Introduction

Earthworms are the key representatives of soil megafauna, which play a salient role in cycling nutrients and creating soil structure and are hence designated as ecosystem engineers [1]. They act as allogenic or physical engineers because they are habitat builders for other soil fauna [2]. The earthworms possess incredible antitumor, antimicrobial, antibacterial, and other therapeutic properties [3] and provide relevant information on soil quality, microclimate, nutritional condition, and the concentrations of toxic components in soils [4]. Studies on ecotoxicology are used to determine how toxic pesticides are to soil biota. Earthworms have been chosen as a model species for ecotoxicological evaluation and they’re one of the finest critters for assessing the impacts of toxins in the soil. Several guidelines proposed Eisenia fetida/andrei for earthworm toxicity testing because it has a large dataset on the effects of numerous substances. The process of choosing which species to use involves selecting E. fetida as the recommended test species. The abundance of the species in organic matter of soil overshadows its property of being not-so-typical soil species. Like any other true soil-dwelling animal, it is susceptible to chemicals [5].

In 1965, chlorpyriphos was approved as an organophosphate insecticide for the first time. It was re-registered by the United State Environmental Protection Agency (US EPA) in 2006. It is the most extensively utilized agricultural insecticide globally, accounting for half of all insecticidal use worldwide [6]. Earthworms are extremely sensitive to expired pesticides (Table 1). An increase in enzymatic and non–enzymatic antioxidants was observed in exposure to environmental pollutants [23]. The commonly used insecticide chlorpyriphos (CPF) is involved in mitochondrial membrane depolarization, production of apoptotic protein molecules, and generation of Reactive Oxygen Species (ROS), ultimately causing apoptosis [24].

Table 1 Impacts of chlorpyrifos (different formulations) on earthworms
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Pesticides and fertilizers are consistently used in conventional agriculture to preserve crops, increase crop yields, and boost productivity. However, overusing agrochemicals can cause soil degradation [25], and pesticide accumulation can have deleterious effects on non-target organisms that are important to agriculture but are not intended to be used as fertilizers, like earthworms [26]. The extensive use of agrochemicals can lead to the accumulation of these compounds in soil and water resources, which can have negative impacts on human health and the environment. Therefore, agrochemical remediation is an important process that involves the removal or reduction of these compounds from contaminated soil and water. This can be achieved through various methods, such as phytoremediation, bioremediation, and chemical treatments. Phytoremediation involves the use of plants to absorb and accumulate agrochemicals from contaminated soils, while bioremediation involves the use of microorganisms to break down and degrade these compounds. Chemical treatments, on the other hand, involve the use of chemical agents to either bind or degrade agrochemicals. The choice of remediation method depends on the specific contaminants, the type and extent of contamination, and other site-specific factors.

Biochar, the complex product of biomass transformation, has been produced and utilized for numerous years and is commonly known as “charcoal” (produced from woody biomass). Carbonization degrades biomass segments while retaining a significant part of their carbon content. The possessions make a difference; the product becomes more carbonaceous and easier to utilize as a reserve in the technical processes [27]. The properties of biochar can be studied in various aspects, together with the presence and composition of an element, pH value, and porosity. Types of biochar depend on the material biomass from which biochar is formed. Firstly, biomass is used for manufacturing biochar; it consists of various diversities of feedstock such as urban green waste, critters manure, paper items, rice bran, unwanted agricultural material, etc. Biochar has multiple advantages increasing soil nutrients, reforming soil, and stopping soil infertility issues because of climate switching (Table 2).

Table 2 Impacts of biochar on soil organisms in pesticides spiked soil
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Fly ash comprises silica, calcium, arsenic, zinc, nickel, magnesium, lead, chromium, and other toxic metals. Fly ash has possible agricultural applications. Fly ash also enriches the soil with plant nutrients, micro as well as macronutrients [37]. Fly ash is generally low in nitrogen content along with lesser bioavailability of phosphorus. However, vermicomposting of fly ash, cow dung, and waste paper combination using E. fetida earthworms led to a boost of 88.1% in available phosphorus for plant utilization [38]. However, increasing fly ash content slowed down worm growth. The survivability of the earthworms was affected to such an extent in the presence of fly ash that in fly ash concentrations greater than 20%, the earthworms failed to survive [39].

