INTRODUCTION
Background and Rationale
The rapid growth of the coconut industry has led to increased waste production, which poses significant environmental risks if not managed properly Technological advancements often contribute to environmental damage, threatening ecological balance and impacting living organisms Human activities, particularly those resulting in water pollution, are major contributors to environmental degradation The indiscriminate discharge of industrial and municipal waste into water bodies disrupts fragile ecosystems and poses health risks to humans Industrial waste, often varied in composition depending on manufacturing processes, significantly contributes to the extinction of aquatic life Key pollutants include sewage, industrial waste, and agricultural chemicals such as fertilizers and pesticides, with insecticides like cypermethrin being among the most commonly used in agriculture.
Cypermethrin is a type of pyrethroid insecticide, widely utilized in agriculture, gardens, and industrial areas for pest control This class of insecticides is also effective in treating ectoparasitic diseases, such as lice, in various animals including sheep, cats, and dogs Pyrethroids share structural similarities with pyrethrins, natural compounds derived from chrysanthemum plants that serve as an effective means of pest management.
Over the past 25 years, pyrethroids have emerged as a leading class of insecticides in agriculture due to their low toxicity to mammals and lower application rates As a result, they are increasingly replacing organophosphates and carbamates in arable crops and forestry across Europe and North America (Hartnik, Sverdrup and Jensen, 2008).
In recent years, water pollution from insecticide compounds has become a significant issue in developing countries that rely on agriculture for economic growth Pyrethroid insecticides, particularly cypermethrin, are widely used in farming and contribute to rising toxic chemical levels in aquatic environments Known for its high toxicity, cypermethrin disrupts the nervous systems of insects and is often transported to surface waters through runoff, erosion, and aerial spray drift This poses risks to benthic and epi-benthic species, as well as fish and other organisms that feed on benthos Due to its harmful effects on aquatic life, the use of cypermethrin is now restricted in crop applications and large-scale farming practices.
2009) Histopathological response of fish exposed to pollutants has been used as the sensitive biomarkers
To mitigate cypermethrin insecticide residues, activated charcoal derived from agricultural waste, particularly coconut shells, can be utilized With the advancement of coconut technology in Indonesia, the production of coconut waste, including shells, has significantly increased Dried coconut shells are composed of 33.61% cellulose, 36.51% lignin, 29.27% pentosans, and 0.61% ash (Shelke et al., 2014), indicating their potential effectiveness in addressing insecticide residue issues.
Coconut shells, due to their high carbon content and hardness, are an excellent raw material for producing activated carbon, which is effective in waste adsorption (Ratnoji and Singh, 2014) The application of activated charcoal in rice fields enhances the population of beneficial bacteria, such as Azotobacter, which aids in nitrogen fixation, particularly around food crop roots This process also increases the microbial diversity, including pesticide-degrading bacteria like Citrobacter sp and Enterobacter sp Moreover, utilizing activated charcoal in agriculture helps minimize insecticide residues in soil, water, and crops, thereby reducing surface water pollution that can harm aquatic life and human health Additionally, it contributes to decreasing agricultural waste and adds value to agricultural by-products (Harsanti et al., 2013).
Fish are effective bio-accumulators of both organic and inorganic toxicants, serving as primary protein sources and bio-indicators of heavy metal contamination in aquatic ecosystems Oreochromis niloticus, a popular species among aquaculturists, is favored for its ability to thrive in diverse environmental conditions, rapid growth, successful reproductive strategies, and adaptability to various trophic levels These characteristics not only make it a valuable commodity in aquaculture but also contribute to its success as an invasive species in subtropical and temperate regions.
In regions where fish is a staple food, individuals are significantly impacted by fish consumption Both terrestrial and aquatic food chains can accumulate harmful environmental pollutants, reaching toxic levels that pose risks to the health of both humans and aquatic organisms.
Fish serve as vital indicators of water pollution levels, with histopathological analysis acting as a biomarker for monitoring marine environments through the assessment of fish health By examining critical metabolic organs, this analysis can facilitate early diagnosis of illnesses in fish (Zulfahmi, Affandi, and Lumban Batu, 2015) Notably, Nile Tilapia, a species of freshwater fish, is particularly sensitive to water pollution.
This study investigates the effectiveness of coconut shell activated carbon in mitigating the effects of water pollution caused by hazardous substances like cypermethrin, particularly on fish health, as assessed through histopathological analysis.
Objectives
This study investigates the toxicological impact of cypermethrin on Nile Tilapia, evaluating the potential of coconut shell as an adsorbent for the pollutant Additionally, it examines the histopathological changes in the gills of Nile Tilapia exposed to varying concentrations of cypermethrin and the treatment with the adsorbent.
