, [D] intestine and [G] gill. ×200H&E staining;
Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
4
dose-dependent decrease correlated with the decrease in feed intake.
The results on the elevated plasma MDA and TNO indicated that dietary
OA was rapidly absorbed and disrupted the balance between oxidants
and antioxidants in O. niloticus, causing the liver and kidney to produce
excessive free radicals and metabolic and tissue damage. The heightened
MDA and TNO in OA-fed O. niloticus suggested a decrease in the activity
of antioxidant enzymes, leading to diminished antioxidant capacity
corroborating the ndings of Bardhan et al. [31] noted in orfenicol
(FFC)-administered O. niloticus. The outcome of this study contradicted
several prior studies that observed a reduction in MDA following
oxytetracycline (OTC) administration [32,33]. With dose cessation, the
MDA recouped on days 14 and 35 POD in the 1 ×and 3 ×groups,
respectively, indicating the adaptive responses of sh. As nitric oxide
(NO) is a reactive nitrogen species critical in the redox biology of he-
patocytes, it plays a signicant role in inducing oxidative stress in sh
[31]. The elevated TNO might believably be one of the primary factors
contributing to the inamed liver, particularly in the overdosed group.
Following the cessation of OD, O. niloticus exhibited a recovery, sug-
gesting their ability to mount adaptive responses against OA-induced
stress and to initiate bioresponses to overcome the abnormalities.
During dosing, a dose-dependent build-up of OA residues was
observed in the liver and kidney tissues. The presence of residues in sh
caused stress, physiological disorders, and organal changes as was noted
in the kidney and liver histopathology and plasma MDA and TNO levels.
Depletion occurred more rapidly in the liver and kidney tissues, with the
liver becoming residue-free on day 14 POD in the recommended dose
Fig. 2. Histopathological changes in the liver of oxolinic acid (OA)-fed Oreochromis niloticus at 12 mg (1 ×) and 36 mg/kg biomass/day (3 ×) for 7 consecutive days.
[A] 1 ×group on day 7 of OD, [B] day 14 POD, [C] day 35 POD, [D] 3 ×group on day 7 of OD, [E] day 14 POD, and [F] day 35 POD. OD: OA-dosing; POD: Post-OA-
dosing. Cytoplasmic vacuolation (CV), cytoplasmic degeneration (CD), cellular hypertrophy (CH), karyolytic nuclear abnormalities (KL) and necrosis of liver tissue
(N). ×200H&E staining; Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
5
group. In the overdosed group, residues were noticeable in the kidney
samples on day 35 POD, thereby showing adverse safety concerns
regarding the abuse of antibiotics. Chen et al. [17] noted that most
quinolones are primarily eliminated through renal excretion and liver
metabolic processes, involving both glomerular ltration and tubular
secretion. This aligns with the ndings of faster elimination of residues
from liver and kidney tissues at the recommended dose. Overall, the
O. niloticus in the current study displayed comparably low accrual and
high removal efciency of OA residues. Depletion results of OA in
O. niloticus are consistent with the depletion of OA in several other
species [17,3436]. The depletion pattern described in this work pro-
vided evidence in favour of more sensible OA use in O. niloticus.
The OA-dosed O. niloticus liver tissues showed structural
irregularities, more or less, similar to the alterations noted in several sh
species treated with chloroquine [37], noroxacin [38], OA [25,39],
ciprooxacin [40], and uoroquinolone [41]. Their ndings suggested
that quinolone exposure/administration could lead to diverse conse-
quences of aberrant metabolic processes, potentially leading to liver
failure. The glycogen-type vacuolation was the most signicant and
prominent histopathological aberration, indicating an increased de-
mand for energy to overcome the OA-induced stress. It further supported
the histological observations with OTC [42], FFC [31], and OA [25].
