Acute Toxicity of Methyl Parathion on Freshwater Catfish, Heteropneustes fossilis (Bloch) Under Laboratory Conditions

 

Chandra Shekhar Ramanuj Raman

Research Scholar, Dept of Zoology, JP University, Chapra, Bihar, India.

*Corresponding Author E-mail: ramanujramanc@gmail.com

 

Abstract:

Methyl parathion, a highly toxic organophosphorus insecticide, is widely employed in agriculture. Runoff from these fields frequently pollutes freshwater ecosystems, endangering non-target aquatic life. This study assessed the acute toxicity of methyl parathion on the freshwater catfish Heteropneustes fossilis (Bloch) in a controlled laboratory setting. Healthy fish were subjected to varying concentrations of methyl parathion in static bioassay systems over a 96-hour period. Mortality rates, behavioral changes, and toxic symptoms were documented at 24, 48, 72, and 96-hour intervals. The median lethal concentration (LC₅₀) was calculated using probit analysis. Fish exposed to the chemical displayed hyperactivity, erratic swimming, and excessive mucus production, loss of balance, breathing difficulties, and surfaced frequently before dying. Previous studies have reported the 96-hour LC₅₀ value for H. fossilis to be approximately 7.0 ppm, indicating the species' high sensitivity to organophosphate pesticides. These findings reveal that methyl parathion poses substantial ecological threats to freshwater fish populations and can negatively impact aquatic biodiversity. The study underscores the importance of stringent regulation of pesticide application near aquatic habitats.

 

Graphical Abstract for Methyl parathion activity

 

KEYWORDS: Acute toxicity, Methyl parathion, LC₅₀, Organophosphate pesticide, Heteropneustes fossilis, Freshwater fish.

 

 

 

1. INTRODUCTION:

Freshwater ecosystems face growing threats from human-made pollutants, with pesticides being a major category of these contaminants. The global increase in agricultural activities has led to a significant rise in pesticide use, resulting in their infiltration into rivers, ponds, lakes, reservoirs, and wetlands through processes like runoff, drift, and leaching.

 

Organophosphorus pesticides (OPs) are widely utilized due to their broad-spectrum efficacy against agricultural pests. However, their widespread application raises significant environmental issues. Numerous studies have shown that OP pesticides negatively impact aquatic life by inhibiting acetylcholinesterase activity, inducing oxidative stress, disrupting endocrine functions, and impairing physiological processes. Methyl parathion [O,O-dimethyl O-(4-nitrophenyl) phosphorothioate] ranks among the most hazardous organophosphate insecticides. Despite restrictions on its use in several countries, residues are still detected in agricultural watersheds. Fish are particularly vulnerable to methyl parathion due to their constant exposure to contaminated water and their capacity to accumulate pesticide residues1.

 

The freshwater catfish Heteropneustes fossilis (Bloch), also known as stinging catfish, is prevalent across India, Bangladesh, Nepal, Pakistan, and Southeast Asia. Acute toxicity studies are crucial for understanding how aquatic organisms respond to toxic substances and for setting environmental safety standards. Previous research has documented physiological, biochemical, and behavioral changes in H. fossilis after exposure to organophosphate compounds such as methyl parathion, malathion, chlorpyrifos, dimethoate, and monocrotophos1,2,3.

 

This study aimed to assess the acute toxicity of methyl parathion on Heteropneustes fossilis under controlled laboratory conditions, focusing on mortality rates and behavioral changes resulting from exposure.

 

2. MATERIALS AND METHODS:

2.1 Experimental Fish and Acclimatization:

In the Saran district of Bihar, India, healthy freshwater catfish, Heteropneustes fossilis (Bloch), were sourced from local fish farms. These fish were transported to the laboratory in containers with aeration and allowed to acclimate for a period of 15 days before the experiments began. During this acclimation phase, the fish were kept in 100-liter glass aquariums filled with dechlorinated tap water, under controlled laboratory conditions. The fish had an average length of 12–15 cm and weighed between 20–25 g. They were fed a commercially available pelleted diet with about 32% crude protein, administered once daily at 2% of their body weight. Feeding was halted 24 hours before the start of toxicity tests and continued to be suspended during the experimental phase to reduce metabolic fluctuations and prevent water contamination. Only fish that were healthy, active, and free from any signs of disease, injury, or stress were chosen for the bioassay.

