1. INTRODUCTION:

Worldwide aquatic water bodies are frequently polluted with a Discharge of 100,000 different chemicals¹and pernicious insecticides into the aquatic system are significant causes, which include non-target organisms Including fish². Agriculture dates back 10,000 years³ and one of the sources of contamination of water is because mainly it provides food as nutrition and includes raw material for shelter, fuels medicines etc., for their daily needs. Almost all we get food supply comes from the land, and less than 1% is from an aquatic ecosystem⁴.

As the human population grows the requirement for these resources also increases. Pollution is the presence in the form of a pollutant in the air, water and food which cause harm to living organisms in the Environment⁵. The more we use longer significant it remains in the soil and further, these chemicals can enter and contaminate water through direct application, and runoff and causes detrimental effects on aquatic animals like fishes⁶.

Toxicity evaluation is to determine the adverse effect on human and animal health and environmental threats can be properly assessed⁷. Afidopyropen is a novel insecticide, derived from the natural product pyripyropen A^8,9 that acts as a TRPV channel modulator in chordotonal organs of target insects. Fishes are biological indicators that indicate poor water quality through their abnormal behaviour¹⁰. Behaviour is evidence for changeable life activities of animals^11,12,13. A behavioural response was the first sign of an organism to environmental changes. Changes in the behaviour of fish, due to toxicant stress can be used as a biological indicator of pollution¹⁴.

AChE is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system AChE which hydrolyzes the neurotransmitter acetylcholine and pseudocholinesterase or butyrylcholinesterase (BChE). Many pesticides inhibit acetylcholinesterase activity¹⁵, and exhibit neurotoxicity as results in the accumulation of acetylcholine in nerve tissues and brain, and other organs, which shows alterations in the behaviour of fish. After pesticide exposure measurement of acetylcholinesterase activity (AChE), is often decreased. Decreases in the activity of acetylcholinesterase can affect the growth, survival, feeding, and behaviour of fish exposed to different contaminants^16,17.

Inhibition of AChE leads to an increase in the ACh accumulation in the brain causing overstimulation of cholinergic receptors and loss of cognitive functions¹⁸. As a result, it declines control over neural and muscular. Cholinesterase activity is used as a diagnostic tool for ecotoxicological risk evaluation studies and in environmental contamination monitoring programs of aquatic systems¹⁹. The present study aimed to determine the acute toxicity of Afidopyropen and its effects on ACh and AChE activity along with the behavioural study of fingerlings Cyprinus carpio.

2. MATERIALS AND METHODS:

2.1. Test chemicals:

Commercial insecticide afidopyropen trade name Sefina 400ml (50g/L DC). volume was procured from Garg Agro centre Malout, Panjab. The remaining chemicals used in the present study were of analytical grades and were procured from SRL Pvt Ltd Company India.

2.2. Test Animal:

Healthy Cyprinus carpio were obtained at the fish farm of the State Department, Department of Fisheries at BR Project (BRP), Shimoga, District: Shimoga, Karnataka, India. Mortality Cyprinus carpio with a mean length of 4.8±0.20cm and weight of 3.9±0.51g was acclimatized for a week in a huge cement tank (4×3×4 feet) During this period commercial pallet was given and health status was monitored, prior to the test. Ten healthy fishes were exposed to Afidopyropen per concentration (0.4-1.6mg/L). Throughout the experimental duration, the oxygen concentration and photoperiods 12 hrs dark 12hrs light were maintained in Control and treated groups in the test chamber as per 203 OECD guidelines²⁰. The water quality parameters were controlled as follows: Temperature - 25±2°C. pH-7.03±0.2. Dissolved oxygen-7.9±0.8mg/L. Total hardness - 30.2±3.2mg as CaCO3/L. Salinity; Nil. Specific gravity - 1.001. Calcium - 17.29± 0.92mg/L. Phosphate - 0.031±0.004mg/L. Magnesium-0.75±0.4mg/L.