Research Questions and Hypotheses
This study addresses the pollution issue caused by insecticide contaminants in water by utilizing an adsorbent material derived from waste biomass, specifically coconut shells.
1 How does cypermethrin affect the gills of Nile Tilapia?
2 How does cypermethrin affect the gills of Nile Tilapia after adsorbent is given?
H0 (Null Hypotheses): Exposure to cypermethrin contamination with the treatment of adsorbent will not result in changes in gills histology of Oreochromis niloticus
HA (Alternative Hypotheses): Exposure to cypermethrin contamination with the treatment of adsorbent will result in changes in gills histology of Oreochromis niloticus Hypotheses Question 2:
H0 (Null Hypotheses): Exposure to cypermethrin contamination with the treatment of adsorbent will not result in changes in gills histology of Oreochromis niloticus after adsorbent is given
HA (Alternative Hypotheses): Exposure to cypermethrin contamination with the treatment of adsorbent will result in changes in gills histology of Oreochromis niloticus after adsorbent is given.
Limitations
- There is a lack of experimental methodology using coconut shell as an adsorbent to adsorb cypermethrin insecticide
- An uncertain fish conditions affects the time of the toxicity testing and adsorbent experiment
- As well, the lack of experimental results on the histopathological effects on the gills tissue of Nile Tilapia which caused by cypermethrin in literature
LITERATURE REVIEW
Cypermethrin Compound
In Table 1 shows the physical and chemical properties of cypermethrin insecticide
Table 1 Physical and Chemical Properties of Cypermethrin
Cyano(3-phenoxyphenyl)methyl 3-(2,2- dichloro ethenyl)-2,2- dimethylcyclopropanecarboxylate
Arrivo, Cymbush, Cymperator, Cynoff, Ripcord, Basathrin, Demar, Grand, Starcyp Chemical Formula C22H19Cl2NO3
Physical State Viscous semi-solid
Conversion Factors 1mg/m 3 = 0.059ppm pH 7 and below (in water)
Toxic Effect of Cypermethrin on Organisms
The toxicity classification of cypermethrin via the designated routes of exposure shows in Table 2
Table 2 Toxicity Classification of Cypermethrin
Inhalation of cypermethrin can lead to significant respiratory irritation, with symptoms including hypersensitivity, pneumonitis, pleuritic chest pain, non-productive cough, and shortness of breath, particularly when directly sprayed or during the packing process.
Ingestion Ingestion of food contaminated by cypermethrin insecticide effects on human will have symptoms such as nausea, diarrheal, vomiting and epigastric pain or even death
Using shampoos containing cypermethrin can lead to dermal allergic reactions in dogs, resulting in symptoms like burning, numbness, and itching Farmers often utilize these products based on their plant-derived properties, but it's important to be cautious of potential skin irritations that may arise from their use.
Source: Javed et al., 2015 Cyano(3-phenoxyphenyl)methyl3–(2,2–dichloroethenyl)–(2,2 dimethylcyclopropanecarboxylate) which has known as cypermethrin is an active
9 substance one of type II synthetic pyrethroid insecticide similar to natural pyrethrin isolated from chrysanthemum flowers, which has high insecticidal activity (ATSDR,
Cypermethrin is a potent pyrethroid insecticide widely used in agriculture and animal husbandry for effective pest control Classified as a Schedule 6 poison, it typically contains over 90% active ingredient in its technical grades While cypermethrin exhibits low vapor pressure and water solubility, it is highly soluble in various organic solvents Established analytical methods allow for the detection of cypermethrin in commercial products, food residues, and environmental samples Although it poses minimal risk to mammals and birds, cypermethrin is highly toxic to fish and aquatic life, necessitating careful application away from water sources to prevent contamination.
Cypermethrin is not soluble in water and adsorbed extensively and rapidly in water-sediment systems According to WHO (1989) cypermethrin was synthesized in
Cypermethrin, a highly active synthetic pyrethroid insecticide, was first marketed in 1977 and is effective against a variety of pests in agriculture, public health, and animal husbandry It is one of the most widely sold pyrethroids in agriculture, often replacing more toxic organophosphates Primarily used to combat foliage pests and certain surface soil pests like cutworms, cypermethrin is not recommended for soil-borne pests due to its rapid degradation in soil.
Cypermethrin used to control many pests including lepidopterous pests of cotton, fruit, and vegetable crops and is available as an emulsify-able concentrate or wet-able
Cypermethrin, widely used in various applications, poses a significant risk to aquatic environments due to its potential discharge When cypermethrin enters water bodies, it disrupts the habitat of aquatic organisms, particularly fish, which can react to both physical changes and pollutant concentrations This sensitivity makes fish valuable indicators in biological testing (Authman et al., 2015).
