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A STUDY OF BIOCHEMICAL CHANGES IN THE TISSUES OF
ZEBRAFISH (DANIO RERIO) ON EXPOSURE TO
ORGANOPHOSPHORUS PESTICIDES
Abeer Ghazie A. Al Safi
Pollution Engineering Department , Engineering Technical College,University of
South Technical, Basrah, Iraq.
(Received 12 February 2016, Accepted 26 May 2016)
Keywords: Toxicity, Pesticides, Zebrafish .
ABSTRACT
The aim of this study was to investigate the acute toxicity of Organophosphate
(Malathion and Atrazine), and determined the accumulation of these pesticides in the
whole body of zebrafish, and some biochemical responses in its brain and muscle.
The results showed that MAL and ATR could be rapidly accumulated in the fish body
shortly after being exposed to acute concentrations of the toxicants and exhibited a
dose-dependent manner. This was also validated by the response of antioxidant
defense, superoxide dismutase and catalase, both exhibited significant increase in
activity. Acetylcholinesterase activity of zebrafish was also shown inhibited in a
dose-dependent manner.
INTRODUCTION
An increasing number of pollutants currently exist in aquatic ecosystems, pose a
seriously threaten to the environment and directly affect the health of organisms, as
well as human consumers (1). The important sources of water pollution are industrial
effluent, domestic, sanitation, drainage and pesticides that pollute rivers and major
water sources(2) and the bioaccumulation of pollutants in aquatic organisms resulting
from water pollution cause growing risk to wildlife and humans(3). The
organophosphate (OP) compounds are one of the most widely used pesticides in the
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field of agriculture and public health, which represents 50% of the global insecticides
use because of their biodegradation promptly and non persistent nature (4).
Associated mechanism of OP toxicity is showed as the inhibition of
acetylcholinesterase (AChE), which resulting in accumulation of acetylcholine in the
cholinergic receptors of the peripheral and central nervous system (5). In
ecotoxicology, it is strongly recommended to use cellular and biochemical parameters
to assess the exposure / effects of chemicals (6). AChE has been used as a
bioindicator of exposure to OP pesticides in non-target species (6), However,
oxidative stress became increasingly important molecular mechanism in OP-induced
toxicity. Some biomarkers of antioxidant more common include superoxide dismutase
(SOD) and catalase(CAT) (7). System antioxidant defense plays a critical role in
maintaining homeostasis of the cell. Under normal physiological conditions, the
reactive oxygen can be removed from the metabolism of extraneous chemicals in the
body well by the regime of antioxidant defense. Some of these enzymes can be
regarded as good molecular bioindicators for pollutant-mediated oxidative stress and
can also indicate the magnitude of the response in populations exposed to pollutants
such as pesticides, metals and other xenobiotics (8). Fish are used widely to assess the
health of aquatic ecosystems because of the accumulation of contaminants in the food
chains, which is responsible for the negative effects and death in aquatic systems (9).
Zebrafish is one of the vertebrates that is most important model for studies in basic
physiology. Despite its size is small, the analysis at the level of the whole organ,
tissue, or intact organism is possible, zebrafish also has several properties that make
of vertebrate useful model to study the toxicology(6).
The aim of study was performed to determine the acute toxicity of OP ((Malathion
(MAL) and Atrazine (ATR)) on zebrafish, and described important physiological
problems associated with the accumulation of toxicants in the body of zebrafish and
showed the effect of this toxicant on AChE inhibition. Additionally, antioxidant
defense, lipid and protein stores (energy reserves) in muscle and brain, which indicate
major physiological responses and biomarkers, were also estimated. The effects of OP
(MAL and ATR) on the environment are not well known which mainly resulting from
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human discharges and more information is required to better understand the
importance of these pollutants.
MATERIALS AND METHODS
Fish samples that used in this experiment was zebrafish, which were collected from
various weights (0.295 to 0.847) g and with a total length ranged from (3.1 to 4.6) cm.
The fish samples put in aquarium have 4 L dechlorinated tap water of pH (7.6),
temperature (25-28)°C and illumination 12:12 light: dark, with the availability of
oxygen during the study period. Fish was purchased from the Institute of
hydrobiology Chinese academy of Science. Test solution was replaced every two days.
Test run for 96 hours, the fish were not fed during the experimental period.
