Introduction

In the recent past, an increasing use of pesticides in agricultural fields has resulted in a significant decrease in the quality of plants and edible commodities leading to the destruction of soil fertility. Soil development and environmental performance have been adversely impacted by insecticide use on a regular basis. The repetitive and excessive application of insecticides and fertilizer pollutes soil and groundwater, affecting both target and non-target inhabitants like earthworms (Tiwari et al., 2019).

Earthworms, the ecosystem engineers, are good model system for studying the influence of the organic matter, structure, and microbial community population among the soil invertebrates. Using earthworms as bioindicators of land use provide useful insights concerning soil fertility and ecosystem health (Cao et al., 2017). The epigeic category of earthworms, such as Eudrilus eugeniae (E. eugeniae), Eisenia fetida (E. fetida), and Perionyx excavatus (P. excavatus), is the most well suited for vermicomposting technology worldwide because of their ravenous feeding nature and high fertility. Among the epigeic category, E. eugeniae is the most reliable and suitable species for use in global environmental sanitation (Karmegam et al., 2021). Among them, E. eugeniae is effectively employed in breaking down complex organic waste products and vermifiltration technology. Several sorts of industrial effluents and sludge were also been treated using E. eugeniae and Lumbricus rubellus (Yuvaraj et al., 2021b). E. eugeniae earthworm has also been employed in detoxification of coir pith through vermicomposting along with Gliricidia sepium (Jayakumar et al., 2022).

Biomarkers can be used effectively to assess the health condition of an organism’s biological systems. The impact and influence of insecticide on soil organisms can be investigated through the effective incorporation of various biomarkers in earthworms (Mekahlia et al., 2016a). Especially, earthworm biomarkers including cholinesterase (ChE), acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST), and lipid peroxidase (LPO) can also be employed to detect and evaluate pollutants that affect the ecosystem (Tiwari et al., 2016).

AChE is a key neurotransmitter enzyme among earthworm biomarkers that is prominently featured in the transmission of nerve impulses in the nervous system inhibited by metal contaminants. Similarly, the other biomarkers CAT, GST, and LPO are phenomenal antioxidants that protect living organisms from the harmful effects of highly reactive oxygen species (ROS) (Saint-Denis et al., 2001). Lipid peroxidation triggered by ROS may negatively impact on proteins, lipids, and DNA. It also increases membrane rigidity and permeability while decreasing cell fluidity in bio membranes (Suresh et al., 2010). Glutathione S hydrolase (GSH) is a very important intracellular antioxidant in the xenobiotic metabolism including detoxification (Anand & Suresh, 2014).

Carboxylesterase (CBE) is a multi-gene enzyme that catalyzes the hydrolysis of esters, amides, thioesters, and carbamates. It is implicated in the detoxification of contaminants such as pesticides (Sanchez-Hernandez et al., 2015). In addition to that, Pb significantly suppresses the biosynthesis of haem protein Cytochrome P450 (Cyt P450) in earthworms by affecting the activity of d-aminolevulinic acid synthetase, d-aminolevulinic acid dehydratase, and haem oxygenase (Saint-Denis et al., 2001). Biomarker enzyme analysis of chlorpyrifos, cypermethrin, and their combination on earthworms was previously reported to demonstrate the impact of these pesticides on earthworms and its morphological transformations (Tiwari et al., 2019). Furthermore, the abnormalities in protein profiling, cellular enzyme variations, and testicular histomorphology are analyzed to better understand the effect of Dimethoate on the earthworm Eisenia kinneari (Leena et al., 2012). In-depth histological analysis of the earthworm E. eugeniae has put the spotlight on the crucial role of the clitellum in the regeneration process (Paul et al., 2022; Selvan Christyraj et al., 2019). Thus, it is essential to investigate the effects of pollutants on E. eugeniae earthworms at different segments such as pre-clitellum, clitellum, and post-clitellum.

Quinalphos, one of the 234 pesticides registered in India, is one of the 76 WHO Class II pesticides. Directorate of Plant Protection, Quarantine, and Storage, Government of India, records show that the consumption of QOI in India between 2005–2006 and 2009–2010 was approximately 6329 metric tonnes (Srivastava et al., 2016). Currently, Quinalphos, Ethion, and Monocrotophos were extremely hazardous pesticide that is banned to be used on tea by European Union. In contrast, quinalphos is widely employed in India’s tea fields to control a wide variety of pests (Bhattacharyya & Kanrar, 2013). In rats, quinalphos has been shown to have toxic effects on the testis and male accessory glands also increased uterine wet weight, and an estrogen-like effect on vaginal cornification were observed in rats (Kitamura et al., 2005). The toxicity effects of the quinalphos (25%) [diethoxy-quinoxalin-2-yloxy-sulfanylidene-λ5-phosphan; PubChem CID: 26124] organophosphate insecticide has not been reported earlier in earthworms. Previous research (Kitamura et al., 2005) has shown that pesticides can have devastating impact on animal models. Hence, the present research is focused to identify the toxicity impact and its biomarker responses of the QOI on non-target beneficial organism E. eugeniae earthworms.