 

Throughout both the acclimatization and experimental phases, water quality parameters were consistently monitored. The temperature was kept at 26 ± 2°C, the pH at 7.3 ± 0.2, dissolved oxygen levels at 6.8 ± 0.4 mg L⁻¹, and total hardness at 135 ± 10 mg L⁻¹ as CaCO₃. A 12-hour light and 12-hour dark photoperiod was maintained throughout the study.

 

2.2 Experimental Design:

Technical-grade methyl parathion with a purity of 99% was sourced from a certified agrochemical supplier. A stock solution was created and then diluted to achieve the required experimental concentrations. Acute toxicity assessments were carried out following4,5.

 

The experiment included one control group and six treatment groups, each with varying concentrations of methyl parathion. Each treatment was replicated three times. Ten fish were randomly placed into each aquarium, which contained 50 L of the test solution. The exposure levels for the control (0 ppm), T1 (2 ppm), T2 (4 ppm), T3 (6 ppm), T4 (8 ppm), T5 (10 ppm), and T6 (12 ppm) were maintained throughout the study. The exposure lasted for 96 hours under static non-renewal conditions, with no food provided during this period. Continuous aeration was maintained, except when observing mortality.

 

2.3 Acute Toxicity Bioassay:

The acute toxicity bioassay adhered to established protocols for assessing fish toxicity. To reduce selection bias, fish were randomly distributed into treatment groups at the experiment's onset.

 

The mortality percentage for each concentration was determined using the following formula: Mortality (%) = (Number of dead fish / Total number of fish exposed) × 100

 

The median lethal concentration (LC₅₀) values for methyl parathion were assessed at intervals of 24, 48, 72, and 96 hours using probit analysis, as outlined by Finney6. Mortality percentages from each concentration were converted into probit units and plotted against the pesticide concentration's logarithm. Regression equations were then formulated, allowing for the estimation of LC₅₀ values along with their 95% confidence intervals from the regression line. The concentration-response relationship was analyzed to assess the sensitivity of Heteropneustes fossilis to methyl parathion exposure (Figure 1).

 

Figure 1: Probit regression curve for 96h (LC50) estimation.

 

2.4 Statistical Analysis:

Experimental results were presented as the mean along with the standard error (SE). To assess differences between treatment groups, a one-way analysis of variance (ANOVA) was employed, followed by Tukey's multiple comparison test when suitable. Before conducting the analysis, the data were checked for normal distribution and equal variance. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were executed using SPSS (Version 26.0) R statistical software. Probit analysis was conducted with the EPA Probit Analysis Program or similar statistical tools.

 

3. RESULTS AND OBSERVATIONS:

3.1 Acute Toxicity and Mortality Response:

Heteropneustes fossilis exhibited mortality rates that varied with both the concentration of methyl parathion and the duration of exposure during the experiment. The control group experienced no deaths over the 96-hour exposure period, validating the experimental setup. Conversely, mortality rates rose steadily as both the pesticide concentration and exposure time increased.

 

At the lowest concentration of 2 ppm, mortality reached only 20% after 96 hours, while concentrations of 10 and 12 ppm resulted in complete mortality (100%). Intermediate concentrations led to corresponding increases in mortality, demonstrating a distinct dose-response relationship (Table 1).

 

Table 1: Percentage Mortality of Heteropneustes fossilis Exposed to Different Concentrations of Methyl Parathion

Concentration

(ppm)

Mortality (%)

after 24 h

48 h

72 h

96 h

Control

0

0

0

0

2

0

10

10

20

4

10

20

30

40

6

20

40

50

60

8

40

60

70

80

10

60

80

90

100

12

80

100

100

100

 

The mortality data demonstrated that methyl parathion exerts significant acute toxic effects on H. fossilis even at relatively low concentrations (Figure 2).

 

Figure 2: Mortality at different concentrations (96 h)

 

The mortality pattern clearly indicates enhanced toxicity with increasing concentration and exposure duration, suggesting cumulative physiological impairment resulting from methyl parathion exposure.