2.3. Determination of 96 hrs LC₅₀:

Using a semi-static bioassay method for acute toxicity test Healthy and uniform sized fish, were distributed into the test chamber, and fish were starved 24 hrs prior to the start of the experiment. Ten fish were placed in each of the test chambers containing 10L water (n=10/test chamber). whereas control fishes were kept in dechlorinated water medium. All aquaria were equipped with air stones and maintained a constant temperature of 25±2°C thought throughout the experiment. The experiment lasted for 96 hrs. Mortality data were recorded every 24 hrs. To determine the LC₅₀ value for 96 hrs, Finney’s probit method²¹ was followed and Observations of behavioural response of Cyprinus carpio were observed at 6 hrs, and every 12 hrs. After the end of the exposure period, two fish from each experimental and control group were randomly sampled for ACh and AChE analysis.

2.4. ACh Determination:

Determination of ACh content in control and experimental samples was estimated by the method of hestrin as described by augustinsson²². The brain tissues were isolated, sliced, weighed and transferred into to tubes that already were kept in a boiling water bath for 5 minutes to inactivate the acetylcholinesterase (AChE) enzyme activity and to release bound ACh as described by Vasantha et al²³. The optical density of the sample was measured at 540 nm in a UV– visible double beam spectrophotometer model 2205 (Systronic India Ltd) using the reagent blank. ACh content was expressed as µmoles of ACh/gm wet weight of tissue.

2.5. Determination of AChE activity:

Acetylcholinesterase activity was conducted after 96 hrs exposure. Acetylcholinesterase activity was determined by following the method of Ellman et al²⁴. With minor modification. AChE reacts with the substrate acetylthiocholine iodide Due to thiocholine iodide reacting with 5,5’-dithiobis (2-nitrobenzoic acid DTNB) to form a yellow compound the maximum absorbance of this compound is 412nm. (U) was defined as one unit of activity as μmol of hydrolyzed substrate per minute. The specific activity was expressed as units of activity per mg of protein using UV– visible double beam spectrophotometer model 2205 (Systronic India Ltd). Protein contents in supernatants of fish brain tissue homogenates were determined according to the method of Lowry et al²⁵, using bovine serum albumin (BSA) as standard. The AChE specific activity was in µmoles of ATChI hydrolyzed/min./mg protein. AChE Percentage inhibition was calculated using the equation²⁶

AChE inhibition (%) = ^Abscontrol - ^Abssample×100.

^Abscontrol

2.6. Statistical Analysis:

To determine Ach and AChE enzyme activities in the brain tissue are presented as mean±SE of six fish per group. The statistical significance among the different concentrations of Afidopyropen, one-way analysis of variance (ANOVA) was used followed by Tukey’s test. was used to perform statistical analysis where the level of significance was set up at p<0.05 Statistical analysis was performed using SPSS version 25.

2.7. Ethical Committee:

All procedures carried out in this study were done in accordance with the Institutional Animal Ethics Committee's guidelines (IAEC). The animals were used and maintained according to the guidelines imposed forth by the committee for the purpose of control and supervision of experiments on animals (CPCSEA), located in New Delhi, India throughout the experiments.

3. RESULTS:

The results obtained from acute 96 hrs toxicity by the semi-static experiment of afidopyropen are summarized in Table 1. At 0.4 mg/L fish showed almost normal, no mortality in it. Minimum mortality of 10% and 20% occurred at 0.6, and 0.8 mg/L concentrations respectively. At 1.0, 1.2 and 1.4 mg/L caused 50%, 70% and 80% mortality respectively. The mean LC₅₀ value of fingerlings was found 1.0 mg/L by the use of Finney’s probit Analysis Method. (Fig.1), shows a sigmoid curve against log concentration, the plot of Finney’s adjusted probit and LC₅₀ results. The highest concentration i.e., (1.6 mg/L) caused 100% mortality (Table 1). The behavioural response of the fish in the control aquarium was normal, both after 24 and 96 hrs.

There is not much alteration occurred in the behaviour of the fish exposed to the lowest concentration (0.4 mg/L) which was exhibited throughout the experiment. At the highest concentrations such as faster opercular movement, mild hyperactivity, rapid jerking movements, and loss of equilibrium. they become hyperactive and move to the corner of the test chamber, behavioural effects such as loss of balance, jerk movement, swirling movement, mild increase in mucus secretion was observed on the body, surfacing behaviour, rapid opercular movement, loss of equilibrium were observed (Table 2).

