Received: 12/07/2024 Accepted: 29/10/2024 Published: 18/01/2025 1 of 7
https://doi.org/10.52973/rcfcv-e35488 Revista Cientíca, FCV-LUZ / Vol. XXXV
ABSTRACT
Dimethoate (DMT) pesticide is one of the chemicals used to
protect some agricultural areas from harmful organisms. DMT
residues released directly or indirectly to the environment
cause serious problems in nature. DMT residues mixed with the
aquatic environment adversely affect aquatic organisms and
this effect is carried to humans through the food chain. In this
study, oxidative stress responses induced by DMT pesticide in
Pontastacusleptodactylus were investigated. For this purpose,
oxidative stress and antioxidant parameters Thiobarbituric acid
reactive substances (TBARS), Glutathione (GSH), Superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidase
(GPX) caused by dimethoate (DMT) pesticide in P. leptodactylus
at 17.5, 35, and 70 mg·L
-1
concentrations at 24 and 96 hours
were investigated. Results were determined using ELISA kits.
No signicant difference was observed in GSH levels and SOD
activities compared to control. Statistically signicant differences
were observed between decreases in CAT and GPx activities and
increases in TBARS levels. SPSS 24.0 package program one–way
ANOVA (Duncan 0.05) was used in the evaluation of biochemical
analyzes. As a result, it was determined that DMT caused oxidative
stress formation in P.leptodactylus and caused changes in enzyme
activities.
Key words: Dimethoate, Pontastacus leptodactylus, oxidative
stress, antioxidant, biomarkers
RESUMEN
El pesticida dimetoato (DMT) es uno de los productos químicos
utilizados para proteger algunas áreas agrícolas de organismos
nocivos. Los residuos de DMT liberados directa o indirectamente
al medio ambiente causan graves problemas en la naturaleza.
Los residuos de DMT mezclados con el medio acuático afectan
negativamente a los organismos acuáticos y este efecto se
transmite a los humanos a través de la cadena alimentaria. En este
estudio, se investigaron las respuestas al estrés oxidativo inducidas
por el pesticida DMT en Pontastacus leptodactylus. Para ello, se
investigaron el estrés oxidativo y los parámetros antioxidantes
Sustancias reactivas al ácido tiobarbitúrico (TBARS), glutatión
(GSH), superóxido dismutasa (SOD), catalasa (CAT) y glutatión
peroxidasa (GPX) causados por el pesticida dimetoato (DMT)
en P. leptodactylus en concentraciones de 17,5; 35 y 70 mg·L
-1
a las 24 y 96 horas. Los resultados se determinaron utilizando
kits de ELISA. No se observaron diferencias signicativas en los
niveles de GSH y las actividades de SOD en comparación con el
control. Se observaron diferencias estadísticamente signicativas
entre disminuciones en las actividades de CAT y GPx y aumentos
en los niveles de TBARS. Se utilizó ANOVA unidireccional del
programa SPSS 24.0 (Duncan 0,05) en la evaluación de los análisis
bioquímicos. Como resultado, se determinó que el DMT provocó
la formación de estrés oxidativo en P. leptodactylus y provocó
cambios en las actividades enzimáticas.
Palabras clave: Dimetoato, Pontastacus leptodactylus, estrés
oxidativo, antioxidante, biomarcadores.
The effect of Dimethoate on oxidative stress and antioxidant responses
of Pontastacus leptodactylus
El efecto del dimetoato sobre el estrés oxidativo y las respuestas
antioxidantes de Pontastacus leptodactylus
Ayşe Nur Aydın1 , Hilal Bulut2* , Osman Serdar3
1Central Fisheries Research Institute, Ministry of Agriculture and Forestry, Republic of Türkiye. Trabzon, Türkiye.
2Firat University, Fisheries Faculty. Elazig, Türkiye.
3Munzur University, Fisheries Faculty. Tunceli, Türkiye.
*Corresponding author: hhaykir@rat.edu.tr
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INTRODUCTION
Environmental pollution is the term used to describe all types
of unnatural damage done to the environment by humans.