Materials and methods

Culturing of earthworms

E. eugeniae earthworms were acquired from the vermicomposting unit were brought into the laboratory and acclimatized for 6 weeks using the mixture of cow dung manure, leaf residues, and soil (8:1:1 w/w) (Liu et al., 2021) with the suitable temperature of 25–30 °C and 60% moisture level in a soil pot. Cow dung manure and organic wastes were restored every 7 days as the earthworms feed on the mixture and harvest vermicompost.

Contact filter paper test

A 48-h contact filter paper test was carried out for testing the acute toxicity of chemicals exposed with earthworms, as recommended (OECD—Organization for Economic Co-operation and Development, 1984). After that, a plastic Petri plate was layered with 90 mm-sized Whatmann No. 1 filter paper. Fine holes were made in plastic Petri plates for ventilation. Insecticide was dissolved in deionized water for the preparation of different range of concentrations 0.7812 µg cm−2, 1.5672 µg cm−2, 2.3437 µg cm−2, 3.1250 µg cm−2, and 3.9060 µg cm−2 were prepared and 1 mL from each concentration was loaded into filter paper. For the control plates, 1 mL of deionized water was loaded and rotated for uniform distribution. All the treated and control plates were allowed to air dry and then moistened with 1 mL of deionized water. Adult earthworms with well-developed clitellum were fasted 4 h on moist filter paper for cleaning the gut. After that, the earthworms were divided into 6 groups in plates and exposed with various concentrations of QOI for 48 h in the dark condition. In that, freshly prepared QOI was restored and filter paper was moistened with 1 mL of deionized water for every 12-h period. Concurrently, the same procedure was followed for control earthworm plates. After 48 h of the treatment, the dead and alive earthworms were identified with the respective concentrations for the analysis of LC50 value (insecticides) and 95% Fiducial limits were calculated using probit analysis (R.H.D., 1952). The morphological and behavioral changes of earthworms were observed and recorded.

Artificial soil toxicity test

The earthworms were exposed to QOI in artificial soil as described (OECD—Organization for Economic Co-operation and Development, 1984). The artificial soil was prepared using 70% industrial sand, 20% kaolin clay and 10% sphagnum peat moss and the pH were adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. Different test concentrations of QOI were prepared with deionized water and applied in artificial soil. The 35% moisture was maintained throughout the experiment. For the control group, 1 mL of deionized water was loaded into the artificial soil instead of QOI. Adult earthworms were placed on each concentration group which contains 1 kg of artificial soil maintained at 20 ± 1 °C under continuous light exposure (400 to 800 lx). Preliminary testing concentrations was determined with higher concentration range of 0.1, 1.0, 10, 100, and 1000 mg kg−1 of QOI on artificial soil for assessing mortality rate for 7th and 14th day period. Following that, lower concentration ranges of QOI (0.4, 0.8, 1.2, 1.6, and 2.0 mg kg−1) soil and a control were used to determine LC50. The overall analysis for 14 days of insecticides LC50 value and 95% Fiducial limits were calculated using probit (R.H.D., 1952).

Biochemical analysis of QOI-treated earthworms

Sample preparation

Control, 1/20th (5%) and 1/10th (10%) of LC50 doses of QOI were exposed to earthworms for 48 h. Earthworms were sacrificed and tissues from pre-clitellar, clitellar, and post-clitellar regions were excised and homogenized with 0.05 M phosphate-buffered saline (PBS; pH: 7.0) in ice-cold condition using a chilled mortar and pestle and centrifuged at 10,000 rpm at 4 °C for 15 min. The supernatant was maintained at − 20 °C for total protein estimation and biomarker enzyme assays (Samal et al., 2017).

Total protein and enzyme assays

AChE, SOD, CAT, GST, LPO, and glutathione-S-hydrolase (GSH) enzyme activities and estimation of total protein concentration levels were performed using methods followed by Tiwari et al (2019). Enzyme assays such as CBE (Thompson, 1999) and Cyt P450 (Omura & Sato, 1964) were performed based on standard protocols.