There are several studies on histopathological effects of cypermethrin on organisms Histopathological alterations give a reliable, efficient measurable index of low-level toxic stress to a wide range of environmental contaminants
A study by Ojutiku et al (2014) investigated the toxicity and histopathological effects of cypermethrin on juvenile Clarias gariepinus, exposing the fish to six acute concentrations (0.025mg/l to 0.125mg/l) over 96 hours The 96-hour LC50 for cypermethrin was determined to be 0.060mg/L Notable gill alterations included destruction of gill lamellae, epithelial hyperplasia, and hypertrophy across all exposure levels At the lowest concentration, gill necrosis, congestion, and haemorrhages were observed, while the highest concentration resulted in severe gill necrosis, extensive vacuolation, and further infiltration and haemorrhages within the gill epithelia.
Rahayu, Zulfatin and Nuriliani (2001) conducted a research about histopathological test of gill tissue, liver and intestine towards Nile Tilapia using λ- cyhalothrin as pollutant The variations concentration of λ-cyhalothrin were 3, 6, 9 and
A study investigating the effects of λ-cyhalothrin on Nile Tilapia revealed significant histopathological damage to the gills, liver, and intestine The gill injuries included hyperplasia, necrosis, edema, secondary lamella fusion, and epithelial cell removal, while liver damage manifested as hemorrhage and leukocyte infiltration Intestinal damage showed signs of fatty degeneration, edema, and necrosis The severity of these damages varied from slight to severe, indicating that λ-cyhalothrin adversely affects Nile Tilapia health Additionally, a separate study on the impact of cadmium (Cd) on the gills of catfish (Clarias batrachus) demonstrated that exposure to increasing concentrations of Cd (0, 1, 2, and 4 ppm) over 14 days resulted in escalating gill damage, with control fish showing only 13.9% damage compared to 45.7%, 71.6%, and 82.1% in fish exposed to 1, 2, and 4 ppm of Cd, respectively.
Histopathological changes due to some chlorinated hydrocarbon pesticides in the tissues to Cyprinus carpio performed by using the concentration of Aldrin, Dieldrin,
The study examined the effects of BHC and DDT on test fish, with pollutant concentrations ranging from 0.002 to 1.000 mg L-1 The fish were exposed to these concentrations for 20 to 30 days, with fresh test solutions introduced every 24 hours All pesticide experiments were conducted in replicates to ensure reliable results.
Histopathological examinations revealed significant alterations in vital organs, with gills exhibiting swelling and thickening of filaments Additionally, the liver displayed abnormal fatty degeneration, while the kidneys showed large vacuolated cells in the glomeruli (Satyanarayan et al., 2012).
Test Species – Oreochromis niloticus
Binomial name : Oreochromis niloticus (Linnaeus, 1758)
Oreochromis niloticus, commonly known as the Nile tilapia, is a highly favored fish species among aquaculturists due to its significant advantages Its ability to thrive in diverse environmental conditions, rapid growth rate, effective reproductive strategies, and adaptability to various feeding levels make it a vital aquaculture commodity These same characteristics contribute to its success as an invasive species in subtropical and temperate regions.
Coconut Shell
Coconut plantation industry is one of the most popular sectors in society in Indonesia even the world The processing of coconut itself has many processes which
13 each of process will certainly produce waste One of the wastes which usually produce from the coconut is coconut shell
Coconut shells, a byproduct of the coconut processing industry, pose environmental challenges due to their large quantities and difficulty in natural degradation Comprising 33.61% cellulose, 36.51% lignin, and 29.27% pentosans, dried coconut shells serve as a promising raw material for producing activated carbon Their high carbon content and hardness make them an excellent source for activated charcoal, which is effective in adsorbing waste Studies show that activated coconut shell charcoal can significantly reduce pesticide residues, such as chlorpyrifos in water by approximately 50%, and effectively manage residual levels of harmful substances like lindan and aldrin in crops.
Activated Carbon
Activated carbon, known for its high porosity and extensive surface area, is primarily derived from coconut shells due to their optimal natural structure and low ash content This form of carbon can be activated through chemical or physical processes, resulting in high-density and high-purity products that are virtually dust-free and resistant to attrition Its applications are diverse, including gas and water purification, gold and metal extraction, medicine, sewage treatment, and air filtration in gas masks and respirators.
There are some characteristics to determine whether the activated carbon is good or not according to SII 0258-88 as can be seen in Table 3
Table 3 Characterization of Activated Carbon (SII 0258-88)
3 The missing part on the heating of
4 Adsorption of I2 Mg/gram Min 750
6 Adsorption of Methylene Blue Ml/gram
7 Speed of Bulk Type g/ml 0.30-0.35
Source: Scientific Communication and Information Center, 1997
Single Drum Carbonization
Carbonization is a pyrolysis process that involves the incomplete combustion of raw materials to produce charcoal, with the carbonization temperature varying based on the base material The quality of charcoal is significantly influenced by the burning process, where uncontrolled combustion leads to the outer shell burning first and continuing to ignite the unburned material.