Environmental pollution has an impact on all living organisms,
including humans, at different times during their existence, from
conception to death. Even though it is up to each individual to lessen
these effects, environmental pollution is steadily rising as a result of
nancial worries [1]. Numerous new types of pollutants have entered
the aquatic environment at the same time that global economic
activity is expanding quickly [2, 3]. Natural water resources are
susceptible to chemical intrusion from runoff, agricultural elds,
and home and industrial wastewater [4, 5]. In agriculture as well
as non–agricultural contexts including businesses, athletic elds,
and other urban green spaces, pesticides including insecticides,
herbicides, and fungicides are frequently used [6, 7]. Aquatic flora
and animals, as well as the environment, are negatively impacted
by pesticides in water [8]. Despite having a low concentration in the
water matrix, persistent pesticides are more dangerous because
of their high stability and bioaccumulative properties. The primary
cause of high bioaccumulation in aquatic creatures is the high water
solubility of particular pesticide groups [9].
Examining the harmful and destructive effects of physical or
chemical agents on living things is known as toxicology Ödün and
Serdar [10]. Finding the concentration at which a drug damages
aquatic species is the goal of aquatic toxicology experiments in
this context [11]. Oxidative stress, also known as oxygen free
radicals and other reactive oxygen species (ROS), has grown
to be a signicant area of research for environmental toxicity
investigations in biological systems. Different pollutants cause
different mechanisms of toxicity, including oxidative damage to
proteins, Deoxyribose Nucleic Acid (DNA), and membrane lipids
as well as adjustments to antioxidant enzymes. As the initial
line of defense, endogenous antioxidants including glutathione
peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT)
may efciently eliminate generated free radicals Bhattacharjee and
Sil [12] and shield cells from oxidative injury. The most efcient
biomarker in defense against oxidative stress in both vertebrates
and invertebrates Batista et al. [13] is CAT activity, which is located
in biological tissues and breaks down hydrogen peroxide into
oxygen and water to protect tissues from oxidative damage [14].
The redox status of the cell is altered by reduction [15, 16].
Due to their susceptibility to pollution contamination in aquatic
environments, it has been reported that crustaceans are species
utilized for evaluation [17, 18]. Crustaceans are suited for use as
water pollution biomarkers [19]. Its long life cycle, wide distribution
and sedentary lifestyle make it a good bioindicator for heavy metals
and organic pollutants. In crustaceans, the gill is a multifunctional
and complex organ that is in close contact with the ambient water.
Contaminants can accumulate signicantly in the gills, and due to
its lipophilicity, DMT can easily penetrate through the gill, causing
oxidative stress and tissue damage [20, 21]. In this Country, it
is widely used in the ght against insecticide and acaris pests in
products such as olives (Olea europaea), pistachios (Pistacia vera),
plums (P. domestica), apples (Malus domestica) and peaches (Prunus
persica) by farmers already licensed is used. Dimethoate is one
of the organophosphate insecticides widely used against various
pests in many crops, and many studies have been conducted on
the toxicity of dimethoate to aquatic and terrestrial organisms [22].
In this study, the effects of pesticide DMT, which is a frequent
environmental pollutant, on Thiobarbituric acid reactive substances
(TBARS), Glutathione (GSH) levels, and Superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GPX) enzyme
activities in the hepatopancreatic tissue of freshwater lobster P.
leptodactylus were investigated.
MATERIALS AND METHODS
Test organism
P. leptodactylus, a freshwater lobster used in the test, was
acquired from a business engaged in aquaculture.
Adaptation of test organisms to the laboratory environment
P. leptodactylus and water from the location where it was
obtained were transported to the lab in plastic boxes. It was
brought as quickly as possible to the lab to lessen the strain on
the living things. They spent about a month getting used to the
lab environment. 15 ± 2°C for the ambient water temperature;
a constant photoperiod of 14:10 light to dark hours. Stock tank
abiotic variables (dissolved oxygen: 11.52 ± 0.87 mg·L
-1
; pH:
8.14 ± 0.4; electrical conductivity: 478 ± 76 S·µcm
-1
; salinity:
0.3 ± 0.02 µg·L-1) were tested daily with a YSI professional plus
brand multiparameter device and changes were recorded. Every
day, the water quality in each aquarium was changed to prevent
further stress from deteriorating water. Feeding took place once
every day. Each tank had its leftover food and waste removed,
and freshwater was added every day to replenish the aquatic
ecosystem. PVC pipes were added to stock tanks as shelter for
the well–being of the craysh.