2.1 Experimental water and stock solution
The standard analytic of Atrazine (ATR) (2-chloro -4-(ethylamino) -6-
(isopropylamino) - S-triazine) and Malathion MAL) (O, O-dimethyl dithiophosphate
of dietyl mercaptosuccinate) with a purity of (97% and 95%) respectively was
obtained from Xingyinhe chemical engineering co.ltd. A 1000 mg L-1 stock standard
solution of each ATR and MAL was prepared by dissolved in (80% aceton).
2.2 Acute toxicity testing
All experiment was conducted for a period of 96h. Each test concentration was
used 3 replicates. Each aquarium contained 4L of exposure solution and ten fish. The
exposure solution was always aerated and fully renewed every 48h. Water quality
parameters (pH and temperature) were measured daily. During the acute toxicity
experiment the number of dead fish was counting every 24h and the dead fish were
removed from the aquarium as soon as possible. Fish were divided into two groups:
first group without treatment (control group with acetone just for organophosphorus
pesticides), whereas the second group were treated with a series of different
concentration of organophosporus insecticide and herbicide (0, 2.5, 5, 10 and 20 mg
L-1), the mortality rate was determined at the end of the 96h by use of Finney’s Probit
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Analysis LC50 determination method Finney (10) and were recorded daily during the
test period. A second test was using tissues organs for biomarker analysis (10) fish per
treatment were used). At the end of the test, the live fish were killed by ice water.
Heads and muscles were isolated in frozen in 2 ml tubes.
Table (1) LC50 values (with 95% confidence limit) of MAL and ATR in (mg L-1) of
zebrafish estimated by Finney.
2.3 Bioacumulation test
Bioaccumulation of MAL and ATR in the whole body tissue of zebrafish, to
determine the pesticides in water after filtrated it using 0.22μ m filtration membrane
and stored at 4°C before analysis by high-performance liquid chromatography
(HPLC). Pesticides were extracted from fish tissue depending on the method
described by Zhao (11), use whole body of zebrafish except its fins, after crushed 2g
wet weight of sample were extraction the pesticides and the extract was also filtered
with 0.22μ m filter and analysis by HPLC, this system contained the SSI
2300-525HPLC: Detector: Variable dual wavelength 525 UV detector; column:
Apollo C18 column (250mm × 4.6 mm, 5μm) ALLTECH company; the Series III
pump Cschrom Plus chromatography workstation; column temperature: 30°C.
Pesticide detections were made in the UV region (λ = 210 nm for MAl and λ = 254
nm for ATR). The composition of mobile phase for MAL consisted of acetonitrile
Chemical LC50 value 95% confidence
limit
R²
MAL
5.292
4.8201-5.8104 0.964
ATR 9.567 8.366-10.941 0.922
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(70%) plus water (30%) with pH 3.5 (30%) and for ATR it was acetonitrile (35%)
plus 0.025 M dipotassium hydrogen phosphate (pH 3.0 with acetic acid) (65%). The
flow rate and injection volume for both the pesticides was 1.0 mL/min and 20μL
respectively. The retention times for MAL and ATR were 5.2 and 11.08 min,
respectively. Under the chromatographic conditions was performed quantification of
pesticides by using external standard was described above.
2.4 Biochemical analysis
At end of each exposure period, the muscle and brain tissues of zebrafish were
homogenized to 1/10 (w/v) ratio of cold physiological saline solution NaCl (0.86%)
using a mortar and pestle, then centrifuged at (10min, 8000r/min) in 4°C, the
supernatant was used for biochemical analysis.
AChE activity was determined at 412nm wavelength of spectrophotometer by
method of George (12). SOD and CAT activities were determinated by method of Can
(13), SOD was use NBT photochemical reduction reaction, then calculate absorbance
values in 560nm with a micro plate reader. For quantitative determination of CAT
activity by use 30% hydrogen peroxide that was diluted by 7.4 phosphate buffer to a
final concentration of 65 micromolar of hydrogen peroxide per milliliter of sodium
potassium phosphate buffer (PH7.4, 60mmol L-1) as substrate solution and
ammonium molybdate solution (32.4mmol L-1), then measure absorbance at 450nm.
The activity of enzymes was defined as units of activity per milligram of protein.
Total protein content was measured by dependent on the procedure of Bradford (14),
at 595nm and using bovine serum albumin as standard and the standard calibration
curve was set as (y=0.0051x- 0.0013, R2=0.9993). The total lipid content was
determined by using 1g dry weight for whole body of zebrafish and extracted the lipid
by used the soxhlet for 6h by method of AOAC (15).