ACh content gradually increases to 1.47%, and 11.44% at both the 0.4 and 0.6 mg/L concentrations respectively. whereas the levels of ACh markedly increase by 19.30%, and 40.50% at 0.8,1.0 mg/L respectively (Fig.2) as compared to the control. the ACh content showed a positive correlation (Figures 2 and 3). Effect of Afidopyropen on the activity of AChE in the brain of the Cyprinus carpio after 96 hrs exposure to different concentrations caused significant inhibition activity has been observed (Fig. 3). After exposure to the toxicant, AChE activity decreased to (-1.19%), (-4.50%) at both the 0.4 and 0.6 mg/L concentrations respectively, as compared to the control. However, in these two concentrations, we did not find much alter brain AChE activity. AChE activity decreased significantly by (-20.39%) (96 hrs) and a maximum decrease of (-52.90%) was found at 0.8 mg/L. At 1.0 mg/L respectively.

Table 1: Determination of LC₅₀ value of the effect of concentrations of afidopyropen to Cyprinus carpio mortality for 96 hrs

Sl. NO	Concentration of pesticide mg/L	Log concentration of pesticide	No of fish exposed	No of fish alive	No of fish dead	Percent kill	Probit kill
1	0.4	2.3479	10	10	0	-------	-------
2	0.6	2.4879	10	9	1	10	3.70
3	0.8	2.5911	10	8	2	20	4.16
4	1.0	2.6737	10	5	5	50	5.00
5	1.2	2.7429	10	3	7	70	5.25
6	1.4	2.8027	10	2	8	80	5.52
7	1.6	2.8556	10	0	10	100	5.84

Table 2: Effect on the behaviour of Cyprinus carpio exposed to different concentrations of the afidopyropen up to 96 hrs

Parameters	Control	Afidopyropen (mg/L)
Parameters	Control	0.4	0.6	0.8	1.0	1.2	1.4	1.6
Schooling behaviour	-	-	-	+	++	++	+++	++++
Erratic swimming movements	-	-	-	+	++	++	++	+++
Hyperactivity	-	-	+	+	++	++	++	+++
faster opercular movement	-	-	+	+	+	++	++	+++
rapid jerking movements	-	-	+	++	+++	++	++	++
swirling movement	-	-	-	+	++	++	++	+++
surfacing to gulp air	-	-	-	+	++	++	++	++++
burst swimming,	-	-	+	+	++	++	++	+++
shrinking to the bottom	-	-	-	+	++	++	+++	++++
loss of equilibrium	-	-	+	+	++	++	++	+++
secretion of more mucus	-	-	-	+	++	++	++	++++

The increase or decrease in the level of behavioural parameters is shown as. -, none (0%); + mild (less than 10%); ++, moderate (10 to 50%); +++, severe (> 50%).

Figure 1. Toxicity evaluation of afidopyropen to Cyprinus carpio. The graph shows a sigmoid curve between the percent mortality of the fish against the log concentration of afidopyropen.

Figure 2. Effect of afidopyropen on ACh release from the brain (µmol/gm wet weight) of Cyprinus carpio exposed to different concentrations for 96 hrs. Values are mean ± SEM, bars are not sharing a common superscript letter (a, b, c, d) differ significantly at p < 0.05. Results are expressed as a percentage of elevation (as compared with control).

Figure 3. Effect of afidopyropen on the brain Specific activity of AChE (µmol/min/ mg protein) of Cyprinus carpio exposed to different concentrations for 96 hrs. Values are mean ± SEM, bars are not sharing a common superscript letter (a, b, c, d,) differ significantly at p < 0.05. Results are expressed as a percentage of inhibition (as compared with control)

DISCUSSION:

Toxicity evaluation will continue to be an essential part of toxicology. Evaluation of LC₅₀ values provides a useful means of comparing the relative toxicity of toxicants to organisms under specified conditions. Before assessing physiological changes in animals, it is necessary to determine the LC₅₀ concentration of contaminants. According to Finney’s Probit Analysis Method, the 96-hrs LC₅₀ value of Afidopyropen in Cyprinus carpio was found 1.0mg/L. The results reveal that the LC₅₀ value of afidopyropen for fingerlings decreased with an increase in time. The control group showed normal behaviour throughout the experiment. However, in this study, behavioural responses at 0.4 mg/L showed no abnormal behaviour and likely harmless concentration of afidopyropen for the fingerlings of Cyprinus carpio which can be considered safe. In terms of environmental significance, the data suggest that the concentration of afidopyropen in the aquatic system beyond this limit may be hazardous for Cyprinus carpio fingerlings. The behavioural changes observed in the exposed fingerlings like rapid opercular movement, swirling movement, burst-swimming, coughing, and jerking movement can be directly related to the central nervous system due to the inhibition of cholinesterase activity²⁷.

