Assessment of Neonicotinoid Insecticide Imidacloprid LC50 And Their Toxicity Parameters Against Earthworm (Eisenia Fetida)


 Because of their high biomass in the soil, earthworms are used as bio-indicator species for assessing soil toxicity against pesticides. The regular observed sensitivity to relatively low pesticide concentrations exits in soils is a significant ecological observation. Insecticide residues harm the flora of beneficial invertebrates and harm the physiological functions of earthworms, resulting in death. They affect morphological parameters as well as internal organs, and eight different imidacloprid concentrations (0.050 µl/cm2, 0.100 µl/cm2, 0.150 µl/cm2, 0.200 µl/cm2, 0.250 µl/cm2, 0.300 µl/cm2, 0.350 µl/cm2, 0.400 µl/cm2) were prepared with water during the procedure. To establish the LC50 value, earthworms of Eisenia fetida were exposed to various concentrations of imidacloprid using the usual paper contact toxicity method, and their toxicity levels are established. The mortality percentage was estimated after 24 hours of imidacloprid exposure, and a dosage of 0.195 µl/cm2 resulted in 50% mortality of earthworms. When higher concentrations of imidacloprid were used, negative effects were observed. For ecotoxicological evaluations, the following morphological and behavioural changes were observed during the experiment: Preclittelar bulging, body constriction, blackening of the body, segment swelling, oozing of coelomic fluid, body constriction, cuticle rupture, and oozing of fluid from the body are all common side effects.


Introduction
Overuse of pesticides and fertilisers in cultivation has poisoned soil to dangerous levels in recent decades. This causes a difference in the aeration and fertility of the soil, resulting in an imbalance between the ora and fauna that live there ( Van et al. 2009).
Soil is made up of minerals, organic substances, and the ora and fauna that dwell there. As a result, the ecosystem's principal consumers and decomposers, the fauna, are largely reliant on soil quality maintenance (Handrix 2000). Earthworms are a great bioindicator for assessing the state of the soil ecosystem's health. According to previous studies, pesticide application endangers their lives by exposing them to pesticide-contaminated soil (Jadhav and David 2017).
Imidacloprid is systemic pesticides that acts as an insect neurotoxin and is a member of the neonicotinoid family of compounds that damage insects' nervous systems. To evaluate a pesticide's ecological risk, a projected non-effective concentration and predicted effect concentration values must be established in order to determine the toxicity exposure ratio.
With regard to target species, various toxicity assessment tests have been established, and effects have been characterised based on exposure time. Pesticide impacts on soil fauna can be assessed using a variety of methods. To examine the impact of pesticides on soil species, standard laboratory toxicity assays for Hypoaspis aculeifer, Folsomia candida Eisenia andrei, Enchytraeus albidus, and Eisenia fetida, were created in Europe (Jansch et al., 2006). Imidacloprid, acetamiprid, nitenpyram, clothianidin, and thiacloprid are nicotinic acetylcholine receptor agonists with varying degrees of toxicity in various species. According to Kai Wang et al., (2015) Worms avoided tropical arti cial soil treated with insecticides imidacloprid and thiametoxam, as well as those treated with the fungicides captan and carboxin plus thiram, in an experiment conducted by Paulo et al., (2013), and at the lowest concentrations, more worms were found in the contaminated compartments than in the control, indicating that worms preferred low-pesticide soils to high-pesticide soils. Worms, on the other hand, preferred tropical fake soil treated with pronil at all concentrations tested to tropical arti cial soil treated with higher concentrations of these three pesticides.

Material And Methods
Fully developed clitellated earthworms were exposed to neonicotinoid insecticides viz. imidacloprid to assess toxicity parameters at the Department of Zoology, College of Basic Science and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar from July to September 2020.

Selection of test earthworm
Chaudhary Charan Singh Haryana Agricultural University, Hisar, has a vermiculture section in the Department of Zoology and from this vermiculture unit collected fully formed clitellated healthy earthworms of Eisenia fetida with an average body weight of 700 mg. The earthworms were brought to the vermiculture laboratory, where they were cultured in plastic tubs with a 60:40 mix of cow dung and organic manure, with adequate moisture (60-65 percent) maintained by sprinkling water on the substrate as required, and they were covered with gunny bags to protect them from pests and maintain moisture levels.