2.5 Statistical Analysis
The bioconcentration factor (BCF) of MAL and ATR in zebrafish was
estimated using the following equation: BCF = Cf/Cw
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Where Cf is the concentration of toxicant in fish and Cw is the concentration of
MAL and ATR in the exposure solution. One-way analysis of variance (ANOVA)
was performed to determine statistical significance among and between groups
followed by Dennett’s test, using the graphPad program. The criterion for significance
was set at (p < 0.05).
RESULTS
3.1 Bioaccumulation of toxicants and total lipid of whole body of zebrafish
Based on the results of acute toxicity, zebrafish were exposed to different
concentrations of OP (MAL and ATR), and bioaccumulation in whole body of fish
were detected during 96h (Figure 1). The results showed that for both treatments, the
BFC was increased with increased the toxicants concentration. The maximum values
to BCF was in higher MAL and ATR concentration (8.467×103 and 2.007×104)
respectively. Total lipid content was significantly increased in 1st concentration for all
toxicants compared with control groups (p<0.01), However, there was slight reduction
in total lipids content with increased concentration of toxins (Figure 2).
Figure (1) Bioaccumulation factor (BCF) of MAL and ATR exposure to
different concentrations to whole body of zebrafish during 96h. Values
are presented as a mean ± SD.
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Figure (2) Total lipid contents % dry weight for whole body of zebrafish in different
acute concentration of toxicants (A) MAL and (B) ATR mg L-1. Values
are presented as a mean ± SD. The asterisk represents a statistically
significant difference when compared with control groups; * at p<0.05,
** at p<0.01 and *** at p<0.001 levels.
3.2 Effects of toxicants on AChE activities of zebrafish tissues
The AChE activity in muscle tissue was significantly induction (p<0.01) in higher
concentration of MAL and ATR by (49.366 and 54.398 %) respectively compared
with control groups for 20 mgL-1, after 96h (Table 2). While in the brain tissues of
zebrafish were significantly induction (p<0.05), the maximum slip was in 5 mg L-1 of
MAL by (29.114%), while in 20 mg L-1 of ATR by (9.653%), compared with control
groups (Table 2).
3.3 Effects of toxicants on antioxidant enzymes activities of zebrafish tissues
The result demonstrated significantly increased (p<0.01) in CAT activities in
muscle tissues after exposure to different acute concentrations for 96h of toxicants.
The maximum increased of CAT activities was apparent with a treatment
concentration of 20 mgL-1, when CAT activity increased by (502.74 and 433.35 %)
respectively, compared to the control groups. as well as observed significantly
increased (p<0.05) for SOD activities in muscle tissues for both pesticides in the last
concentration by (185.454 and 109.463 %) respectively. While it was observed that
different apparent with a treatment concentration in brain’s tissues, that the maximum
increased of CAT in brain tissues for both pesticides by (108.652 and 130.103% )
respectively for 5 mgL-1 after 96h compared with control groups (Table 2).
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3.4 Effects of toxicants on Protein contents in zebrafish tissues
The protein content in muscle tissue was significantly depression (p<0.01) with
increased of toxins of MAL and ATR, specialties in higher concentration of pesticides
by (18.369 and 35.152%) respectively compared with control group for 20 mgL-1,
after 96h (Table 2). The significantly decreased of protein content was also observed
in brain’s tissue of zebrafish (p<0.01). But the processes were different from that
observed in muscle, Maximum decreased of protein content in 20 mgL-1 for ATR, the
decreased about (33.683 %) respectively.
Table (2) Changes in biochemical parameters after exposed to different concentrations
of MAL and ATR for 96h in muscle and brain of zebrafish
Chemical
con.
MgL-1
AChE a CATb SODb Protein content c
Muscle Brain Muscle Brain Muscle Brain Muscle Brain
MAL
0 1.1526±0.11 0.237±0.02 69.172±1.52 320.502±3.54 8.607±0.36 18.903±0.10 13.294±0.91 8.428±0.66
2.5 0.477±0.013* 0.506±0.22 166.074±2.17** 250.255±1.94* 11.75±0.53* 10.088±0.64** 7.116±2.814 12.727±4.67
5 0.303±0.03** 0.069±0.04 109.405±3.11** 348.235±2.28** 8.14±0.45 13.521±0.37* 4.363±2.73 10.577±0.13
10 0.538±0.11** 0.135±0.05 200.581±1.01* 300.099±2.05** 9.763±0.54 15.723±0.51** 3.992±4.37 9.139±3.77
20 0.569±0.04** 0.353±0.06
347.758±1.95**
173.536±2.50** 15.962±0.68* 21.567±0.40** 2.442±3.19 9.326±0.83
ATR 0 1.1526±0.107 0.259±0.02 69.152±1.52 320±3.54 8.507±0.35 18.863±0.61 6.819±4.175 17.632±1.67
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The standard deviations values in the same row with different significantly compared to the control; *
at p<0.05 and ** at <0.01 levels.