Vacuolations suggested diverse biochemical changes, including hin-
dered protein synthesis and ionic regulation, decreased energy levels,
enzyme denaturation, alterations in substrate utilisation, and disruption
of microtubules in the liver [37]. The severe damage to the liver was due
Fig. 3. Histopathological changes in the kidney of oxolinic acid (OA)-fed Oreochromis niloticus at 12 mg (1 ×) and 36 mg/kg biomass/day (3 ×) for 7 consecutive
days. [A] 1 ×group on day 7 of OD, [B] day 14 POD, [C] day 35 POD, [D] 3 ×group on day 7 of OD, [E] day 14 POD, and [F] day 35 POD. OD: OA-dosing; POD:
Post-OA-dosing. Degeneration of renal epithelium (DRE), hydropic swelling (HS), necrotized areas (N), nephrocalcinosis (NC) and vacuolation (V). ×200H&E
staining; Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
6
to the hyperactivity of the nucleus, which can be indicated by cellular
hypertrophy, nuclear degeneration, alteration of hepatocytes, and ne-
crosis. As the liver is the primary source of exoenzymes such as alanine
aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline
phosphatase (ALP) bound to the membranes of hepatocytes, the damage
to the liver tissue may release these membrane-bound proteins from the
impaired hepatocytes. In several earlier studies, sh subjected to anti-
biotics such as OTC, FFC, OA and others had higher ALT, AST, and ALP
levels, which indicated their hepatotoxic effect [25,31,42,43]. On day
35 POD, the liver tissues displayed insufcient improvement, charac-
terized by the persistence of vacuolation with glycogen-type features,
despite the below detectable levels of residues. Nonetheless, the rec-
ommended dose group improved tissue histoarchitecture suggesting
their ability to initiate physiological responses to overcome these
abnormalities. The recovery of cellular hypertrophy was only close to
normal even on 35 days of POD, thus suggesting the persistence of
OA-induced stress in the liver.
The present study recorded signicant and dose-dependent histo-
pathological changes in the kidney on day 7. Nephrotoxic effects of OA
were recognized by the degeneration of renal epithelium, hydropic
swelling and vacuolation, which corroborated previous studies on sh
treated with ciprooxacin [40], chloroquine [37], OA [25], FFC [31]
and OTC [44]. Degeneration is recognized as a non-specic condition
that acts as an initial indication of necrosis [45]. Furthermore, the
heightened presence of disintegrated renal epithelium and vacuolation
indicated that vacuolation precedes disintegration [45]. On day 7,
scanty nephrocalcinosis was observed, which may be associated with a
decrease in calcium and impaired reabsorption [25]. As the kidney is
Fig. 4. Histopathological changes in the spleen of oxolinic acid (OA)-fed Oreochromis niloticus at 12 mg (1 ×) and 36 mg/kg biomass/day (3 ×) for 7 consecutive
days. [A] 1 ×group on day 7 of OD, [B] day 14 POD, [C] day 35 POD, [D] 3 ×group on day 7 of OD, [E] day 14 POD, and [F] day 35 POD. OD: OA-dosing; POD:
Post-OA-dosing. Splenic necrosis (SN), increased sinusoidal space (ISS) and hemosiderin deposits (H), ×200H&E staining; Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
7
involved in the calcium and phosphate homeostasis processes, its dis-
turbances may lead to mineral deposition within the renal tubules [46].
Inammation of the renal tubules can occur in higher vertebrates, and it
can be acute or chronic, depending on the primary cell type or cell
response involved [45]. The hydropic swelling of epithelial cells could
potentially be the source of inammation of the renal tubules. Although
subtle, the increase in hydropic swelling was dose-dependent. These
changes proclaimed a dose-dependent nephrotoxic effect of OA in
O. niloticus. However, the changes were reversible as observed on day 35
POD. The sh administered OA displayed noticeable changes in the
spleen tissue. A mild to moderate increase in sinusoidal space and
splenic necrosis, indicating the toxic effects of OA on the sh spleen. It
possibly affected the spleens functioning as the sinusoids reportedly
remove damaged and old erythrocytes as well as transfer leucocytes
[47]. Splenic necrosis occurs when the blood supply to the spleen is
reduced, resulting in tissue ischemia and, nally, necrosis [40]. In
Oncorhynchus mykiss congested cells, foci of myocyte necrosis, and small
clusters of melanocytes were noticed upon enrooxacin treatment [48].
Hemosiderin is an iron-storing pigment that is derived from the
breakdown of erythrocytes [49]. In the current study, hemosiderosis was
found due to the toxicity of OA, indicating the reduction of erythrocytes.
Reduced erythrocytes and hemosiderosis have a link with splenic ne-
crosis [49]. The recommended dose group experienced mild splenic
alterations that returned to normal after the medication was terminated,
demonstrating the tolerance of OA at the recommended dose. Further,
the study suggested that OA has mild to moderate toxic effects on the
spleen of O. niloticus and may cause reversible histopathological
alterations.