Literature survey

The effect of biochar on the mortality of Eisenia fetida in the artificial soil test with chlorpyriphos was investigated earlier in the lab [40]. Rice straw, used to make biochar, is a common agricultural crop residue. After two weeks of pesticide exposure at the median lethal concentration (LC50), changes in mortality were observed. The LC50 was calculated for CPF using probit analysis and was found to be 99.806 mg/kg (active ingredient).

Once the LC50 had been determined, artificial soil was prepared and spiked with freshly prepared biochar from rice straw at various doses on the same day as of pesticide exposure. To assess the impact of various dosages of biochar on the LC50 of the pesticide, a 14-day artificial soil test was repeated. In different trials, for CPF, each with three replicates, a control was established using simply LC50 treatment. To test the impact on mortality at LC50 from 50% to lower, various concentrations of RSB300 (rice straw biochar) and RSB500 were coupled with LC50-contaminated artificial soil. On the 14th day, mortality was noted, and the dose with the greatest reduction in death was noted.

The finding cited that RSB500 added at 3% and 5% showed a reduction in mortality from 50% below. It was reduced from 50 to 37.5% and 30% at 3% and 5% for CPF RSB500. The newest fly ash was collected from the National Thermal Power Corporation (NTPC), Jhajjar, and Haryana. Fly ash concentrations were chosen to match the biochar concentration. Post-acclimatization of animals in laboratory conditions, animals were randomly selected into 7 Groups, with three replicates, each containing 10 animals (Table 3).

Table 3 Experimental design for the objectives
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Research gap

In previous research, the highlight areas were the effects of chlorpyrifos on non-targeted soil fauna and there are a lot of scopes for exploring the mitigation strategies. Additionally, there is a pressing need to address agricultural waste. Rice straw-derived biochar and NTPC-derived fly ash posed exempted potent solutions for mitigation of chlorpyrifos stress in E. fetida. The underline of the study was to quantify the stress level and investigate the efficacy of both the amendments (4 treatment groups) provided. To find the best concentrations and application techniques of amendments to provide earthworms with the most stress-relieving benefits. Examine the processes that underlie the stress reduction in E. fetida following amendments. This involves examining the amendment’s function in soil detoxification, absorption, and modification of chlorpyrifos availability. Evaluate the long-term sustainability of improved soil health and stress mitigation. Ascertain the additions’ long-term effects on the soil ecosystem’s sustainability. Convert study results into useful suggestions for environmentally friendly farming methods. Give farmers and policymakers information about the possible application of biochar additions to reduce stress caused by chlorpyrifos and improve soil health.

Materials and methods

Collection and rearing of earthworm

The study was conducted on vermicomposting worm, E. fetida. The worms have high reproductive rates and are easy to culture on various media in the laboratory. E. fetida was procured from Bhoojevan Organic Pvt. Ltd. Location-Najafgarh, Delhi (28.6090° N, 76.9855° E). Worms were collected in a wooden crate with soil and organic cow manure. Worms were reared by OECD Guidelines no. 207 (1984), ISO (1993,1998), and tropical artificial soil was used as a medium at Rohtak (28.8955° N, 76.6066° E).

The medium’s temperature was kept between 25 ± 20 ℃ and relative humidity was kept at 80%. The nutritive medium was added to the artificial soil and given a gentle mix. After stabilization of the medium for 24 h, the collected worms were cleaned and washed in distilled water and released in the Tropical artificial soil augmented with the nutritive medium.