As a result, many shells become ash and others have not been burned so that the low yield of charcoal is 22.5% (Lindayanti, 2006)
The combustion process in a controlled air supply system is optimized by regulating airflow into the combustion tube By closing the air supply hole in the burned shell portion of the charcoal and opening the top row's opening, combustion occurs only where airflow is permitted This method continues until the air hole on the top row is reached, resulting in minimal ash and a small amount of uncharred shell Consequently, this leads to a higher yield of charcoal, achieving an impressive 31.58%.
Activation of Activated Carbon
Activated carbon can be activated through various methods, including chemical, physical, and physicochemical activation Each method offers distinct advantages and disadvantages, allowing for customization based on specific requirements.
Carbon activation involves breaking the carbon chain of organic compounds using various chemicals, including alkali metal hydroxides, carbonate salts, chlorides, sulfates, phosphates of alkaline earth metals, and notably ZnCl2, along with inorganic acids like H2SO4 and H3PO4 Effective chemical solutions for carbon activation include ZnCl2, H2SO4, KOH, and CaCl2 While mineral activators can be challenging to wash out, they offer the benefit of shorter activation times and greater yields of activated carbon The choice of activator influences pore formation, with factors such as activator concentration and temperature playing critical roles.
The activation process involves breaking carbon chains in organic compounds through heat, steam, and CO2, typically conducted in a furnace at temperatures between 800-900°C While low-temperature oxidation with air is exothermic and challenging to control, high-temperature activation using steam or CO2 is endothermic, making it more manageable Research indicates that using nitrogen gas as a medium for activation yields superior activated carbon compared to air, as nitrogen is inert and minimizes carbon combustion and oxidation during further heating (Gumelar, 2015).
Physics-Chemical Accumulation combines physical and chemical activation processes to produce high-quality activated carbon, outperforming traditional methods However, the reliance on complex laboratory equipment poses challenges, particularly for rural communities The process begins with carbonizing organic materials into charcoal, which is then subjected to physical activation through pyrolysis using CO2 or nitrogen vapor for a specific duration Following this, the charcoal undergoes chemical activation by soaking in a predetermined chemical solution (Hanum, 2009).
The quality of activated charcoal is significantly affected by the type of raw material used; denser, hard raw materials yield higher adsorption capabilities compared to lighter materials with lower specific gravity.
(2013) the quality of activated charcoal is influenced by:
Inherent water content refers to the quantity of water present in activated charcoal after the carbonization and activation of raw materials This water can be both chemically bound and influenced by external factors like climate, grain size, and the screening process Understanding this aspect is crucial for assessing the hygroscopic properties of activated carbon Additionally, the presence of volatile matter plays a significant role in determining the overall characteristics of activated charcoal.
Volatile Matter is a value that shows the percentage of the amount of flying substances contained in charcoal H2, CO2, CH4, and vapors that condense like CO2 and
Activated charcoal contains ash, which represents the mineral matter that remains after the carbonization process and is not removed during activation This mineral content is crucial for understanding the composition of activated charcoal.
The determination of tethered carbon content is essential for assessing the carbon levels in activated charcoal post-carbonization and activation Factors such as water content, ash content, and the presence of volatile substances significantly influence the amount of bound carbon in activated charcoal, impacting its adsorption properties.
The most important active charcoal property is absorption In this case, there are several factors that affect adsorption absorption, namely: i Absorption Properties
Activated charcoal's adsorption capacity varies by compound, with increased adsorption observed for similar molecular structures, such as those found in a homologous series Additionally, temperature plays a crucial role in influencing the effectiveness of activated charcoal in adsorbing different substances.
The temperature of the adsorption process is significantly influenced by the viscosity and thermal stability of the absorption compound For volatile adsorption compounds, the process is typically conducted at room temperature or, when feasible, at lower temperatures Additionally, the pH level, which indicates the acidity degree, also plays a crucial role in this process.
The adsorption of organic acids increases with a decrease in pH, particularly through the addition of mineral acids, which effectively enhance this process by lowering the pH levels.
20 ionization of organic acids Conversely, if the pH of the organic acid is increased by adding alkali, the adsorption will decrease as a result of the formation of salt.
MATERIALS AND METHODS
Place and Time
From May to September 2017, an experiment focusing on adsorbent adsorption and toxicity testing was conducted at the Aquaculture Laboratory of Sriwijaya University, Inderalaya Campus Additionally, histopathological examinations were performed at the Barokah Laboratory, located at km 3.5 in Palembang.