Protease activity

One gram of control and treated earthworm samples were homogenized and mixed with 1 mL PBS (pH 7.0) and incubated at 22 ± 2 °C for 24 h. One percent (w/v) skimmed milk agar was prepared and poured in Petri plates. After solidification, holes of 3 mm diameter were punched. 75 µL of samples were loaded in wells and incubated for 37 °C overnight (Jeyaprakasam et al., 2021).

Neutral red retention time assay (NRRT)

NRRT assay was performed according to the method described (Weeks & Svendsen, 1996). The coelomic fluid of control, and treated earthworms were collected gently by drawing action using a syringe containing 20 µL of Ringer’s solution. 20 µL of collected fluid was placed on a glass slide and 20 µL of neutral red stain was added to the slide. Coverslip was placed onto a glass slide and observed under a light microscope (Meiji Techno, Japan). The slide was viewed and cells were counted every 10 min, and the time taken for 50% of the lysosomes to discharge neutral red into the cytoplasm was determined.

Histopathology study

The 5% and 10% LC50 groups were exposed with QOI on earthworms along with the control. The pre-clitellum, clitellum, and post-clitellum region of these earthworms were cut and rinsed with distilled water and fixated with 10% formaldehyde solution. Then, the fixed tissues were dehydrated with a series of isopropyl alcohol which was transferred into xylene for cleaning and fixed with paraffin wax. Transverse sections of 3-micron size were cut and stained with hematoxylin and eosin. After that, the slides were observed under light microscope for examining the histopathological modifications (Rico et al., 2016).

Statistical analyses

The median lethal concentration (LC50) was calculated by using the statistical method “probit analysis” for assessing the toxicity of insecticides by Finney’s probit analysis. The consistent presentation of toxicity data was provided by calculating the 95% confidence limits. A one-way analysis of variance (ANOVA) was performed on a Microsoft Excel spreadsheet to analyze all of the data. Using one-way analysis of variance and covariance, the differences between the treatments were assessed for their statistical significances (ANOVA). At a p < 0.05 significance level, the findings were considered significant. All the experiments were repeated three times and evaluated.

Result

Contact filter paper test

In the contact filter paper test, the control earthworms were healthy, normal, and not showing any mortality during the experimentation time of 48 h. In QOI-treated plates, the percentage of mortality rates was found as 16.6%, 33.3%, 33.3%, 50%, and 100% in respective to 0.78, 1.56, 2.34, 3.12, and 3.90 µg cm−2 concentrations. The LC50 value of QOI for E. eugeniae by 48 h of contact filter paper test was found to be 3.56 µg cm−2 which was statistically evaluated as extremely toxic with 95% of fiducial confidence limit interval 2.00–6.34 (Table 1).

Table 1 Lethal concentrations (LC1-99) of QOI for earthworm, E. eugeniae by contact filter paper test
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Artificial soil toxicity test

In the artificial soil toxicity test, the control earthworms were healthy and normal and without any mortality during the experimentation period of 14 days. After 7 days, in QOI-treated plates, the percentage of mortality was found to be 0%, 0%, 16.6%, 16.6%, and 33.3% corresponding to 0.4, 0.8, 1.2, 1.6, and 2.0 mg. kg−1 concentrations. After 14 days, in QOI-treated plates, the mortality percentage was found to be 0%, 33.3%, 50%, 83.3%, and 100% corresponding to 0.4, 0.8, 1.2, 1.6, and 2.0 mg kg−1 concentrations. The LC50 value of QOI for E. eugeniae by 14-day exposure period was 1.05 mg kg−1 in artificial soil toxicity test with 95% fiducial confidence limit interval 0.85–1.30 (Table 2).

Table 2 Lethal concentrations (LC1-99) of QOI for earthworm, E. eugeniae by artificial soil toxicity test
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Estimation of total protein concentration

The protein concentration was found to be 41.99 ± 1.33 mg mL−1 in control pre-clitellum, 78.99 ± 1.68 mg mL−1 in 5% pre-clitellum, 112 ± 1.86 mg mL−1 in 10% pre-clitellum, 83.99 ± 1.29 mg mL−1 in control clitellum, 101.29 ± 1.63 mg mL−1 in 5% clitellum, 117 ± 1.48 mg mL−1 in 10% clitellum, 26 ± 1.29 mg mL−1 in control post-clitellum, 35.99 ± 1.40 mg mL−1 in 5% post-clitellum, and 43.99 ± 1.39 mg mL−1 in 10% post-clitellum. The concentration of total protein in different regions of earthworms were observed to be increasing in a dose-dependent manner. The total protein concentration was found to be elevated in 10% LC50 value of QOI-treated samples when compared to control samples (Suppl. Figure 2).