a Activities are expressed as mmol/min/mg protein.
b Activities are expressed as U/mg protein.
cContents are expressed as mg/g .
DISCUSSION
The pesticides and chemical pollutants appears to play a major role in the loss of
fish resources and impact on the ecosystem aquaculture and human health risk
assessment, and this led to the study of acute toxicity and accumulation of pesticides
in the organisms to obtain more and more attention(11). The present study showed
that MAL and ATR could be rapidly accumulated in fish shortly after their exposure
to acute concentrations of toxicants and the highest BCFs of toxins was in fish were
(8.467×103 and 2.007×104) respectively in 20 mgL-1 for MAL and ATR after 96h
exposure. The results suggested that both pesticides had probably to accumulate
rapidly in fish or other organisms, which was in agreement with some previous
reports. A study by Zhao(11) showed that exposed zebrafish to pyrimorph fungicide
could be rapidly accumulated in fish shortly after their exposure to a sublethal
concentration of pyrimorph, and the highest BCFs of its were 1.07 × 102 (144 h) and
23.1 (96 h) after exposure to 2.00 and 0.25 mg L−1 of pyrimorph respectively. Sun (16)
was determined the accumulation of HC Orange No. 1 in liver of goldfish (Carassius
auratus), and referred to the accumulation in fish tissue shortly after the start of the
exposure and reached the maximum level at a 24h exposure.
Enzymatic changes considered as important signs of a hazardous chemical
materials (17). Many studies have shown that inhibition of AChE after OPs exposure,
they were also used to measure the activity of this enzyme in the fish to monitor
neurotoxicity of the OPs. These pesticides inhibit the activities of this enzyme was
dose and time-dependent, as well as the difference in the rate of inhibition depends on
2.5 1.011±0.65 0.374±0.25 283.443±2.43** 312.516±1.78 7.005±0.703 9.233±0.52** 3.997±2.73 7.387±3.95
5 0.716±0.03 0.583±0.34 102.472±1.75** 416.328±3.06** 2.094±0.64*** 10.862±0.61** 4.256±2.73 14.766±3.15
10 0.358±0.04 0.782±0.17 268.195±3.29** 362.806±1.98** 8.046±0.67 22.329±0.47** 4.284±4.37 10.725±0.90
20 0.627±0.01 0.025±0.39 299.667±2.36** 338.022±2.14** 9.312±0.57 22.986±0.697** 2.397±3.19 5.939±0.31
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the species and age (18). The inhibition of AChE enzyme activity is widely as a good
biomarker for exposure to toxic OPs (19). This investigation in our study for most of
tested groups with OP (MAL and ATR) was significant inhibition of AChE activity
(p<0.05) in brain and muscle tissues of zebrafish with the increase of concentration.
In agreement with our results. Senger (6) suggested that pesticides can interact
directly with cholinergic receptors at or below the concentrations that inhibit AChE.
Feng (20) directly investigated AChE inhibition of trichlorfon with muscle of (T.
niloticain).
Increase or inhibitions of antioxidant activity under pressure of chemical depend on
the severity and duration of stress applied, as well as susceptibility of the species to
exposure. In this study, SOD and CAT activities showed an increasing trend in the
tissues of zebrafish exposed to MAL and ATR and an increase was more pronounced
in muscle with increased the concentration of toxin especially in high concentration
for all toxicants. It was noted that this increase in SOD activity may be due to the
higher O2
- production (8). Moreover, mechanism of antioxidant defense has great
significant for fish because it works to protect them from free radicals produced by
oxidative stress and other factors and it is the first defense mechanism against
oxidative stress (7) this was agree with Oruc (21) who investigated the increase in
SOD activity caused by chlorpyrifos exposure in adult of (Oreochromis niloticus).