The abnormalities in fish behaviour observed in this study were possibly due to the inhibitory action of afidopyropen on AChE and resulting in the accumulation of Ach at the neuromuscular junction. Okey et al²⁸reported that fish exposed to toxicants usually show some abnormal behavioural such as rapid opercular beating, erratic swimming, mucus secretion, and gulping for air before death. In this regard, our study is similar to that of Singh et al²⁹who exposed common carp to dimethoate and found that carp fingerlings exhibited behavioural abnormalities when exposed to dimethoate. Within a few minutes of being exposed to higher concentrations 1.60mg/L and onward. According to Sharma and Thakur³⁰, abnormal fish behaviour may be a defensive mechanism of fish to avoid toxic damage. It is evident that the alterations in the behavioural patterns were more pronounced with Afidopyropen at increased concentrations (Table 2).

ACh regulates a variety of visceral functions in both vertebrates and invertebrates. In fish, acetylcholine plays an essential role in autonomic transmission³¹. In the present study over a period of 96 hrs the different concentrations of afidopyropen exposure i.e., 0.4, 0.6, 0.8, 1.0mg/L responsible for the accumulation of ACh content in the brain. Mushigeri and David²⁷ were of the view that pesticides bind the active site and inhibit the breakdown of Ach resulting in the blocking of synaptic transmission in cholinergic nerves which arises in the nucleus ambiguous in the brain stem, excess accumulation of acetylcholine causing abnormal behavioural activities of the fish²⁷. Our investigation is supported by Previous researchers who reported that ACh accumulated in the brain and other tissues and damage to the central nervous system might have caused hormonal release that is not under control and will impact the degeneration of many biochemical and physiological functions of an animal³². The function of AChE is to remove the excess accumulation of acetylcholine (ACh), if it’s unable to do its function then there is an inhibition in AChE activity due to xenobiotics, In the present study, concentrations of the insecticide caused concentration-dependent inhibition in AChE activity of fingerlings. Fingerlings exposed to 0.4 and 0.6mg/L for 96 hrs showed the lowest inhibition as compared to the control. A Similar, experiment conducted by Glusczak et al³³ demonstrated that the AChE activity in the brain of Rhamdia quelen was exposed to 0.2 and 0.4mg/L concentrations of glyphosate (Roundup) for 96 hrs was lower than in the control group. Our results are consistent with Rodrigues et al³⁴ have reported that a significant decrease in AChE activity by 20% or more can be considered a clear toxicological effect of xenobiotic exposure. However, brain AChE activity differs according to fish length³⁵. It is clear that Afidopyropen insecticide exposed to 0.8 mg/L showed (-20.39%) possesses inhibitory effects on the AChE activity of Cyprinus carpio from the results shown. Golombieski and Morsy^36,37 reported that, when fishes are exposed to pesticides and some chemicals, AChE activity decreases, and acetylcholine is accumulated and is not broken within synapses which leads to cause behavioural impairments, which could eventually end in death from respiratory failure.

4. CONCLUSION:

This study brings new evidence of afidopyropen toxicity to the fish Cyprinus carpio, which strongly influences behaviour. Taking into account the effects of afidopyropen Acute toxicity evaluation was found 1.0 mg/L, which is very toxic to fish. During afidopyropen exposure could be due to lesser breakdown of ACh because AChE is known to be inhibited and cause behavioural impairments due to action on the enzyme. AChE activity inhibition showed that exposure to lethal concentration for a period of 96 hrs adversely affected the brain. The inhibition (%) in higher concentrations was more significant than in lower concentrations. It is clear that afidopyropen insecticide possesses inhibitory effects on the AChE activity of Cyprinus carpio and tells their role in neurotoxicity. These studies represent biomarkers in the aquatic environment and are needed for future research.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGEMENTS:

The authors express their gratitude for providing financial support from the Karnatak University and the Department of Zoology providing lab facilities for the completion of this work.

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Received on 23.10.2021 Modified on 15.01.2022

Research J. Pharm. and Tech 2023; 16(1):67-72.

DOI: 10.52711/0974-360X.2023.00012