Morphological alterations

Morphological changes observed with QOI-treated earthworms were as follows: part of body coiling and clitellar shrinkage in 0.78 µg cm−2 concentration, whole body coiling in 1.56 µg cm−2 concentration, thinning of body and body swelling in 2.34 µg cm−2 concentration, mucosal fluid release and fragmentation of body in 3.12 µg cm−2 concentration, and segmentation of body and bleeding in 3.90 µg cm−2 concentration, respectively. There were no morphological alterations observed in the control group of earthworms during the experimentation of 48 h (Fig. 1).

Fig. 1
figure 1

Morphological characteristics of QOI-treated and untreated E. eugeniae earthworm. Represents the morphological observations of E. eugeniae earthworms treated with QOI. a Control, b 0.78 µg cm−2, c 1.56 µg cm−2, d 2.34 µg cm−2, e 3.12 µg cm−2, and f 3.90 µg cm−2. CS clitellar shrinkage, BC body coiling, WBC whole body coiling, BS body swelling, TB thinning of body, FB fragmentation of body, RMF release of mucosal fluid, BB bleeding from body

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Biomarker enzyme assays

AChE activity

The specific activity of AChE was expressed as nM ATI hydrolyzed/min/mg protein. The AChE activity was found to be 451.46 ± 5.16 units in control pre-clitellum, 276.15 ± 6.43 units in 5% LC50 pre-clitellum, 188.28 ± 6.54 units in 10% LC50 pre-clitellum, 443.93 ± 6.03 units in control clitellum, 211.29 ± 6.38 units in 5% LC50 clitellum, 187.78 ± 6.13 units in 10% LC50 clitellum, 69.45 ± 4.79 units in control post-clitellum, 51.46 ± 4.61 units in 5% LC50 post-clitellum, and 28.03 ± 4.07 units in 10% LC50 post-clitellum. Earthworms treated with QOI showed a significant decrease in the specific activity of AChE in various parts of the body (pre-clitellar, clitellar, and post-clitellar) (Fig. 3a). Ten percent of LC50 of QOI elicited maximum inhibition of AChE activity in all segments [inhibition of AChE (10% LC50 > 5% LC50 > control)]. The AChE activity decreased region-wise in a dose-dependent manner (pre-clitellar < clitellar < post-clitellar).

SOD activity

The specific activity of SOD was expressed as U/min/mg protein. The SOD activity was found to be 61.2 ± 1.41 units in control pre-clitellum, 66.0 ± 1.89 units in 5% LC50 pre-clitellum, 127.7 ± 2.71 units in 10% LC50 pre-clitellum, 63.6 ± 2.11 units in control clitellum, 76.6 ± 2.43 units in 5% LC50 clitellum, 90.5 ± 1.99 units in 10% LC50 clitellum, 44.3 ± 2.00 units in control post-clitellum, 47.6 ± 2.16 units in 5% LC50 post-clitellum, and 58.3 ± 1.65 units in 10% LC50 post-clitellum. Vital inhibition in the activity of SOD was observed in various segments of the body (pre-clitellar, clitellar, and post-clitellar) of earthworms exposed to QOI in comparison with control (Fig. 3b). The maximum increase in SOD activity was found to be in 10% of LC50 (10% LC50 > 5% LC50 > control). In various body segments, maximum activity was observed in post-clitellar (post-clitellar > clitellar > pre-clitellar) in a dose-dependent manner.

Fig. 2
figure 2

Histological analysis of QOI exposed to E. eugeniae earthworms. Represents the histopathological analysis of QOI exposed to E. eugeniae earthworms; a, e, i Tissue layers such as epithelial tissue, circular muscle tissue, longitudinal tissue and chloragogen tissues were in a proper structure; b, c, h, l inflammation and rupturing in tissue layers; j damaged epithelial tissue; f bulginess in chloragogen tissue; d, g, k, m chloragogen tissues were disintegrated

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LPO activity

The specific activity of LPO was assessed by the exposure of TBARS contents to QOI to earthworms and expressed as nM TBARS released/min/mg protein. The LPO activity was found to be 156.8 ± 2.0 units in control pre-clitellum, 238.6 ± 3.0 units in 5% LC50 pre-clitellum, 378.3 ± 2.8 units in 10% LC50 pre-clitellum, 114.8 ± 2.2 units in control clitellum, 222.3 ± 2.6 units in 5% LC50 clitellum, 238.3 ± 2.9 units in 10% LC50 clitellum, 38.9 ± 2.0 units in control post-clitellum, 53.8 ± 2.3 units in 5% LC50 post-clitellum, and 82.9 ± 2.9 units in 10% LC50 post-clitellum regions of the body were observed in a dose-dependent manner (Fig. 3c). The 10% LC50 of QOI resulted in the maximum increase in lipid peroxidation (10% LC50 > 5% LC50 > Control).