Most of the increase in CAT activities in the muscles and brain zebrafish observed in
this study may be a response to H2O2 produced by activity SOD, because the CAT is
responsible for detoxification of H2O2 in the water. This refers to a positive
correlation between of SOD and CAT to the muscles and the brain in this study,
should be accompanied by increased activity SOD with increase in the production of
H2O2, which led to higher activity CAT, and chemicals used in this study cause
significant elevation (P<0.05) in the activities of the SOD and CAT in zebrafish after
exposure. This agreed with the investigation of Sharbidre (7) showed increase in CAT
and SOD levels in brain and gills of guppy fish (Poecilia reticulate) after 96h
exposure of chlorpyrifos in all treatment groups.
Changes in the biochemical parameters such as proteins and lipids are important to
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signify the sensitivity of organs to pollutants by changing their function. The lipids
are one of the most important sources of energy and structural components in the
body of fish. Biological studies have revealed for lipids the importance of lipids
during periods of stress. Where that stress increases the overall energy consumption,
reduce the availability of energy for other processes strongly expensive. Thus, it
invokes compensatory metabolic changes in the tissues of animals through the
modulation and modification of the quantity and quality of various metabolites,
including the lipids (22). In the present study, it was proved the increase in the total
lipids content in the first concentrations for OP (MAL and ATR) in the analyzed
tissues of the zebrafish body exposed to acute concentrations compared with control
groups. There are some different factors which may effect on the lipid contents of the
organisms, such as age, sex, and food supplies. It was noted that the increase in lipid
contents may due to the exposure of animals to conditions stressful, and the
biotransformation of other organic components such as carbohydrates and proteins to
lipids, and also to resist the toxicity made by various chemicals. Nandurkar and
Zambare (23) found the increase in lipid contents in both the selected models,
(Lamellidens corrianus and Parreysia cylindrical) after acute and chronic exposure of
chloramphenicol. At the same time, the increased lipid content in fish may also
cause damage, because the organisms with high lipid content have bio-concentration
of these chemicals and this property leads to bioaccumulation and thus, to a
detrimental effect on the human food chain(24).
CONCLUSION
The results of the current study showed that (OP) pesticides were highly toxic to
zebrafish. Studies indicated that the acute toxicity as a first step in determining the
water quality requirements to fish and so reveal toxin concentrations (LC50) that cause
fish mortality even in the short exposure. It can be concluded from this study that the
zebrafish is very sensitive to these toxicants and their mortality rate was dependent on
the concentration of the toxins. The ability of zebrafish to bioaccumulation of these
toxins in a dose-dependent manner is a strong indicator of suitability as a model in
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biomonitoring programs. As well as the zebrafish has a number of properties that
makes them useful sentinels for environmental monitoring. Biological monitoring
using a series of assays having different endpoints. It could allow a sensitive approach
to predict the potential risk of pesticides which are useful in the formulation of the
“safe levels” of such bioaccumulative chemicals. However, this study has been
ignored many of the factors in the experiment as fish that live in the environment
where they face different circumstances. Therefore, there is a need to further study to
answer whether induction of these parameters can be useful as early biomarkers of
toxicants in the environment practical for fish.
دراسة التغیرات الکیمیائیة الحیویة فی أنسجة سمکة الصغیرة المخططة بعد تعریضھا للمبیدات
الفوسفوریة العضویة
عبیر غازی عزیز الصافی
قسم ھندسة البیئة والتلوث، الکلیة التقنیة الھندسیة ، الجامعة التقنیة الجنوبیة، البصرة، العراق
الخلاصة
ان الھدف من ھذه الدراسة تحقیق السمیة الحادة لمبیدی ( الملاثیون و الأترازین )، و تحدید تراکم ھذه
المبیدات فی کل جسم السمکة ، و بعض الردود الحیویة فی الدماغ و العضلات.
وأظھرت النتائج أن الملاثیون و الأترازین سریعة التراکم فی جسم السمکة و بعد فترة وجیزة من التعرض
لتراکیز الحادة من المواد السامة و بطریقة تعتمد على الجرعة .وتم تحقیق ذلک من ردود فعل الدفاعات المضادة
نتیجة لزیادة الکبیرة لفعالیتھا الدفاعیة . وقد تبین تثبیط ، catalase و superoxide dismutase ، للأکسدة
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للسمکة بطریقة تعتمد على الجرعة . Acetylcholinesterase نشاط انزیم
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