Chemical substance supply
DMT pesticide containing 400 g·L-1 dimethoate active ingredient
was purchased from a commercial (Koruma) company that markets
agricultural products.
Determination of sublethal concentrations
DMT is applied at a rate of 80 to 140 g·L
-1
in agricultural
regions [23]. Sublethal concentrations were established, as in all
toxicological investigations, by taking into account the application
concentrations discovered in our DMT application study, the release
rates to the environment, and the application concentrations
compared to their values in this range.
Research design
Each experimental group was placed in a glass tank with a
capacity of 80 L with 30 L of water with the specied properties
and 6 model animals were placed. The control group was created
with no pesticide, the second group with a pesticide concentration
of 17.5 mg·L-1, the third group with a pesticide concentration of
35 mg·L
-1
and nally with a pesticide concentration of 70 mg·L
-1
(FIG.1). These conditions (exposure time) were maintained
for 96 hours (h). A summary of the experimental design is
presented below.
»
C1 Group (Control); administration group: no DMT was present.
Eect of dimethoate antioxidant responses of Pontastacus leptodactylus / Aydın et al._______________________________________________ _________________________________________________________________________________________________Revista Cientica, FCV-LUZ / Vol.XXXV
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C1 C2 C3 C4
0
10
20
30
40
50
60
70
80
TBARS (µM)
24h 96h
e
d* d
d
c
c
ba*
FIGURE 1. Experimental applications of Pontastacus leptodactylus exposed to
DMT. (All experiments in the experimental study were performed three times)
FIGURE 2. TBARS (µM) levels of Pontastacus leptodactylus exposed to dimethoate,
Dierent letters on the column are statistically signicant (P<0.05). * indicates
time (24 and 96 h) dierence
»C2 Group; was exposed to DMT concentrations of 17.5 mg·L-1
for 24 and 96 h.
»
C3 Group; was exposed to DMT concentrations of 35 mg·L
-1
for 24 and 96 h.
»
C4 Group; was exposed to DMT concentrations of 70 mg·L
-1
for 24 and 96 h.
and all exposure groups (A, B, C, D) (a,b,c,d,e P<0.05). Two–tailed
independent t–test was applied to compare differences between
exposure times (24 and 96 h) in the same control and exposure
groups (P<0.05).
RESULTS AND DISCUSSION
In the literature, there are many scientic studies investigating the
effects of pollutants on aquatic organisms with various biomarkers.
Oxidative stress represents the potential for mismatch between
ROS production and removal and damage to tissues and cellular
components and is generally accepted in toxicology studies [8].
Thiobarbituric acid reactive substances level
Different concentration and time dependent TBARS levels of DMT
are given in FIG. 2. It was determined that there was a statistically
signicant increase (P<0.05) in the TBARS level in the C3 and C4
groups in the treatment groups exposed to DMT compared to the
control, and there were statistically signicant increases in the
C1 and C4 groups at the 96
th
hour in terms of duration (24 and
96 h) has been done.
Biochemical evaluation
In the experimental groups (including 3 replicate groups
conducted simultaneously), three test organisms were
randomly chosen from the aquarium. The animals from which
the hepatopancreas samples would be taken were placed in
ice water for 30 min, underwent cold shock treatment, and had
a 0.5 g sample of the organ removed from each living item. To
assess antioxidant properties, the samples were weighed (BEL
engineering, M214Ai model precision scale, Italy), homogenized
(Daihan brand, Hg–15D Digital ultraturax model, Korea), and 1/5
w/v of PBS buffer (phosphate–buffered saline solution) was added.
The samples were centrifuged (NUVE brand, NF1200R model
centrifugal, Turkiye) for 15 min at 54,000 G. Until measurements
were taken, supernatants were stored at -86°C in a deep freezer
(Daihan brand, Wisd ultra freezer model, Korea). SOD, CAT, and
GPx activities as well as GSH and Thiobarbituric acid reactive
substances (TBARS) levels were measured using an ELISA
(Agilent brand, BioTek 800 TS Absorbance Reader, USA) reader.
In this investigation, the biochemical reaction was determined by
measuring the GSH, TBARS level, SOD, CAT, and GPx activities.
The SOD, CAT, and acetylcholinesterase (AChE) kits utilized in
the study were bought from a business called CAYMAN. TBARS:
10009055, GSH: 703002, CAT: 707002, SOD: 706002, and GPx:
703102 are the respective catalog numbers for this kit.