Fig. 3
figure 3

Specific activity of biomarker enzymes of QOI-treated and untreated E. eugeniae earthworm. Represents the specific enzyme activity of AChE (a), SOD (b), LPO (c), and CAT (d). For quinalphos-treated E. eugeniae earthworm group, * represents p < 0.05 control vs 5% LC50; ** represents < 0.05 control vs 5% LC50 and 10% LC50

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CAT activity

The specific activity of CAT was expressed as nM of H2O2 depleted/min/mg protein. The CAT activity was found to be 287.75 ± 3.88 units in control pre-clitellum, 538.5 ± 3.98 units in 5% LC50 pre-clitellum, 589.5 ± 4.58 units in 10% LC50 pre-clitellum, 365.5 ± 3.95 units in control clitellum, 565.7 ± 3.8 units in 5% LC50 clitellum, 622.5 ± 4.75 units in 10% LC50 clitellum, 404.75 ± 3.34 units in control post-clitellum, 552.6 ± 3.77 units in 5% LC50 post-clitellum, and 594.3 ± 4.43 units in 10% LC50 post-clitellum regions of earthworms exposed to QOI for 48 h (Fig. 3d). The effect of CAT activity was similar to those of SOD activity, where the significant highest increase in CAT activity was found to be in 10% of LC50 (10% LC50 > 5% LC50 > control). In various body segments, maximum activity was observed in post-clitellar (post-clitellar > clitellar > pre-clitellar) in a concentration-dependent manner.

GST activity

The specific activity of GST was expressed as nM CDNB conjugates/min/mg protein. The GST activity was found to be 7.58 ± 0.45 units in control pre-clitellum, 17.3 ± 0.75 units in 5% LC50 pre-clitellum, 36.56 ± 0.74 units in 10% LC50 pre-clitellum, 10.18 ± 0.80 units in control clitellum, 23.46 ± 0.74 units in 5% LC50 clitellum, 25.2 ± 0.79 units in 10% LC50 clitellum, 5.3 ± 0.48 units in control post-clitellum, 8.16 ± 0.59 units in 5% LC50 post-clitellum, and 17.8 ± 0.73 units in 10% LC50 post-clitellum were observed in QOI exposed to earthworms (pre-clitellar, clitellar, and post-clitellar regions) for 48 h (Fig. 4a). The GST activity was found to be maximum in 10% LC50 of the pre-clitellar region of E. eugeniae earthworms, followed by 5% LC50 and control. GST activity was observed in a concentration-dependent manner with a maximum increase in pre-clitellar followed by clitellar and post-clitellar. The GST activity of various segments of earthworms.

Fig. 4
figure 4

Represents the specific enzyme activity of GST (a), GSH (b), CBE (c), and Cyt P450 (d). For quinalphos-treated E. eugeniae earthworm group, * represents p < 0.05 control vs 5% LC50; ** represents < 0.05 control vs 5% LC50 and 10% LC50

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GSH activity

The specific activity of GSH was expressed as nM GSH consumed/min/mg protein. The GSH activity was found to be 293.2 ± 1.38 units in control pre-clitellum, 236.26 ± 1.66 units in 5% LC50 pre-clitellum, 204.16 ± 1.49 units in 10% LC50 pre-clitellum, 239.56 ± 1.34 units in control clitellum, 212.13 ± 1.68 units in 5% LC50 clitellum, 202.9 ± 1.42 units in 10% LC50 clitellum, 246.43 ± 1.57 units in control post-clitellum, 199.6 ± 1.70 units in 5% LC50 post-clitellum, and 178.26 ± 1.54 units in 10% LC50 post-clitellum segments was observed with earthworms exposed to QOI (Fig. 4b). The levels of GSH decreased with increase in concentration of QOI. The maximum decrease in GSH level was observed in 10% LC50 concentration (control > 5% LC50 > 10% LC50). The levels of GSH were observed in a dose-dependent manner (pre-clitellar > clitellar > post-clitellar).