Procurement of test neonicotinoid insecticides
The technical grade of imidacloprid (95.3%) was obtained from the pesticide market of Hisar, Haryana.
Treatments have been created. Table 1 shows the control and imidacloprid doses provided to the test earthworm Eisenia fetida.
Experimental set up for assessing the LC50 of imidacloprid The earthworms were rinsed in distilled water and placed on wet lter paper for two hours to clean their gut contents. Whatmann lter papers were held to conform correctly to the size of the vials to prevent side overlapping, and at bottomed glass vials 8 cm long, 3 cm diameter, medium grade 0.2 mm thick were used. Eight different imidacloprid concentrations (0.050 µl/cm 2 , 0.100 µl/cm 2 , 0.150 µl/cm 2 ) were tested. Each vial of lter paper was poured with one ml of insecticide and then rotated horizontally to ensure that the insecticide and control were evenly distributed throughout the lter paper (having 1 ml of deionized water only). They then inoculated one earthworm per vial with various concentrations of imidacloprid, as well as a control, and covered all vials with muslin. To reduce mortality, proper aeration, a temperature range between 20 and 25 degrees Celsius, and su cient moisture levels were maintained during the experiment. To maximise the e ciency of the experiment, all treated vials were laid horizontally and shielded from light exposure. Eight replicates of each concentration, as well as a control, were held.

Determination of LC50 of insecticides against adult Eisenia fetida
The corrected mortality was calculated after determining the percent mortality (Abbott, 1925), and the data collected during the experiment was subjected to Probit analysis (Finney, 1971) as suggested by the standard paper contact toxicity procedure.
Percent mortality = Corrected mortality = Post treatment assessment of earthworms exposed to imidacloprid insecticide At various time intervals after treatment, morphological and behavioural changes were reported. After 24 hours of exposure, the mortality rate was tested. Earthworms that did not respond to stimuli were deemed dead, and the observed mortality was utilised to calculate the percent mortality rate. The adjusted mortality was calculated after subtracting the control mortality. The LC50 value was calculated using the collected data.

Results
Experiments were carried out on the Eisenia fetida to measure the toxic effects of neonicotinoid insecticide imidacloprid from various pesticide groups. The LC50 values of imidacloprid insecticides on E. fetida were measured, and post-treatment behavioural and morphological changes caused by imidacloprid insecticide exposure were recorded at various intervals of time. It was discovered that as pesticide concentrations increased, the mortality of earthworms increased as well. Low doses of imidacloprid at a concentration of 0.050 µl/cm 2 resulted in the lowest mortality, and such concentrations are considered negligible. At a concentration of 0.400 µl/cm 2 of imidacloprid, 100 percent mortality was observed ( Table 2 & Fig. 1). Table 3 shows the LC50 values of imidacloprid, which are 0.195 µl/cm 2 . The LC50 value of a pesticide is described as the concentration at which it kills 50% of the earthworms under investigation.

Morphological observations
When earthworms were given a dose of imidacloprid at a concentration of 0.050 µl/cm 2 , their bodies curled and coiled, as opposed to the control, which moved slowly. It was discovered that increasing the concentration of imidacloprid caused further damage to the earthworm's body.
At higher concentrations of imidacloprid, morphological changes such as swift, aggressive, slow and sluggish movement, coiling around itself, curling of the body, hyperactivity, raising of the body, and at 0.400 µl/cm 2 the earthworms displayed restlessness, zigzag, and curling movement were observed. Table 4 and Fig. 2 show the morphological observations made in relation to imidacloprid.

Behavioural observations
According to behavioural research, imidacloprid at a concentration of 0.050 µl/cm 2 did not cause any changes in earthworms. In the case of control, similar observations were made.
When higher concentrations of imidacloprid were used, negative effects such as preclittelar bulging, body constriction, blackening of the body, segments swelling, oozing out of coelomic uid, body constrictions, cuticle rupture, and oozing out of uid from the body were observed at 0.400 µl/cm 2 had showed blackening of body, oozing out of coelomic uid and dark pigmentation. Table 4 and Fig. 2 show the behavioural changes in response to imidacloprid.