The histological alterations and impairments identied in intestinal
tissues of O. niloticus due to OD in varying degrees were dose-dependent
and attributed to the direct toxicity of OA. Epithelial layer degeneration
was observed in all OD groups, ranging from normal to mild, throughout
the dosing and post-dosing periods, possibly due to the drug rejection by
the sh intestinal lining. A mild increase in degeneration of epithelium
proclaimed that the harmful potential of OA on the intestinal tissues was
the least. The mild to moderate increase in the loss of absorptive vacu-
oles seen throughout the OD duration indicated poor feed absorption
and, as a result, reduced feed intake. The loss of absorptive vacuoles was
Fig. 5. Histopathological changes in the intestine of oxolinic acid (OA)-fed Oreochromis niloticus at 12 mg (1 ×) and 36 mg/kg biomass/day (3 ×) for 7 consecutive
days. [A] 1 ×group on day 7 of OD, [B] day 14 POD, [C] day 35 POD, [D] 3 ×group on day 7 of OD, [E] day 14 POD, and [F] day 35 POD. OD: OA-dosing; POD:
Post-OA-dosing. Moderate loss of absorptive vacuoles (LAV), mildly degenerated epithelial layer (DE), mildly degenerated epithelial layer (DE), necrotized area (NA),
mildly swollen lamina propria (SLP), and mucinous degeneration (MD), ×200H&E staining; Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
8
most observed in all OD groups throughout the experimental period,
revealing the reduction in energy charges as the intestine expends a lot
of energy during digestion and absorption [50,51]. The loss of such
energy charge in the intestinal villi may be responsible for epithelial cell
destruction. The lamina propria is a vascularized connective tissue that
exists beneath the epithelium and is composed of nerves and leukocytes
[52]. An increase in lamina propria thickness indicated heightened
immunity and the host responses to drug absorption [52]. The
dose-dependent alterations, like mucinous degeneration and necrotized
area in the intestinal villi upon OD, were only normal to mild. Kitamura
et al. [53] stated that quinolones can reduce mucus-producing goblet
cells, and promote intestinal disruption and inammation. The main
function of intestinal goblet cells is the production of mucin and the
formation of mucus layers for innate defence [54], and their reductions
may result in intestinal damage and breach of innate defences. In the
intestine of O. mykiss, enrooxacin treatment caused an increased
number of eosinophilic granular leukocytes [48]. Except for the persis-
tence of mucinous degeneration and swollen lamina propria, all other
aberrations recovered to normal, demonstrating tolerance of O. niloticus
to OA at the recommended dose.
Modications in the gill morphology can potentially suffocate sh by
disrupting normal respiratory function and preventing an adequate
Fig. 6. Histopathological changes in the gill of oxolinic acid (OA)-fed Oreochromis niloticus at 12 mg (1 ×) and 36 mg/kg biomass/day (3 ×) for 7 consecutive days.
[A] 1 ×group on day 7 of OD, [B] day 14 POD, [C] day 35 POD, [D] 3 ×group on day 7 of OD, [E] day 14 POD, and [F] day 35 POD. OD: OA-dosing; POD: Post-OA-
dosing. Epithelial hyperplasia (EH), thinning of secondary lamellae (Th), thickening of secondary lamellae (TF), curling of secondary lamellae (C), vacuolation (V),
erosion of secondary lamellae (E), lamellar hyperplasia (LH), chloride cells (CC), swollen tips (ST), fusion of secondary lamellar laments (F), and mild lamellar
hyperplasia (LH), ×200H&E staining; Scale Bar: 100 µm.
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
9
supply of oxygen and inorganic ions from achieving the shs normal
metabolic processes [40]. Xu et al. [55] reported that quinolones inter-
fere with sh respiratory and osmoregulation activities, and essential
gill functions to induce gill inammation and oxidative stress. This study
documented several structural anomalies in the recommended dose
group on day 7 with increased intensity in the overdosed group, similar
to sh treated with chloroquine [37] and ciprooxacin [41]. Epithelial
and secondary lamellar hyperplasia may develop from some defence
cells, such as macrophages and leucocytes, as part of a compensatory
mechanism of tissue healing [56]. The curling and swollen tips of sec-
ondary lamellae were noticed during the dosing period, leading to
improper oxygen uptake and anoxia. The swelling of secondary lamellar
laments may cause the reduction of oxygen consumption efciency of
sh gills [31,57]. On day 35 POD, except for epithelial hyperplasia,
other anomalies returned to normal or imperfectly recovered, with mild
alterations. A comparison of the results of the present study with pre-
vious publications on quinolone compounds on varied sh species is
presented in the supplementary Table. This study, thus, demonstrated
that OA can cause mild to moderate structural and functional damage in
the vital organs of sh, which is a cause for concern.