CBE activity

The specific activity of CBE was expressed as U/min/mg protein. The CBE activity was found to be 206.73 ± 2.54 units in control pre-clitellum, 181.13 ± 2.00 units in 5% LC50 pre-clitellum, 135.3 ± 2.05 units in 10% LC50 pre-clitellum, 208.46 ± 2.44 units in control clitellum, 159.2 ± 2.21 units in 5% LC50 clitellum, 151.13 ± 1.39 units in 10% LC50 clitellum, 182.73 ± 2.00 units in control post-clitellum, 159.7 ± 2.03 units in 5% LC50 post-clitellum, and 146.63 ± 1.74 units in 10% LC50 post-clitellum segments of earthworms exposed to QOI. The maximum inhibition of CBE enzyme was found in 10% LC50 concentration followed by 5% LC50 and control. The CBE inhibition levels were observed in a concentration-dependent manner where maximum inhibition was found in the region of pre-clitellar followed by post-clitellar and clitellar (Fig. 4c).

Cyt P450 activity

The specific activity of Cyt P450 was expressed as U/min/mg protein. The Cyt P450 activity was found to be 164.3 ± 1.96 units in control pre-clitellum, 155.5 ± 1.86 units in 5% LC50 pre-clitellum, 121.3 ± 1.56 units in 10% LC50 pre-clitellum, 85.9 ± 1.52 units in control clitellum, 68.03 ± 1.31 units in 5% LC50 clitellum, 64.03 ± 1.31 units in 10% LC50 clitellum, 133.3 ± 1.18 units in control post-clitellum, 97.6 ± 1.18 units in 5% LC50 post-clitellum, and 76.5 ± 1.19 units in 10% LC50 post-clitellum regions of earthworms exposed to QOI. The maximum decrease in the Cyt P450 was recorded in the 10% LC50 concentration of QOI (10% LC50 > 5% LC50 > control). The activity of the Cyt P450 enzyme was found maximum in pre-clitellar followed by post-clitellar and minimum in the clitellar region in a dose-dependent manner (Fig. 4d).

Protease activity

The zone of clearance in skimmed milk agar indicates the proteolysis activity. A zone of proteolysis was observed in 10% LC50 and 5% LC50 concentrations of pre-clitellar, clitellar, and post-clitellar regions. Nevertheless, there is no zone of clearance observed in control earthworms (Suppl. Fig. 1).

NRRT assay

The NRRT assay was performed to analyze the cellular damage in the earthworms through the 50% neutral red dye retention from the lysosome to cytosol. The earthworm cells retaining neutral red dye was indirectly proportional to the concentration of QOI with a maximum decrease obtained in 10% LC50 dosage of insecticide. Cells retaining the neutral red dye was found to be maximum in control untreated earthworms and minimum in 5% LC50 treated in a time dependent manner (control > 10% LC50 > 5% LC50) (Suppl. Fig. 3, Suppl. Video 1).

Histopathological studies

In control earthworms, the epithelial tissue, circular muscle tissue and longitudinal tissue were in proper structural arrangement and the chloragogen tissue layer outgrowth was visible towards the longitudinal tissue. While 5% LC50 treated earthworms shown significant structural architectural changes like inflammation and rupturing of circular and longitudinal tissues in pre-clitellum, bulginess of chloragogen tissue in clitellum, damaged epithelial layers and disintegration of chloragogen tissues in post-clitellum regions. Instead in 10% LC50 treated earthworms, inflammation and rupturing of epithelial, circular, and longitudinal tissue layers and disintegration of chloragogen tissues occurred in all the regions of earthworm (Fig. 2).

Discussion

In the present study, the toxicity effect of the QOI on exotic earthworm E. eugeniae has been explored through biomarker enzyme analysis and histological characterization. Based on the LC50 values, the severity level of pesticide toxicity has categorized into 4 groups [super toxic (< 1.0 µg cm−2), extremely toxic (1–10 µg cm−2), very toxic (10–100 µg cm−2), moderately toxic (100–1000 µg cm−2), and relatively nontoxic (> 1000 µg cm−2)] (Roberts & Wyman Dorough, 1984). In which, the LC50 toxicity effect of the QOI on E. eugeniae (3.56 µg cm−2) falls in extremely toxic (Tiwari et al., 2019). Pesticides such as Carbaryl (4.21 µg cm−2), Pyridaphenthion (3.84 µg cm−2), Azoxystrobin (2.72 µg cm−2), and Picoxystrobin (3.15 µg cm−2) were found to have similar toxic levels with the E. fetida earthworms as study model (Wang et al., 2012). Meanwhile, we reconnoitred the artificial soil toxicity test to evaluate the lethality of earthworms in an artificial natural environment. Comparable results have been obtained with other toxicity studies, such as the cadmium-atrazine-lambda-cyhalothrin mixture (18.47 mg kg−1), cadmium-chlorpyrifos-lambda-cyhalothrin-abamectin mixture (7.42 mg kg−1), cadmium-atrazine-chlorpyrifos-lambda-cyhalothrin-abamectin mixture (10.26 mg kg−1), carbaryl (15.2 mg kg−1), flonicamid (9.32 mg kg−1), pyridaben (6.30 mg kg−1), prodiamine (6.79 mg kg−1), mesotrione (5.11 mg kg−1), metsulfuron-methyl mixture (6.23 mg kg−1), azoxystrobin (9.53 mg kg−1), ipconazole (5.06 mg kg−1), and tebuconazole (6.78 mg kg−1), and carbendazim (2.0 mg kg−1) with the E. fetida (Rico et al., 2016; Wang et al., 2012; Yu et al., 2019). According to this present research, it was revealed that the artificial soil test of the LC50 value (1.05 mg kg−1) of the QOI has shown greater toxicity when compared with the clothianidin insecticide (6.06 mg kg−1) and pyridaphenthion organophosphate pesticide (9.26 mg kg−1) on E. fetida (Wang et al., 2012).