Equipments and Materials
Table 4 Equipments for Toxicity and Adsorbent Test
Table 5 Materials for Toxicity and Adsorbent Test
Methods
Black Nile Tilapia fish were sourced from Balai Benih Ikan Indralaya in Ogan Ilir, specifically selected for their size, ranging from 6 to 8 cm The chosen fish exhibited active swimming behavior, were free of illness, and showed no physical defects To ensure their readiness for experimentation, the fish underwent a 7-day acclimatization period in laboratory conditions (Edy, 2001).
In an experiment involving 40 fish, the subjects were randomly divided into four chambers, each holding 10 liters of water with 10 fish per chamber Three of these chambers were exposed to varying concentrations of a test chemical (0, 2 ml/L, 4 ml/L, and 6 ml/L), while one chamber served as a control group without exposure To ensure accurate results, the fish were not fed during the toxicity assessment, and the lethal concentration for 50% of the fish (LC50) was calculated using probit analysis in SPSS.
The carbonization procedure of coconut shells using single drum with controlled air supply (Hadi, 2011) is as follows:
1 Coconut shells were dried in the sun till dry and cleaned from dirt
2 A design bans with controlled air supply system were prepared
3 The holes on the controlled air were left opened, the coconut husk is used as an angler at the first combustion
4 Insert coconut shells up to ẳ drum parts, the holes of controlled air which is located in the centre and top of the drum were closed
5 When the fire was perfectly alive added the coconut shells slowly until the drum full
6 Drum closed and let the chimney opened
7 Observed the burned shells at the bottom through the controlled air, if it has become the embers then closed the holes after that the centre control hole was opened and so on
8 Chimneys and control holes were slapped with clay so no air enters the drum
9 Fire will be extinguished by itself ± 1.5 hours after closure due to the absence of air
10 Open the drum cover and the temperature in the drum decreased then take out the formed charcoal
The procedure for activating charcoal chemically (Gumelar, 2015) is as follows:
1 The charcoal smoothed with a crusher and sieved with a mesh size of 50
2 Immersed the charcoal powder in CaCl2 solution for 18 hours
3 Rinsed with distilled water to pH 7
4 Dried the charcoal powder in an oven with a temperature of 100 o C for 24 hours
5 The activated charcoal then will be analysed
The determination of ash content in activated carbon is essential for assessing the quantity of oxides present, as a higher oxide level indicates increased ash content According to the SII-0258-88 standard, the maximum allowable ash content is 10% The gravimetric method, as outlined by AOAC (2005), is employed to measure ash content through a systematic procedure.
• The porcelain cup is put in the oven for 30 minutes and cools in a desiccator for 15 minutes then weigh the cup
• Samples of approximately 2 grams are weighed in a cup of known weight
• The cup containing the sample was put into a furnace at 600 o C for 3 hours
• Samples and cups are transferred to the desiccator for 15 minutes, then weigh
• Calculation of ash weight can be calculated by equation (1)
Ma: Mass of the sample before
Mb: Mass of the sample after
Water content refers to the quantity of water present in activated carbon, which is crucial for understanding its hygroscopic properties This measurement helps assess the residual moisture in activated carbon following the activation process using an activator Understanding the standard water content is essential for evaluating the effectiveness and performance of activated carbon in various applications.
25 carbon according to SII No 02258-88 which is a maximum of 10% Water content Activated carbon is measured by an oven-based method (AOAC, 2005), the methods are:
• The porcelain cup is put in the oven for 30 minutes and cools in a desiccator for
15 minutes then weigh the cup
• Samples of approximately 2 grams are inserted into the cup that has been known to weigh and weigh, the sample is dried in an oven at 105 o C for 24 hours
• Samples and cup are transferred to the desiccator for 15 minutes, then weigh
• The water content of the sample is determined by the weight of the water that evaporates, the percentage of water content can be calculated using equation (2)
Ma: Mass of the sample before
Mb: Mass of the sample after
3.3.6 Adsorbent Experiment Using Fish as Bio indicator
This experiment investigates the effectiveness of coconut shell as a natural adsorbent for cypermethrin Probit analysis using SPSS determined that the lethal concentration (LC50) of cypermethrin is 3.8 ml/L The toxicity testing for batch experiments utilized varying concentrations, including C, C-20%, C+20%, C-10%, and C+10% ml/L.
The study involved 300 Nile Tilapia, divided into six chambers with five repetitions, each containing 10 liters of water mixed with cypermethrin and 40 grams of activated carbon from coconut shells, which had been allowed to precipitate for 24 hours A control group was maintained without exposure to the test solution, while the other groups were subjected to the treatment After the 24-hour precipitation period, the water containing cypermethrin and activated carbon was separated using a siphoning method (Jubaedah, 2017).