 

3.2 Determination of LC₅₀ Values:

The median lethal concentration (LC₅₀) values estimated through probit analysis declined progressively with increasing exposure duration (Figure 3).

 

Figure 3: LC50 values at different exposure durations.

 

The LC₅₀ value for 24 hours was determined to be 10.8 ppm, while it dropped to 7.0 ppm after 96 hours. This decline in LC₅₀ values over time indicates a heightened toxic effect due to extended exposure, highlighting the cumulative toxicity of methyl parathion in experimental fishes.

 

3.3 Morphological and Behavioral Changes:

During the exposure period, several external changes were noted. Fish subjected to methyl parathion exhibited excessive mucus secretion on their body surfaces and gills. The amount of mucus produced increased with both the concentration of the pesticide and the length of exposure. Fish exposed to higher concentrations displayed darkened body coloration, fin erosion, decreased fin movement, and sluggish behavior. Fish have near death lost their balance entirely and remained still at the bottom of the aquaria.

 

Behavioral observations indicated that methyl parathion caused significant toxic symptoms in the exposed fish. These symptoms appeared quickly after exposure and became more severe with higher concentrations and longer exposure times (Figure 4).

 

Figure 4: Behavioral responses of H. fossilis.

 

Fish subjected to lower pesticide concentrations initially showed heightened swimming activity, frequent surfacing, and slight agitation. At moderate levels, they displayed erratic swimming, rapid gill movements, and a loss of schooling behavior. When exposed to higher concentrations (8-12 ppm), severe behavioral disruptions were noted, including loss of balance, excessive mucus production, breathing difficulties, lethargy, and settling at the bottom. Behavioral abnormalities emerged before death, serving as sensitive indicators of toxic stress. The severity of these impairments was directly linked to both pesticide concentration and mortality rate.

 

3.4 Gill Histopathology:

Histological analysis of gill tissues showed concentration-dependent structural changes. In control fish, gill architecture consisted of well-organized primary and secondary lamellae with intact epithelial lining. Fish exposed to lower concentrations showed mild epithelial lifting and slight lamellar fusion. Moderate concentrations led to hyperplasia, congestion, and fusion of secondary lamellae. Severe exposure caused extensive epithelial degeneration, necrosis, and disruption of lamellar organization (Figure 5). These lesions indicate impaired respiratory function and reduced gas exchange capacity.

 

Figure 4: Photomicrographs of gill sections (HandE stained) showing dose-reponses.

 

4. DISCUSSION:

The current study unequivocally showed that methyl parathion has pronounced acute toxic effects on the freshwater catfish Heteropneustes fossilis, leading to mortality that varies with concentration, behavioral changes, respiratory issues, and histopathological damage. The decreasing LC₅₀ values with longer exposure times suggest cumulative toxicity and increased vulnerability of fish to prolonged pesticide exposure. Similar acute toxicity patterns have been observed in H. fossilis when exposed to dimethoate, chlorpyrifos, monocrotophos, and other organophosphate pesticides1,3,7.

 

Globally, organophosphate pesticides are acknowledged as significant environmental pollutants due to their widespread agricultural application and frequent presence in freshwater ecosystems8,9,10. Methyl parathion is among the most toxic organophosphorus compounds impacting aquatic life because of its potent acetylcholinesterase-inhibiting effects (Edwards and Tchounwou, 2005). The mortality observed in this study aligns with the known neurotoxic action of organophosphate pesticides. The past research11 explain that acetylcholinesterase inhibition leads to excessive acetylcholine accumulation at synapses, causing continuous neuronal activation, neuromuscular issues, respiratory problems, and eventual death.

 

The concentration-dependent mortality noted in this study aligns with previous research on Heteropneustes fossilis. The previos researches as3 found significant mortality in catfish exposed to dimethoate, while12 observed similar toxic effects with cypermethrin exposure. Similarly, past research13 reported increased mortality and physiological stress in H. fossilis subjected to combined carbaryl and methyl parathion treatments. These findings collectively suggest that freshwater catfish are highly sensitive to pesticide pollution and can serve as effective indicators of aquatic contamination.