Soil bio-indicators, such as earthworms, are effective at reclaiming metal-contaminated soil and mitigating soil metal toxicity (Yuvaraj et al., 2021a). Furthermore, epigeic earthworm species such as E. eugeniae, E. fetida, and P. excavatus function well in heavy metal-containing substrates by digesting complex organic waste and govern the physicochemical and biological characteristics of substrate (Yuvaraj et al., 2021b). Recent scientific studies have shown that earthworms might indeed flourish in polluted soils, but further research is needed to fully grasp the earthworm’s physiological processes (Yuvaraj et al., 2021a).

Analysis of protein biomarkers revealed that QOI treatment elevated protein concentration levels. This may be due to the up-regulation of DNA repair protein molecules such as H2AX (Subbiahanadar Chelladurai et al., 2020) for the development of pesticide stress defense mechanism (Mosleh et al., 2003) and cellular enzyme restoration (Chowdhury et al., 2005; Mekahlia et al., 2016b). Similar, reports have been observed in earthworm Lumbricus terrestris, exposed with herbicide sekator and phosphate fertilizer (Mekahlia et al., 2016b).

The morphological changes such as whole-body coiling, clitellar shrinkage, thinning of body, body swelling, and mucosal fluid release, fragmentation of body and bleeding and segmentation were observed with the QOI-treated earthworms. These may be due to the microscopic damage or trauma caused by variations the ionic potential (Ca++ and K+ levels) in neuromuscular junction, which in turn influence the morphological variation (Kumar et al., 2017). The results of our study evidenced by the earlier findings with the cell wall lesions and rupturing in profenofos treated E. fetida earthworms (Chakra Reddy & Venkateswara Rao, 2008).

In our present findings, the level of AChE activity was found to be decreased in pre-clitellar region rather than other regions, which might be due to the cellular malfunction, toxic stress (Calisi et al., 2013) and increased pesticide residues accumulated in the brain, dorsal brain in the prostomium, and other internal organs, resulting in hyperactivity, neuronal, and muscle dysfunction (Narahashi, 1996; Tiwari et al., 2019). The results of our current investigation are consistent with the findings of a previous study on the effects of atrazine herbicide on E. fetida (Lammertyn et al., 2021).

The exposure of earthworm to pesticide generates ROS including free radicals, single oxygen atoms, and H2O2. This leads to oxidative stress, tissue damage, cellular malfunctioning, lipid peroxidation, and cell death (Cnubben et al., 2001; Tiwari et al., 2019). In our present findings, the LPO level rises dramatically after being exposed to QOI, which may be due to the lack of antioxidant defense and ROS production (Anand & Suresh, 2014). Similar results of LPO and ROS production was reported with the treatment of fomesafen herbicide with E. fetida (Cnubben et al., 2001; Q. Zhang et al., 2014).

The antioxidant defense enzymes SOD and CAT are being released to protect ROS formation from free radicles following QOI exposure (Dazy et al., 2009; Q. Zhang et al., 2013). In our present findings, the level of SOD and CAT found to be increased with the QOI-treated earthworms. This might to enhance the quenching property of the ROS free radicals (O2− and H2O2) to water and oxygen molecules (S. Liu et al., 2011; Sun et al., 2007; Tiwari et al., 2019). Similar reports are identified with the exposure of E. fetida with polycyclic musks (S. Liu et al., 2011).

GST plays significant function in the detoxification of pesticides and xenobiotics (Velki & Hackenberger, 2013). Earthworms treated with QOI were found to have higher GST levels, indicating biotransformation of enzyme-assisted GSH conjugation (Mekahlia et al., 2016b). Because of its properties in cell metabolism, it plays a major role in retaining the integrity of cells. In concurrence with GST, GSH detoxifies the xenobiotics enhancing protection against oxidative stress (Anand & Suresh, 2014).