In the adsorbent experiment, 10 fish were randomly selected from each aquarium To prevent contamination, the deceased fish were meticulously removed, and their gill sections were preserved for histopathological analysis.
Dead fish were removed from the chambers and dissected with a cutter The gills were extracted and placed into a bottle containing a 10% neutral buffered formaldehyde solution, with a volume ten times that of the gills The collected samples were processed with the assistance of technicians from the Anatomical Pathology Laboratory at Barokah Laboratory in Palembang.
The gills were dissected and placed into a tissue cassette for fixation Following fixation, dehydration of the tissues was performed using a series of sequential alcohols, transitioning from 70% to 95% and finally to 100% The subsequent clearing process involved immersing the tissue in xylene for one hour, preparing it for infiltration.
27 paraffin The whole process was performed using automated processing machine for overnight
Tissues were embedded in paraffin wax with a melting point of 60-62°C and subsequently cut using a microtome The tissue sections were floated on a warm water bath to eliminate wrinkles before being transferred onto glass microscopic slides These slides were then placed in a warm oven for approximately 15 minutes to ensure proper adhesion of the tissue sections Finally, the slides proceeded to the staining process.
3 Staining Using Haematoxylin and Eosin
In staining process, there are no stain can be done if it still contains paraffin wax
To effectively remove paraffin wax from tissue slides and enable the penetration of water-soluble dyes, the slides must undergo a deparaffinization process This involves sequentially immersing the slides in xylenes, followed by a series of alcohol solutions (95%, 90%, 80%, 70%, 50%, and 30%) for 5 minutes each, and then rinsing in running tap water for 5 minutes The slides are stained with haematoxylin for 10 minutes, rinsed again for 10 minutes, and then stained with eosin for a duration ranging from 15 seconds to 5 minutes Subsequently, the slides are dehydrated through increasing alcohol concentrations (30%, 50%, 70%, 80%, 90%, and 95%) for 3 minutes each, followed by immersion in 95% and 100% alcohol for 1 to 2 minutes each, repeated 2 to 3 times The slides are then treated with acetone in two changes for 3 minutes each, followed by dipping in xylene (1:1 absolute alcohol) for 3 minutes in two changes Finally, after clearing with xylene, the slides are mounted in Dibutyl Phthalate Xylene (DPX) medium and covered with a cover slip.
The 28 slides were meticulously examined under a microscope and photographed at varying resolutions, both high and low The nuclei displayed a striking blue coloration, while the cytoplasm exhibited various shades of pink A thorough investigation of the histopathological alterations was conducted.
RESULTS AND DISCUSSIONS
Results
LC50 is a lethal concentration required to kill 50% of population A difference in
The determination of LC50 values is influenced by factors such as species, sex, age, size of test animals, and environmental conditions This study aimed to establish the concentration of pollutants for subsequent experiments, with the LC50 value estimated at 3.8 ml/L using probit analysis (SPSS) The toxicity tests on Nile Tilapia revealed that increased concentrations of cypermethrin led to higher mortality rates among the fish, as shown in the data (Table 6) Notably, these results were obtained within a short experimental duration.
Table 6 Effect of Cypermethrin Concentration on Nile Tilapia in Preliminary
Table 6 illustrates how varying concentrations of cypermethrin impact the mortality rates of Nile Tilapia Behavioral changes in the fish were observed within 1 to 2 hours following exposure to cypermethrin, with the timing influenced by the level of the toxicant.
30 display intense activity one hour after exposure The highest mortality was occurred in
In a study on the effects of cypermethrin on Nile Tilapia, it was observed that at a concentration of 6 ml/L, the fish exhibited erratic movements and struggled against water currents The control group (0 ml/L) showed no mortality, while increasing concentrations of cypermethrin correlated with higher mortality rates Specifically, at 2 ml/L, three fish were found dead, and at 4 ml/L, the death toll rose to four fish However, the increase in mortality was not as pronounced at higher concentrations.
At a concentration of 2 ml/L, the effects of cypermethrin were minimal, allowing fish cells to adapt and recover from damage However, at a higher concentration of 6 ml/L, mortality rates spiked, with nine fish found dead This increase in death is attributed to mitochondrial dysfunction, as the fish's cellular tissues could not withstand the high levels of cypermethrin The fish exhibited signs of paralysis due to insufficient oxygen supply to the brain, ultimately leading to death from hypoxia, as their bodies were unable to effectively bind oxygen in the blood.
In a recent experiment, it was observed that nearly all fish died within four and a half hours Notably, Nile Tilapia exhibited significant behavioral changes, particularly at the highest cypermethrin concentration of 6 ml/L, with these changes becoming apparent shortly after exposure.