Statistical analysis
In this study, SPSS version PASW Statistics 24.0 was used for
statistical analysis. One–way ANOVA and Duncan’s multiple range
tests were applied to determine statistical differences in the control
The decrease in Glutamate–Cysteine Ligase Catalytic Subunit
(GCLC) protein synthesis, which provides the production of SOD,
GPX, GST and GSH, leads to a decrease in the antioxidant defense
of the cell and thus an increase in the amount of TBARS. Lidovaet al.
[24], in their study, Procambarus fallax f. virginalis also examined
the oxidative stress parameters of cypermethrin and stated that
TBARS level decreased as a result. Huang et al. [25], examined the
oxidative stress parameters of Procambarus clarkii of Imidacloprid
and observed a signicant increase in increases in MDA levels.
Yüksel et al. [8], examined the bioresponse of malathion pesticide
on Gammarus pulex and as a result, increases in TBARS levels.
Rossi et al. [26], investigated the oxidative stress responses of
the herbicide glyphosate, insecticide bifenthrin, BF and fungicide
azoxystrobin (AZ) and cyproconazole (CYP), mixtures in Markiana
living in rice elds, and as a result, they increses in TBARS levels. It
is thought that the increase in TBARS level in P. leptodactylus with
Eect of dimethoate antioxidant responses of Pontastacus leptodactylus / Aydın et al._______________________________________________
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C1 C2 C3 C4
0
20
40
60
80
100
120
GSH level (µM)
24h 96h
C1 C2 C3 C4
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
SOD activity (U·mL-1)
24h 96h
FIGURE 3. GSH (µM) levels of Pontastacus leptodactylus exposed to dimethoate
FIGURE 4. SOD (U·ml-1) activities of Pontastacus leptodactylus exposed to
dimethoate, dierent letters on the column are statistically signicant (P<0.05)
the effect of DMT is due to the decrease in antioxidant defense in
the cell. It was determined that the TBARS level increased with
the increase in zaman and DMT concentration. The increased
TBARS levels reflect the increase lipid peroxidation (LPO) found
in the present investigation, which may have resulted from an
increase of free radicals as a result of stress condition generated
by pesticide exposure.
Glutathione level
Different concentration and time dependent GSH levels of DMT
are given in FIG. 3. It was determined that there was no signicant
increase in the GSH level in the treatment groups exposed to DMT
compared to the control (P<0.05), and there was no signicant
increase in the GSH level in the comparison in terms of time (24
and 96 h) (P<0.05).
Superoxide dismutase activity
Different concentration and time dependent SOD activity of DMT
is given in FIG. 4. It was determined that there was no signicant
increase in SOD activity in the treatment groups exposed to DMT
compared to the control (P<0.05), and there was no signicant
increase in SOD activity in terms of duration (24 and 96 h)
compared to the control (P<0.05).
Lidova et al. [24], examined the oxidative stress parameters in
the model organism P. fallax f. virginalis exposed to cypermethrin
and stated that the GSH level decreased as a result. Serdar [27],
evaluated the oxidative stress parameters in G. pulex, which was
exposed to DMT, and stated that there was no change in GSH levels.
Söylemez et al. [28], investigated some biochemical responses of
Beta–Cyfluthrin (β–CF) in Dreissena polymorpha and stated that
GSH level decreased as a result. Zhang et al. [29], studied the
protective effects of Melatonin and oxidative damage of Chinese
mitten crab (Eriocheir sinensis) exposed to deltamethrin and
noted that GSH content increased in the organisms exposed to
deltamethrin. Lin et al. [30], investigated the effect of ammonia
on P. clarkii and as a result, decreases in GSH levels observed.
Abd El–Atti et al. [31], examined the oxidative stress parameters
of titanium dioxide in P. clarkii and observed increases in GSH
levels as a result. Similar to the current study, it was reported
that there was a decrease in several aquatic organisms in GSH
content produced by anticholinesterase agents [32]. GSH depletion
is associated with the oxidation of glutathione peroxidases due
to an increase in free radicals and/or direct oxidation of these
compounds [33]. Additionally, decreasing GSH content can be
associated with its role as GST substrate in detoxication reactions.