 

Behavioral changes were among the earliest responses to methyl parathion exposure. Hyperactivity, erratic swimming, increased surfacing, faster opercular movements, and loss of balance was noted within hours of exposure. Previous researches1,14 reported several behavioral changes in similar fish species with chlorpyriphos exposure. These behavioral changes are generally attributed to disrupted neurotransmission due to acetylcholinesterase inhibition and are widely recognized as sensitive indicators of pesticide-induced stress.

 

The observed increase in opercular activity and surfacing behavior in exposed fish indicates severe respiratory impairment. Similar respiratory issues have been documented in various fish species exposed to organophosphate pesticides and are typically linked to impaired oxygen uptake and branchial dysfunction7,15. Although H. fossilis has accessory respiratory organs that aid in aerial respiration, the frequent surfacing behavior noted in this study suggests that methyl parathion exposure significantly affects respiratory efficiency. These physiological responses may represent compensatory mechanisms to maintain oxygen balance under toxic stress conditions.

 

Fish exposed to pesticides often exhibit excessive mucus secretion on their body surfaces and gills, which is a notable response. This mucus production acts as a crucial defense mechanism, minimizing direct contact between toxicants and epithelial tissues. However, an overabundance of mucus can hinder gas exchange and exacerbate respiratory stress. Similar observations were made by recent researchers7,15, who noted increased mucus secretion and respiratory issues in catfish exposed to organophosphates. The excessive mucus production noted in this study seems to be a general defensive reaction to tissue irritation caused by pesticides.

 

Histopathological analysis of gill tissues showed that structural damage worsened with higher concentrations of methyl parathion. Exposed fish exhibited epithelial lifting, lamellar fusion, hyperplasia, congestion, and necrosis. Similar gill lesions have been documented in H. fossilis after exposure to monocrotophos and other organophosphate pesticides7,15. These pathological changes are typically seen as adaptive responses aimed at reducing toxicant absorption. However, these alterations also reduce the respiratory surface area and impair oxygen diffusion, which can lead to physiological dysfunction and death.

 

The toxic effects observed may also be associated with metabolic disturbances caused by methyl parathion exposure. Past research2 found significant changes in carbohydrate metabolism in H. fossilis after acute exposure to methyl parathion, indicating increased energy expenditure and metabolic stress. Enhanced glycogen mobilization and disruption of energy metabolism are common reactions in fish exposed to toxic chemicals and may contribute to the behavioral and physiological abnormalities observed in this study.

 

Recent reviews have highlighted the extensive ecological impact of organophosphate contamination in aquatic ecosystems. Recent research8 pointed out the persistence and widespread presence of organophosphate residues in surface waters, while16,17 documented the environmental distribution, bioaccumulation potential, and harmful biological effects of organophosphorus compounds in aquatic organisms. According to Mukherjee et al.18, organophosphate pesticides pose a significant threat to fish biodiversity by negatively affecting behavior, growth, reproduction, immune competence, and survival. The findings of this study support these conclusions and provide further evidence of the vulnerability of freshwater fish to pesticide pollution.

 

5. CONCLUSION:

The current research illustrates that methyl parathion has a pronounced acute toxic impact on the freshwater catfish Heteropneustes fossilis (Bloch), leading to mortality that varies with concentration and exposure time, significant behavioral changes, respiratory issues, and severe histopathological damage to gill tissues.

 

Histological analysis also showed considerable structural damage to gill tissues, indicating reduced respiratory efficiency and disruption of vital physiological functions. These results highlight the ecological dangers of indiscriminate organophosphate pesticide use in agricultural areas. Additionally, the study points to the effectiveness of Heteropneustes fossilis as a sensitive bio-indicator for detecting pesticide pollution in tropical freshwater settings. 

This study provides crucial evidence that methyl parathion poses a significant threat to freshwater fish and underscores the importance of incorporating ecotoxicological data into environmental management plans to protect aquatic biodiversity and ensure the long-term sustainability of freshwater ecosystems.

 

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Received on 15.06.2026     Revised on 30.06.2026

Accepted on 13.07.2026      Published on 14.07.2026

Available online from July 25, 2026

Research J. Science and Tech. 2026; 18(3):254-260.

DOI: 10.52711/2349-2988.2026.00035

 

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