The measurement of ash content in activated charcoal is essential for determining the mineral content that remains after the carbonization and activation processes This residual mineral matter does not combust during carbonization, highlighting its significance in the overall composition of activated charcoal.
Activated charcoal contains approximately 31% ash, which includes small amounts of potassium, sodium, magnesium, calcium, and other components, indicating its purity Ash is an inorganic residue resulting from the combustion of organic materials (Sangotayo et al., 2017) Analysis reveals that activated carbon has an ash content of 8.7% (Appendix 2) Coconut shells, known for their high lignocellulose content, exhibit significant ash levels, making them ideal for use with acidic activators that effectively react with such materials.
So that the water content becomes low and the activated carbon becomes drier which makes the ash content higher
The water content of activated carbon, which is determined after the carbonization and chemical activation of raw materials, reflects its hygroscopic properties—the ability to adsorb moisture from the environment High moisture levels can indicate lower purity, as excessive water content negatively impacts the adsorption capacity of activated carbon (Pongener et al., 2014) In our analysis, the water content of the activated carbon was found to be 3.62% (Appendix 2).
4.1.4 Adsorbent Experiment using Fish as Bio Indicator
The study demonstrated that after soaking the adsorbent and cypermethrin for 24 hours, a significant change occurred, evidenced by the presence of activated carbon residue at the bottom of the aquarium and the clarity of the water This indicates that the adsorbent effectively reduced the concentration of cypermethrin in the chamber, transforming the previously turbid water into a clear state.
Figure 2 The Condition of Aquarium after 24 hours of Adsorbent Additional Process
In this experiment involving 300 Nile Tilapia, the clear water conditions allowed the fish to survive for approximately 11 hours, an improvement over preliminary tests Despite this extended survival time, some mortality was observed, and the Nile Tilapia exhibited notable physical characteristics and behavioral changes during the study.
Table 7 Effect of Cypermethrin on Nile Tilapia in Five Repetitions using
Mortality of Nile Tilapia in Five Repetitions Total Total
Body fluids are very pale, the body is mushy and watery, bleeding in gills, pale organs, bile
Pale body fluids, pale organs, bile rupture, blackish intestines
Pale body fluids, pale organs, blackish intestines
Pale body fluids, pale organs, blackish intestines, bile rupture
Pale body fluids, pale organs, blackish intestines, bile rupture
Pale body fluids, pale organs, blackish intestines, bile rupture
Table 7 presents the results of cypermethrin treatments with five repetitions, including one control group without adsorbent Nile tilapia exhibited similar characteristics across treatments In the absence of adsorbent, a concentration of 3.8 ml/L cypermethrin resulted in nearly 92% mortality, with average deaths ranging from 9 to 10 fish per repetition, displaying severe symptoms like pale body fluids, mushy bodies, gill bleeding, pale organs, bile rupture, and blackened intestines This treatment also led to the fastest death rate among the groups Conversely, at a concentration of 3.1 ml/L with adsorbent, only 54% of the tilapia died, totaling 27 fish, with an average of 5 deaths per repetition, and exhibiting similar symptoms including pale body fluids and organs, bile rupture, and blackened intestines.
In 3.4 ml/L of cypermethrin concentration with adsorbent treatment, there were 60% of Nile tilapia found dead which contains of 30 fish in total with an equal average is 6 and
In a study on Nile tilapia, 64% of the fish exposed to a cypermethrin concentration of 3.8 ml/L with adsorbent treatment exhibited symptoms such as pale body fluids, pale organs, and blackish intestines, alongside instances of bile rupture Additionally, 72% of the fish in the sample were found dead, highlighting the severe impact of this pesticide on aquatic life.
In a study examining the effects of cypermethrin on Nile tilapia, a total of 36 fish died at a concentration of 4.2 ml/L, with 7 to 8 fish found dead in some repetitions The deceased fish exhibited notable characteristics, including pale body fluids, pale organs, blackish intestines, and bile rupture At a higher concentration of 4.6 ml/L, 78% of the fish succumbed, resulting in an average of 9 dead fish per repetition and a total of 39 fatalities, with similar signs of distress observed.
In an experiment comparing Nile tilapia with and without adsorbent treatment, the results demonstrated a significantly lower mortality rate in the group receiving the adsorbent treatment.
Table 8 Observation of Cypermethrin Effect on Nile Tilapia
Concentration Behaviour of Nile Tilapia (R1 until R5) Mortality Time
3.8ml/L Almost all fish dead four and a half hours
Passive, fish were swimming sideway, some fish dead 11 hours
Some fish were swimming at the bottom, some fish dead 9 hours
Passive, some fish were swimming to the surface, some fish dead 8 hours
Some fish knockdown, some fish passive, some fish dead 7.5 hours
Some fish knockdown, some fish passive, some fish dead, some fish were swimming sideway
In a preliminary test, all fish died within four and a half hours; however, during the experiment with adsorbents, some fish exhibited various behavioral changes Notably, the total death time for certain fish extended significantly, lasting approximately longer than the initial test.