While the SOD enzyme converts the superoxide anion radical
(O2-) to hydrogen peroxide (H2O2). SOD, a group of metalloenzymes,
is the primary defense against the toxic effects of superoxide
radicals in aerobic organisms and catalyses the transformation of
superoxide radicals into H2O2 and O2 which play an important role
in antioxidant system cleaning. The response of the antioxidant
system to oxidative stress in various tissues differs from species
to species due to differences in antioxidant potentials of these
tissues. The changes in SOD activities in P. leptodactylus individuals
exposed to DMT are thought to depend on the concentration of
the pollutant. The decreases in SOD enzyme activity determined
as a function of time and concentration are indicative of a defense
mechanism developed by the model organism against oxidative
stress caused by the pollutant DMT. The toxic effect decreased
with increasing concentration and time. Ghisi et al. [34], examined
the effect of P. clarkii exposed to prometrine on oxidative stress
and antioxidant response and reported that prometrine caused
a decrease in SOD activity. Abdel–Daim et al. [31], examined the
oxidative damage caused by chlorpyrifos in Oreochromis niloticus
exposed to chlorpyrifos and reported that as a result, SOD values
decreased. Nataraj et al. [35], investigated hepatic oxidative stress
in freshwater sh Labeo rohita exposed to Profenofos, and as a
result, they found signicant reductions in SOD activity. Uçkun and
Öz [36], investigated the oxidative stress caused by Azoxystrobin
in Astacus leptodactylus and stated that as a result, the level of
SOD in the gills and muscles decreased. Yang et al. [37], examined
the oxidative stress effects of P. clarkii, which they exposed to the
herbicide atrazine, and stated that SOD were inhibited. Lidovaet
al. [24], in their study, P. fallax f. virginalis also examined the
oxidative stress parameters of cypermethrin and stated that
Eect of dimethoate antioxidant responses of Pontastacus leptodactylus / Aydın et al._______________________________________________ _________________________________________________________________________________________________Revista Cientica, FCV-LUZ / Vol.XXXV
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C1 C2 C3 C4
0
20
40
60
80
100
120
140
160
CAT activity (nmol·min-1·ml-1)
24h 96h
a
ab
ab
ab
bc bc
abc
c
C1 C2 C3 C4
0
50
100
150
200
250
300
350
400
GPx activity (nmol·min-1·ml-1)
24h 96h
ab b
a
b
ab
b
a
b
FIGURE 5. CAT (nmol·min-1·ml-1) activities of Pontastacus leptodactylus exposed to
dimethoate, dierent letters on the column are statistically signicant (P<0.05)
FIGURE 6. GPx (nmol·min-1·ml-1) activities of Pontastacus leptodactylus exposed to
dimethoate, dierent letters on the column are statistically signicant (P<0.05)
SOD activity decreased as a result. Yang et al. [37], examined
Cyhalofop–butyl and pyribenzoxim–induced oxidative stress in
P. clarkii muscle and found decreases in SOD activity. Huang et
al. [25], examined the oxidative stress parameters of P. clarkii of
Imidacloprid and observed a signicant increase in SOD activity.
Serdar [27], evaluated the oxidative stress parameters in G. pulex,
which was exposed to DMT, and stated that there was no decrease
in SOD activity, Zhang et al. [29], examined the oxidative damage
of the Chinese mitten crab (E. sinensis) exposed to Melatonin
deltamethrin and stated that the SOD activity decreased. Lin etal.
[30], investigated the effect of ammonia on P. clarkii and as a result,
decreases in SOD activity were observed.
Catalase activity
The CAT activity of DMT depending on different concentrations
and time is given in FIG.5. The changes in CAT activity of 24 and
96 hour exposure groups (C2, C3 and C4) were determined to be
statistically signicant compared to the control (C1) (P<0.05).
When the changes of the same application groups at different
times (24 and 96 h) were compared, it was determined to be
statistically insignicant (P>0.05).
Labeo rohita exposed to Profenofos, and as a result, they found
signicant reductions in CAT activity.
Uçkun and Öz [36], investigated the oxidative stress caused
by azoxystrobin in A. leptodactylus and stated that as a result,
the level of GSH in the gills and muscles decreased. Yang et al.
