الفهرس 
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Abstract
Pesticides are crucial for pest control in agricultural and domestic sectors throughout
the word. Although their use can increase agricultural productivity and minimize pest
populations, the continued intensive use of pesticides in agriculture has polluted nearby water
bodies and endangered aquatic life, especially fish.
In the real environment, the body is exposed to many compounds at the same time,
rather than just one, especially as the variety and quantity of pesticides increases. Research on
the risks that mixtures of compounds pose to living organisms, especially aquatic animals, is
limited and difficult to predict due to differences in the mode of toxic action, toxicokinetics,
and chemical properties of each compound. Therefore, the present study aimed to evaluate the
combined effects of the two formulated pesticides; ethoprophos (Smart-N, 40% EC) and
bispyribac-sodium (Nominee, 2% SL), commonly found in the environment against Nile tilapia
(O. niloticus).
The results of the present study can be summarized as follows:
1. Toxicity of the tested pesticides on Nile tilapia
The 96 h LC50 values of ethoprophos and bispyribac-sodium were 4.8 and 0.064 mg/L,
respectively. This result indicated that commercial formulation of bispyribac-sodium was more
toxic to Nile tilapia than ethoprophos.
2. Effect of sublethal concentration of ethoprophos and bispyribac-sodium or their
combination on some biomarkers of Nile tilapia
The in vivo effect of 1/50 LC50 of ethoprophos and bispyribac-sodium or their
combination for different time intervals (1, 2, 3 and 4 weeks) on biochemical, genetic and
histological changes in Nile tilapia fish was evaluated.
2.1. Biochemical markers
2.1.1. Acetylcholinesterase (AChE)
The brain AChE activity of all treated fish was lower than untreated ones. Results
showed that the mixture of ethoprophos and bispyribac-sodium was more potent to inhibit
AChE followed by ethoprophos and then bispyribac-sodium.
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2.1.2. Oxidative stress
Lipid peroxidation (LPO)
Treatment of fish with the tested pollutants for 4 weeks revealed a significant elevation
in LPO levels compared to the control value. Among the treatments, the mixture was the most
toxic pollutant in increasing the LPO level, while bispyribac-sodium was the least toxic.
Reduced glutathione (GSH)
The mixture of tested chemicals has proven to be more effective to decrease the GSH
content than ethoprophos or bispyribac-sodium alone, where they decreased GSH levels by
42.94%, 37.72% and 31.14%, respectively after 4 weeks of exposure.
Superoxide dismutase (SOD)
The results showed that all treatments decreased hepatic SOD activity by 65.42%,
47.44% and 84.49% after exposure to ethoprophos, bispyribac-sodium and their mixture,
respectively for 4 weeks.
Catalase (CAT)
The results indicated that all treatments considerably reduced CAT activity in
comparison to the control. Fish treated to ethoprophos, bispyribac-sodium, and the mixture
attained CAT activity of 0.421, 0.435, and 0.399 U/mg protein, respectively at the end of the
experiment.
Glutathione peroxidase (GPx)
Significant depletion in the GPx activity was noticed after one week of fish exposed to
the combination of ethoprophos plus bispyribac-sodium (19.22 µmole GSH/mg protein/min),
followed by ethoprophos (23.57 µmole GSH/mg protein/min) then bispyribac-sodium (26.42
µmole GSH/mg protein/min) compared to control (30.02 µmole GSH/mg protein/min).
Glutathione S- transferase (GST)
Significant elevation in GST activity in all treatment groups compared to the control
one. The activation power of the compounds could be ranked in the following order: mixture >
ethoprophos > bispyribac-sodium.
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2.1.3. Liver function parameters
Alkaline phosphatase (ALP)
The current data illustrated that the three tested substances: mixture, ethoprophos and
bispyribac-sodium induced a significant elevation in ALP activity recording 117.42, 71.47 and
31%, respectively after 4 weeks of exposure.