Discussion
The LC50 value is used to express the chemical analysis of mortality caused by imidacloprid exposure. In ecotoxicological assessments, morphological and anatomical changes in earthworms caused by imidacloprid insecticide exposure can be used as indicators for the presence of toxicants in top soil. Earthworm activity, development, reproduction, and organisational structure are all affected by sub-lethal pesticide doses, which are more closely associated to long-term harmful consequences in ecological systems (Capowiez et In the control group, no behavioural changes were observed. When earthworms were handled with imidacloprid at a concentration of 0.050 µl/cm 2 , similar results were observed. Behavioral changes such as preclittlar bulging with body constrictions and oozing out of coelomic uid with symptoms of clittelar bulging were reported at 0.100 µl/cm 2 and 0.150 µl/cm 2 , respectively.
Imidacloprid concentration of 0.200 µl/cm 2 had showed blackening of the body with segment swelling.
At 0.250 µl/cm 2 , 0.300 µl/cm 2 , 0.350 µl/cm 2 , and 0.400 µl/cm 2 imidacloprid concentrations, additional behavioural changes such as blackening of the body associated with oozing out of coelomic uid, body constrictions with swelling in segments, oozing out of uid due to cuticle rapture, and blackening of the body with concomitant oozing out of coelomic uid causing dark pigmentation were observed.
The rst exposure to insecticide caused morphological changes such as rigid body movements. The earthworms' bodies were instantly shocked and exhibited coiling, which was due to an adapted technique to limit insecticide exposure by curving the body surface area. The insecticide-coated lter paper was less vulnerable to movement like lifting the earthworm's body. The body's surface was reduced as a result of imidacloprid exposure, resulting in hyper coiling. Lack of energy and unnecessary stress caused by insecticide exposure result in extreme lethargy and decreased activity.
Earthworms used swift and zigzag movements to shield themselves from epidermal rapture. Worms behaved violently in order to reduce the insecticide effect. The earthworms eventually displayed slow restless movements, sluggish movements due to tiredness, and a low energy level in the body as a result of the insecticide effect.
When an insecticide comes into contact with the earthworm's skin, it enters the body's coelomic uid, causing toxic symptoms. Imidacloprid penetration tears the cell membranes, causing the segments to swell. Dark pigmentation occurs in worms when body wall tissue is traumatised or microscopic damage is caused by toxicants migrating from the touch surface area. In line with the current research, Reinbeck and Reinecke (2007) discovered that sub-lethal pollutant concentrations can affect earthworm behaviour, development, reproduction, and organisational structure, which are more akin to long-term harmful consequences in the natural environment.
Imidacloprid insecticide causes morphological and behavioural changes in stable E. fetida earthworms, making it a signi cant symbol for worm ecotoxicology. The alterations which were observed due to imidacloprid were also reported by the study of Suneel and Singh, (2017) in earthworm Eisenia fetida after 48 hour exposure to phorate showed curling, coiling and rupture of body wall, increased mucus secretion, coelomic uid ejection, sluggishness, and body surface lesions. Due to atrophy, the earthworm's body wall began to thin, eventually leading to necrosis and erosion of the entire body wall, although no such changes were observed in the control. There were signi cant epidermal, morphological changes observed after 48 hours of exposure to phorate concentrations of 20-40 µl/cm 2 .
Earthworms subjected to the greatest amounts of phorate developed constrictions, swelling in the clitellar area with many ruptures, and punctures in the body wall with many lesions, resulting in body fragmentation and cuticle shedding, comparable to ecdysis in insects and snakes.
The epidermis and cuticle serve as a major barrier between the earthworm's body and the environment, allowing ion transfer and allowing or blocking xenobiotics (Clauss, 2001 With growing concentrations of chlorpyrifos, morphological changes such as coiling, clitellar swelling, mucus release, and bleeding, followed by body segmentation, were observed in chlorpyrifos-treated earthworms. However, morphological changes were found to be more prevalent for cypermethrin than for chlorpyrifos, whereas the in uence of co-exposed pesticides on morphological changes was found to be intermediate (Rishikesh et al. 2019).

Conclusion
The earthworm Eisenia fetida is extremely susceptible to imidacloprid insecticide.
Imidacloprid insecticide causes morphological and behavioural changes in healthy earthworms, making it an important symbol for worm ecotoxicology.
The amount of imidacloprid insecticide that harms earthworms is proportional to its concentration.
The imidacloprid pesticide has a harmful potential for the organism being studied. The observed effects were tissue speci c and dose based. Changes in a pesticide-contaminated climate may jeopardise the life of an eco-friendly non-target organism, the earthworm. The measured LC50 for the following doses of imidacloprid on earthworm Eisenia fetida is 0.195 µl/cm 2 .
Declarations g) Naresh Kumar (NK): Weather parameter during experiment. 5. Competing interests: Authors declare that there are no competing interests.
6. Availability of data and materials: This paper does not qualify for data sharing since no datasets were created or analyzed during the research.

Figure 1
Eisenia fetida mortality at different doses of imidacloprid after 24 hr exposure