[37], examined the oxidative stress effects of P. clarkii, which
they exposed to the herbicide atrazine, and stated that CAT
were inhibited. Yang et al. [37], examined cyhalofop–butyl and
pyribenzoxim–induced oxidative stress in P. clarkii muscle and
found decreases in CAT activity. Huang et al. [25], examined
the oxidative stress parameters of P. clarkii of Imidacloprid and
observed a signicant increase in CAT activity. Yüksel et al. [8],
examined the bioresponse of malathion pesticide on G. pulex
and as a result, determined reductions in CAT activity. Serdar
[27], evaluated the oxidative stress parameters in G. pulex, which
was exposed to DMT, and stated that there was no decrease in
CAT activity. Rossi et al. [26], investigated the oxidative stress
responses of the herbicide glyphosate, insecticide bifenthrin, BF
and fungicide azoxystrobin, AZ and cyproconazole, CYP mixtures
in Markiana living in rice elds, and as a result, they observed
decreases in CAT activity and It is thought that the decreases in
CAT activity in P. leptodactylus with the effect of DMT is due to
the decrease in antioxidant defense in the cell. Gao et al. [38],
investigated the oxidative stress results of Maduramycin in P.
clarkii and stated that there were decreases in CAT activity. Lin
et al. [30], investigated the effect of ammonia on P. clarkii and as
a result, decreases in CAT activity were observed.
Glutathione peroxidase activity
GPx activities in P. leptodactylus exposed to DMT concentrations
over time are given in FIG. 6. A statistically signicant (P<0.05)
decrease was detected in the GPx activity of the 24 and 96 h
exposure groups compared to the control group (FIG. 6).
Huang et al. [25], examined the oxidative stress parameters of
P. clarkii of Imidacloprid and observed a signicant increase in
GPx activity increases. Yüksel et al. [8], examined the bioresponse
of malathion pesticide on G. pulex and as a result, determined
reductions in GPx activity. Serdar [27], evaluated the oxidative
Catalase is the main enzyme for the detoxication of ROS and
it is a very common enzyme found in almost all living organisms
using oxygen. It catalyses the decomposition of hydrogen peroxide
affects the generation of water and oxygen [32, 33]. It can be
said that the decrease in CAT enzyme activity in parallel with the
increasing concentration groups compared to the control group
is an indicator of a defense mechanism developed by the model
organism against oxidative stress induced by the pollutant DMT
and decreases in parallel with the increasing concentration.
Ghisietal. [34], examined the effect of P. clarkii, which they applied
Prometrine herbicide, on oxidative stress and antioxidant response,
and stated that there was a decrease in CAT activity as a result of
the study. Abdel–Daim et al. [31] study, examined the oxidative
damage caused by chlorpyrifos toxicity in Oreochromis niloticus,
and as a result, they determined decreases in CAT values. Nataraj
et al. [35], investigated hepatic oxidative stress in freshwater sh
Eect of dimethoate antioxidant responses of Pontastacus leptodactylus / Aydın et al._______________________________________________
6 of 7 7 of 7
stress parameters in G. pulex, which was exposed to DMT, and
stated that there was nno change in GPx aktivity. Gao et al. [38],
investigated the oxidative stress results of Maduramycin in P. clarkii
and stated that there were decreases in GPx activity.
The oxidative stress and antioxidant responses in this study
were related to the DMT concentrations determined in the
hepatopancreas, which is thought to be dependent on the
concentration and exposure time. Since the data obtained in this
study are consistent with the study data in the literature, it is
thought that it will contribute to the literature.
CONCLUSION
It was determined that DMT has a toxic effect on P. leptodactylus.
When the study’s ndings were assessed, biomarker parameters
(SOD, CAT and GPx activty and TBARS, GSH level) responses of
DMT
pesticide in P. leptodactylus were identified.
In addition,
biochemical parameters such as SOD, CAT, GPx activities and
GSH, TBARS levels have been shown to be suitable biomarkers in
the evaluation of oxidative stress and antioxidant effects of DMT.
Credit authorship contribution statement
Ayse Nur Aydın: Visualization, Validation, Supervision, Formal
analysis, Writing – review & editing. Hilal Bulut: Writing – review &
editing, Writing – original draft, Validation, Resources, Methodology,
Investigation, Formal analysis, Data curation, Conceptualization.
Osman Serdar: Validation, Formal analysis, Data curation,
Resources, Methodology.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
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