The observed GST activity in this study was agreed with the earlier findings with the increased activity of the GST in tetrabromobisphenol-A exposed with E. fetida earthworms (Xue et al., 2009) and also substantiate with the reports of chlorpyrifos pesticide on E. eugeniae earthworms (Tiwari et al., 2016, 2019). Similarly, the increased level of GSH also denoted with the QOI-treated earthworm, and this may be due to the upregulation of GSH metabolic pathway or alternate mechanism for the detoxification of QOI toxicants (Xue et al., 2009). This observation is also agreed with the prior studies with the exposure of the E. fetida with 1-methyl-3-octylimidazolium bromide (Li et al., 2010).

Cyt P450 is an earthworm enzyme family involves in biotransformation of xenobiotic or toxic materials, in which CYP1A2, CYP2C9, and CYP3A4 are key iso-enzymes (Yang et al., 2019). In our present study, the QOI-treated earthworm has shown decreased enzyme production; this might be due to the inhibition/rupturing property of the QOI to the CYP enzyme complex and lysosomal membranes (S. Zhang et al., 2019).

Organophosphate pesticide detoxification is facilitated by the serine hydrolase complex known as CBE (Araneda et al., 2016). In the present study, the level of CBE was found to be decreased with the QOI-treated earthworms; this might be due to the formation of stable covalent bonding of CBE-QOI complex through the nucleophilic compensation between the CBE active site serine residue to the oxidized phosphorous QOI (Sanchez-Hernandez et al., 2009, 2014). Our current findings on CBE substantiate the earlier findings on the exposure of the malathion on E. fetida and Lumbricus terrestris (Henson-Ramsey et al., 2011).

NRRT assay is a sensitive subcellular biomarker for measuring the dose-related pesticide lethality in earthworm model system (Ma et al., 2017). In our present study, the retention factor of the NRRT assay was observed as indirectly proportionate to the QOI concentration, which might be due to the instability of the lysosomal membrane caused through QOI derivative stress and thus reduces the dye retaining capacity (Lionetto et al., 2016). Our current findings are comparable with the earlier reports on Aporrectodea caliginosa earthworms exposed with diazinon and chlorpyrifos organophosphate as dose dependently (Booth & O’Halloran, 2001).

Earthworm proteases are proteinaceous multicomponent which responds relatively to the environmental factors for maintaining the balancing of acid phosphatase and alkaline phosphatase during tissue damage and inflammation (Verma & Pulicherla, 2016). Protease are also involving in upholding the oxidative stress caused by reactive species (ROS), and hydroxyl radical (OH−) (Ismail et al., 1992). In the present finding, the proteases zone inhibition was identified with the QOI-treated earthworm and it might influence the anti-inflammation and anti-oxidative stress property.

The histopathological studies are used to monitor the cellular morphological changes in earthworms with pollutants or toxic substances (Saxena et al., 2014). In the present findings, the structural disintegrations were identified with QOI treated earthworm’s section as in a circular and longitudinal cellular arrangement. This might be due to the accumulation and inculcation of the QOI in the earthworm epidermal layers and leads to cellular destruction, chloragogen deformation and body fragmentation (Lourenço et al., 2011; Saxena et al., 2014). Under pathological situations, a high TBARS level (LPO) is also seen as a reputable marker for appraising the amount of tissue damage (Suresh et al., 2010). The results of the histopathological cellular disintegration, further support our findings on morphological deformations. These observations are further supported by the earlier findings on the exposure of the carbaryl, carbofuran, cypermethrin and fenvalerate in Metaphire posthuma and E. fetida with the circular and longitudinal muscle necrosis, disintegration of ectoderm layers, tissue destruction, deformation in chloragogen and body fragmentation (Gobi & Gunasekaran, 2010; Saxena et al., 2014).

Conclusion

The current experimental study indicates the significant toxic effects of QOI on E. eugeniae. The effects were observed as tissue specific in a dose-dependent manner with ROS production and neurotoxic effects. The contact of pesticide contaminated environment may possess serious risk to the survival, metabolism, and functioning of the E. eugeniae earthworms by altering AChE and antioxidant enzymes including SOD, CAT, LPO and detoxifying enzymes such as GST, GSH, CBE, and Cyt P450. Hence, it is concluded that extensive usage of QOI in the soil environment poses a significant threat to the nontarget eco-friendly earthworm organisms. Further molecular studies need to be analyzed up to cellular level and metabolic process of QOI on earthworm is highly warranted.