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 al., 2005; Reinecke and Reinecke, 2007).
Because of their easy capacity and higher reproduction rates, the Organisation for Economic Cooperation and Development (OECD, 2004) recommended E. fetida are the best earthworm species for ecotoxicological research. The artificial soil test is preferred to the filter paper contact test in the natural habitat of earthworms because pesticides are absorbed mostly via the gut of earthworms in soil (Zhang et al., 2014). Earthworm mortality increased as pesticide concentrations increased, with an LC50 value of 0.195 µl/cm2 for imidacloprid, which was close to the findings of Zang et al., (2000), who found that the neonicotinoid insecticide imidacloprid (LC50 of 0.1 µl/cm2 at 24 hr and 0.034 µl/cm2 at 48 hr) was highly sensitive to earthworms.
Morphological and behavioral changes
Imidacloprid treatment of earthworms resulted in morphological and behavioural alterations. Slow movements were observed in the control group, although increased imidacloprid concentrations resulted in a variety of morphological changes in the treatment groups. At 0.050 µl/cm2 and 0.100 µl/cm2, curling and coiling of the body, as well as rapid movement, coil around itself, were observed. A concentration of 0.150 µl/cm2 resulted in hyperactive, curling of the body, while a concentration of 0.200 µl/cm2resulted in worms moving violently and coiling around themselves. Fast movement by lifting the body, slow sluggish movements, restlessness, zigzag curling movement and highly lethargic reduced movements were recorded at 0.0250 µl/cm2, 0.300 µl/cm2, 0.350 µl/cm2 and 0.400 µl/cm2 concentrations of imidacloprid respectively.
Table 4
Effects of various doses of imidacloprid on morphological and behavioural changes in Eisenia fetida
S. No.
|
Treatments IMD
|
Morphological changes
|
Behavioural change
|
1
|
Control
|
Slow movement
|
No changes observed
|
2
|
0.050 µl/cm2
|
Curling and coiling of body
|
No changes observed
|
3
|
0.100 µl/cm2
|
Fast movement, coil around itself
|
Preclittelar bulging, body constriction
|
4
|
0.150 µl/cm2
|
Hyperactive, curling of the body
|
Oozing out of coelomic fluid, clitellar bulging
|
5
|
0.200 µl/cm2
|
Violent movement, coil around itself
|
Blackening of body, segments swelling
|
6
|
0.250 µl/cm2
|
Fast movement, lifting the body
|
Blackening of body, constrictions, oozing out of coelomic fluid
|
7
|
0.300 µl/cm2
|
Slow, sluggish movement
|
Body constrictions, swelling in segments
|
8
|
0.350 µl/cm2
|
Restlessness, zigzag, curling movement
|
Cuticle rupture, oozing out of fluid
|
9
|
0.400 µl/cm2
|
Highly lethargic, reduced movements
|
Blackening of body, oozing out of coelomic fluid and dark pigmentation,
|
IMD = Imidacloprid |
In the control group, no behavioural changes were observed. When earthworms were handled with imidacloprid at a concentration of 0.050 µl/cm2, similar results were observed. Behavioral changes such as preclittlar bulging with body constrictions and oozing out of coelomic fluid with symptoms of clittelar bulging were reported at 0.100 µl/cm2 and 0.150 µl/cm2, respectively.
Imidacloprid concentration of 0.200 µl/cm2 had showed blackening of the body with segment swelling. At 0.250 µl/cm2, 0.300 µl/cm2, 0.350 µl/cm2, and 0.400 µl/cm2 imidacloprid concentrations, additional behavioural changes such as blackening of the body associated with oozing out of coelomic fluid, body constrictions with swelling in segments, oozing out of fluid due to cuticle rapture, and blackening of the body with concomitant oozing out of coelomic fluid causing dark pigmentation were observed.
The first 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 filter 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 fluid, 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 significant 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 fluid 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 significant epidermal, morphological changes observed after 48 hours of exposure to phorate concentrations of 20–40 µl/cm2.
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). Eisenia andrei avoidance behaviour due to methomyl (Carbamates chemical) toxicity was described by Pereira et al (2010).Insecticide exposure at higher levels resulted in a thick yellow discharge from the body. Worms most likely used extensive coiling to minimise their body surface area and thereby reduce insecticide exposure. Santos et al., (2012) and Ferreira et al., (2015) found that the earthworms curled up and became less responsive to touch. After exposure to imidacloprid and methyl tetra-butyl ether, similar findings were recorded in other earthworm species, E. andrei and Perionyx excavates (Youn, 2005; Yvan et al., 2005).
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 influence of co-exposed pesticides on morphological changes was found to be intermediate (Rishikesh et al. 2019).