5. Conclusion
The present study demonstrated that oral administration of OA in-
duces dose-dependent physiological and histopathological alterations,
signicantly affecting O. niloticus health. The reduction in feed intake,
growth, and survival highlighted the adverse effects of OA overdose. The
study further conrmed that OA administration induces oxidative stress,
as evidenced by elevated MDA and TNO levels, leading to metabolic and
tissue damage in the liver and kidney. While the sh exhibited an ability
to recover post-dosing, the histopathological analysis of the liver,
kidney, spleen, intestine, and gills revealed persistent structural im-
pairments, particularly in the overdosed group. The OA residues peaked
on day 7 of OD and declined with dose suspension. Residue diminution
occurred more rapidly, with the liver becoming residue-free on day 14
POD in the recommended dose group. The residues were detectable in
both groupskidneys on day 35 POD. The accumulation of OA residues
in the liver and kidney tissues further indicated the potential health risks
associated with OA use in aquaculture. The observed depletion patterns
suggested that responsible and controlled use of OA is necessary to
mitigate long-term health implications. The increasing concern
regarding antimicrobial resistance emphasized the importance of using
OA cautiously as second-line therapy in food-producing aquatic animals
when there are no other options available, as it could potentially pose
risks to sh health. Overall, the present study contributed signicantly
by providing a detailed assessment of OAs dose-dependent impact on
sh health, highlighting recovery patterns post-administration, and of-
fering histopathological evidence of tissue damage in the vital organs.
The ndings on OA residue depletion provide valuable insights for food
safety regulations, reinforcing the need for responsible antibiotic use
and the adoption of sustainable aquaculture practices. Nevertheless,
additional research on the long-term effects of OA administration on
sh, the environmental impact of OA residues, the combined toxic effect
of OA with other chemicals or water quality parameters in the aqua-
culture systems, the precise molecular mechanisms driving OA-induced
oxidative stress, and the potentials of vaccines, probiotics or herbal al-
ternatives to improve the disease resistance may yield some directives
on the effective mitigation strategies for bacterial disease control.
Therefore, further research should explore sustainable sh health
management approaches, ensuring aquaculture productivity without
compromising sh welfare and consumer safety.
Table 2
Qualitative assessment of the major histopathological changes in oxolinic acid (OA)-dosed Oreochromis niloticus juveniles at 12 mg (1 ×) and 36 mg/kg biomass/day
(3 ×) for 7 consecutive days in comparison with normal architecture.
Histopathological changes Qualitative assessment on a six-point ordinal scale*
1×3×
7 OD 14 POD 35 POD 7 OD 14 POD 35 POD
Liver
Glycogen-type vacuolation 2.70 ±0.45
a
2.25 ±0.45 1.85 ±0.42 3.30 ±0.22
@
2.75 ±0.36 2.45 ±0.15
Cytoplasmic degeneration 1.25 ±0.25
a
0.85 ±0.20 0.55 ±0.15 1.85 ±0.25
@
1.30 ±0.35 0.85 ±0.20
Cellular hypertrophy 1.85 ±0.25 1.85 ±0.25 1.85 ±0.25 1.50 ±0.26
@
1.20 ±0.20 0.85 ±0.15
Kidney
Degeneration of renal epithelium 1.75 ±0.15
a
0.40 ±06 0.25 ±06 2.45 ±0.20
@
0.65 ±0.21 0.45 ±0.06
Hydropic swelling 1.35 ±0.30
a
0.55 ±0.20 0.35 ±0.08 1.55 ±0.26
@
0.85 ±0.15 0.65 ±0.15
Nephrocalcinosis 1.00 ±0.05
a
0.65 ±0.25 0.50 ±0.22 1.25 ±0.16
@
0.70 ±0.18 0.50 ±0.18
Vacuolation 0.45 ±0.15
a
0.35 ±0.12 0.25 ±0.12 1.30 ±0.10
@
0.75 ±0.22 0.65 ±0.21
Spleen
Splenic necrosis 0.85 ±0.10
a