Aspartate transaminase (AST)
The results showed that the AST activity in fish exposed to the mixture was significantly
higher than in fish exposed to a single compound.
Alanine transaminase (ALT)
ALT activity increased after a week of exposure to ethoprophos, bispyribac-sodium,
and their mixture. It is clear that the effect of the mixture was greater than that of each
compound alone.
2.1.4. Kidney function parameters
The results presented a significant increase in the levels of creatinine and urea in all
treated groups compared to the control group, and the greatest increase was recorded when
treated with the mixture. This increase was time dependent.
2.2. Genotoxic markers
2.2.1. Micronucleus (MN) assay
All treatments revealed a significant increase in the frequency of MN compared to the
control at all tested times (1, 2, 3, and 4 weeks), and a combination has a greater frequency of
MN, followed by ethoprophos and bispyribac-sodium.
2.2.2. 8-hydroxy-2ʹ-deoxyguanosine (8-OHdG)
Exposure of fish to the tested compounds for 4 weeks increased 8-OHdG levels to 1.836,
1.413 and 2.545 folds for ethoprophos, bispyribac-sodium and their mixture, respectively,
compared to the control group. In addition, the mixture was more effective at damaging DNA
than the individual compounds.
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2.3. Histological examination
The ultrastructural effects of the sublethal concentration of tested compounds and their
mixture on the gill tissues of O. niloticus were conducted using SEM. The treated gill for 4
weeks showed alterations such as erosive lesions on the primary and secondary lamellae,
impairment and disturbance of bony ossification of gill filament and lamellae, fusion of
microridges of epithelial cells and excessive mucus secretions on the gill epithelium.
from the present study, it can be concluded that:
In reality, the organism may be exposed to multiple xenobiotics rather than just one.
Nile tilapia fish is an ideal model organism for aquatic contamination due to its high
nutritional value, good flavor and wide distribution in fresh water, in addition to its
commercial importance in many countries, especially Egypt.
The gills are the primary organ in direct contact with aquatic pollutants and play an
important role in the respiratory gas exchange, osmoregulation, metabolism of toxins,
their excretion and/or transport into the bloodstream, and subsequent accumulation in
tissues and organs. Thus, histopathological changes of gills are widely used indicator of
fish exposure to environmental contaminants.
This study showed that while short periods of exposure to high pesticide concentrations
are possible, long-term exposure to low pesticide concentrations is much more
prevalent.
Higher toxicity of the formulated pesticides may result from supposedly inert additives
such as surfactants, as well as from the toxic effects of the pesticides themselves. This
was at least partially due to the ability of surfactant to facilitate the absorption of
pesticides into tissues, which increases the toxicological impact of pesticides.
This study demonstrated the benefits of using a number of simple and sensitive
biomarkers to create a complete picture and provide the best information on the
toxicological effects of pollutants.
The data revealed that the two pesticides either applied individually or together, altered
several parameters, such as oxidative stress response, AChE activity, liver and kidney
function, genetic markers, and gill histological examination.
All tested substances significantly changed the level of antioxidant parameters of fish,
which probably plays an essential role in the mechanism of toxicity of these pesticides.
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Recommendations
Strict legislation regarding the use of pesticides must be applied in developing
countries.
Precautions should be taken to prevent the accidental or deliberate release of
pesticides into water system, by using the pesticides cautiously, in a restricted
manner and at the rate recommended by the manufacturer to minimize their release
into the aquatic system and protect water quality.
In the context of sustainable development, governments and the scientific
community must implement environmental action plans to combat environmental
damage. These efforts are necessary to ensure that the situation gradually improves
in the coming years.
Overall, the results highlight the importance of the responsible authorities in
ensuring the quality of drainage waters that feed fish farms, which can act as a main
source of protein for humans.
Environmental protection is no longer an entertainment or luxury, but has become a
vital national responsibility through laws governing the preservation of good
resources and the environment for future generations