Discussions
The widespread use of insecticides, particularly dangerous types like cypermethrin, poses significant risks to the environment and human health Improper application leads to harmful accumulation in soil, water, and agricultural products, surpassing maximum residue limits and degrading environmental quality This decline can adversely affect aquatic ecosystems, notably impacting fish, whose gills are crucial for breathing and osmoregulation To mitigate insecticide residues, scientists are exploring innovative solutions, such as utilizing activated carbon derived from coconut shells.
This study demonstrates that activated carbon derived from coconut shells exhibits excellent adsorption properties, meeting the quality standards outlined in SII No 02258-88, with a water content of 3.62% and an ash content of 8.7% The results indicate that fish subjected to treatments with the activated carbon adsorbent experienced significantly longer survival times compared to those without the adsorbent Specifically, fish exposed to cypermethrin concentrations without the adsorbent showed a mortality time of approximately four and a half hours, while those treated with the adsorbent survived for nearly 11 hours.
Exposure to cypermethrin concentrations of 3.8 ml/l without adsorbent treatment leads to irregular swimming, weakness, and fish resting at the bottom At 3.1 ml/l with adsorbent treatment, fish display irregular swimming and begin to adapt, also swimming near the bottom At 3.4 ml/l, fish exhibit restlessness, slow movements, and emit waste With 3.8 ml/l and adsorbent treatment, fish struggle to breathe, swim sideways, and show significant lethargy At 4.2 ml/l, fish are hyperactive yet weak, often found at the bottom Finally, at 4.6 ml/l, motor behavior changes drastically, with fish displaying hyperactivity, weakness, and signs of unconsciousness.
Changes in motoric behavior in tilapia vary with different concentrations of insecticide exposure Clinically, contaminated fish exhibit stress symptoms, including instability and a tendency to remain at the bottom of the tank According to Thiery et al (2009), signs of insecticidal toxicity in fish include hyperactive movements, erratic swimming patterns, floating with their stomachs exposed, fluttering, fainting, darker body coloration, decreased appetite, and eventual death This study's observed behavioral changes align with Aliza (2014), which noted that fish experiencing low oxygen levels exhibit accelerated movement and surface breathing Insufficient oxygen in the water significantly affects fish respiration and behavior.
Pesticides pose a significant threat to aquatic ecosystems, as their active ingredients can contaminate water sources and harm fish and other aquatic organisms High concentrations of pesticides can lead to immediate mortality in fish, while lower concentrations may cause harmful accumulation of residues in their bodies over time Fish are particularly vulnerable to pollution from insecticides, which can enter their systems through water absorption and food intake The gills, essential for gas exchange, can be adversely affected by pesticide exposure, as demonstrated by histopathological findings showing fusion of secondary lamellae in Nile Tilapia This fusion disrupts the respiratory function of the gills, likely due to injury that prompts excessive mucus production, leading to the adhesion of secondary lamellae.
Fusion occurs when lamellae undergo swelling or hyperplasia, disrupting the respiratory process This leads to a narrowing of the capillary lumen and causes the cells in the center of the secondary lamellae to shift, resulting in their attachment to the opposite side of the secondary lamellae.
Congestion in treatment figures 9, 7, and 4 is suspected to result from the entry of toxic substances, such as insecticides, into the gills This aligns with the findings of Lujić, Marinović, and Miljanović (2013), which indicate that congestion can cause blood to pool due to circulation disturbances, potentially leading to insufficient oxygen and nutrient supply Notably, congestion is often preceded by cell swelling, characterized by an increase in cell size due to water accumulation.
Epithelial proliferation of secondary lamellae in some treatments (fig 6, fig 7, fig
Excessive epithelial proliferation of secondary lamellae, suspected to be caused by uncontrolled cell division, leads to hyperplasia, particularly in cells that divide rapidly This phenomenon is observed in treatments exposed to cypermethrin insecticides Aliza's research (2014) supports these findings, indicating that such cell proliferation results in changes in fish gills, highlighting the impact of cypermethrin on cellular behavior.
The rupture of secondary lamellae in fish gills, characterized by the loss of epithelial cell pillars, compromises the stability of these structures This damage can lead to respiratory difficulties, as it reduces blood oxygen levels and hampers hemoglobin's ability to bind oxygen, resulting in hypoxia The accumulation of cypermethrin insecticides in fish gills exacerbates this issue by disrupting normal respiratory functions Observations of test animals display hovering behavior at the water's surface prior to death, indicating severe respiratory distress.