0.65 ±0.10 0.45 ±0.11 1.15 ±0.10
@
0.85 ±0.10 0.65 ±0.11
Increased sinusoidal space 1.15 ±0.12
a
0.75 ±0.12 0.55 ±0.16 1.65 ±0.08
@
1.05 ±0.12 0.75 ±0.16
Intestine
Loss of absorptive vacuoles 1.75 ±0.15
a
0.30 ±0.11 0.20 ±0.10 2.10 ±0.05
@
0.47 ±0.10 0.35 ±0.10
Degenerated epithelial layer 0.75 ±0.12
a
0.65 ±0.08 0.45 ±0.08 1.00 ±0.06
@
0.85 ±0.11 0.65 ±0.08
Necrotized area 0.30 ±0.15
a
0.30 ±0.15 0.25 ±0.08 0.85 ±0.20
@
0.85 ±0.18 0.45 ±0.10
Swollen lamina propria 1.35 ±0.08 1.35 ±0.08 1.35 ±0.08 2.10 ±0.05
@
1.75 ±0.15 1.65 ±0.15
Mucinous degeneration 0.65 ±0.10
a
0.45 ±0.08 0.25 ±0.05 0.85 ±0.15
@
0.60 ±0.10 0.40 ±0.08
Gill
Thinning of secondary lamellae 0.55 ±0.24
a
0.35 ±0.15 0.25 ±0.15 0.85 ±0.24
@
0.65 ±0.15 0.35 ±0.10
Epithelial hyperplasia 1.65 ±0.20
a
1.05 ±0.20 0.75 ±0.15 1.85 ±0.25
@
1.25 ±0.20 0.85 ±0.15
Curling of secondary lamellae 0.35 ±0.08
a
0.35 ±0.08 0.25 ±0.10 0.67 ±0.05
@
0.45 ±0.10 0.25 ±0.08
Thickening of secondary lamellae 0.53 ±0.24
a
0.35 ±0.20 0.25 ±0.15 0.53 ±0.24
@
0.53 ±0.24 0.35 ±0.20
Lamellar hyperplasia 0.45 ±0.20
a
0.27 ±0.10 0.20 ±0.10 0.60 ±0.20
@
0.35 ±0.10 0.25 ±0.15
Erosion of secondary lamellae 0.45 ±0.20
a
0.27 ±0.10 0.15 ±0.05 0.65 ±0.10
@
0.35 ±0.20 0.27 ±0.10
Swollen tips of secondary lamellae 0.25 ±0.20
a
0.20 ±0.10 0.20 ±0.08 0.45 ±0.20
@
0.35 ±0.20 0.27 ±0.10
* Qualitative assessment ordinal scale: 0 =No change; 1 =Normal with <5 % of tissues affected; 2 =Mild with 515 % of tissues affected; 3 =Moderate with
1525 % of tissues affected; 4 =Marked with 2550 % of tissues affected and 5 =Severe with >50 % of tissues affected. The qualitative assessment was based on six
observations (mean ±standard deviation) for each organ of the respective group. No changes were noted in the control. The qualitative scores on histopathological
changes between the 1 ×and 3 ×groups on days 7, 14, and 35 differed signicantly (p <0.05). 7 OD: Day 7 OA-dosing; 14 POD: Day 14 post- OA-dosing; 35 POD: Day
35 post-OA-dosing. a, and @: Signicant increase compared to the post- OA-dosing regimen of the 1 ×and 3 ×groups, respectively (p <0.05).
T.J. Abraham et al.
Toxicology Reports 14 (2025) 102020
10
CRediT authorship contribution statement
Nadella Ranjit Kumar: Validation, Resources, Investigation. Das
Ratnapriya: Investigation, Formal analysis, Data curation. Sen Arya:
Writing original draft, Investigation, Formal analysis, Data curation.
Bardhan Avishek: Investigation, Formal analysis, Data curation. Bora
Masud: Investigation, Formal analysis, Data curation. Thangapalam
Jawahar Abraham: Writing review & editing, Supervision, Resources,
Project administration, Methodology, Conceptualization. Patil Pra-
sanna Kumar: Supervision, Methodology, Funding acquisition,
Conceptualization.
Ethics approval
The current study was performed following the guidelines of the
Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Government of India. The experimental protocols
were approved by the ICAR, Government of India, New Delhi, under the
All-India Network Project on Fish Health (F. No. CIBA/AINP-FH/
201516 dated 16.7.2015).
Funding
Indian Council of Agricultural Research (ICAR), Government of
India, New Delhi, under the All-India Network Project on Fish Health,
supported this work vide Grant F. No. CIBA/AINP-FH/201516 dated
02.06.2015.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
The authors thank the Vice-Chancellor, West Bengal University of
Animal and Fishery Sciences, Kolkata, for providing the necessary fa-
cilities for this work.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.toxrep.2025.102020.
Data Availability
Data will be made available on request.
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