For Tropical Artificial soil [21, 41]; 10% Non-decomposed Coconut coir dust/ coconut peat, decomposed Coconut coir dust/ coco peat was used as an alternative for sphagnum peat, along with rice husk dust and saw dust moistened to 50% of their water holding capacity, 20% kaolin clay, 70% fine industrial sand/silica. Calcium carbonate was added, and pH was adjusted to 6 ± 0.5.

Before removing coelomic fluid from earthworms, their gut should be washed to prevent contamination. For that earthworms were examined individually, and 4–5 worms were poured into each jar containing agar gel. Gut-cleaned worms were placed in an extrusion fluid prepared to collect coelomic fluid [42]. A master solution of guaiacol glycerol ether, EDTA, 95% saline, and 5% ethanol was prepared. Earthworms were poured into the Petri plate with extrusion fluid to stimulate the spontaneous release of coelomic fluid.

Total phenolic content (TPC)

Coelomic fluid samples were exposed to the Folin-ciocalteu (FC) reagent procedure [43]. 50 µl of the sample was blended with 3 ml of deionized water, followed by the addition of 250 µl of FC reagent. A few moments later, 20% sodium carbonate was added to the blend. Then samples were incubated in the dark. Gallic acid was used as the standard. Absorbency was read at a wavelength of 765 nm.

Total antioxidant capacity (TAC)

Assay was evaluated using a phosphomolybdate reagent in acidic pH [44]. 0.1 ml of coelomic fluid samples were treated with phosphomolybdate reagent followed by incubation in a boiling water bath for 1 h and 30 min. Absorbency was read at 695 nm after cooling the samples to room temperature. Here the control was a methanolic phosphomolybdate solution, and a gallic acid standard curve was employed for total antioxidant content determination.

Determination of proline quantum

Coelomic fluid samples were incubated after inoculation with ninhydrin reagent and glacial acetic acid [45]. After 1 h, the tubes were cooled sharply to arrest the reaction using an ice bath. Vertex the mixture while adding toluene. The toluene layer was pipette out, and optical density was read at a wavelength of 520 nm. Proline was taken as standard.

ROS content analysis

To prepare the mitochondrial suspension, E. fetida from each concentration was extracted. of each concentration was taken out to prepare the mitochondrial suspension. The protein concentrations were measured using bovine serum albumin (BSA)as standard, and colourimetric measurement was detected at 595 nm to determine the light absorption value. The ROS content was resolved by the Dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescence method [46]. Mitochondria suspension was taken with a 10 µl probe DCFH-DA and mixed with a specific amount of Phosphate Buffered Saline (PBS) buffer to make the final reaction liquid for 3 ml. In a water bath (37 °C), heat this for 20 min. After heating, 1 mol/l HCl was added to terminate the reaction. Finally, the optical density of the reaction solution at 488 nm as the excitation wavelength and 522 nm as the emission wavelength was recorded.

Antioxidant enzymes assay

After gut cleaning, earthworms were homogenized using prechilled mortar and pestle. Potassium phosphate buffer was centrifuged with homogenized content at 4 °C at 10,000 rpm. To study enzymatic activity and protein quantity, the supernatant was employed.

Superoxide dismutase (SOD) activity (EC 1.15.1.1) was calculated by measuring the reduced blockage of nitro blue tetrazolium [47,48,49]. The reaction mixture involved phosphate buffer (pH 7.8, 50 mM), Ethylenediaminetetraacetic acid (EDTA 100 mM), methionine (130 mM), nitroblue tetrazolium (NBT 750 mM), riboflavin (20 mM), and enzymatic fluid. The reaction was allowed to proceed for 20 min, and a 560 nm wavelength was selected to read the absorbency. Peroxidase (POD) activity (EC 1.11.1.7) was measured by adopting the guaiacol mode [50]. A reaction concoction contained 100 mM potassium phosphate buffer (pH 6), 30% H2O2, and guaiacol. After adding enzymatic fluid, an absorbance reading was taken at 470 nm. Ascorbate peroxidase (APX) activity (EC 1.11.1.1) reaction was started by the addition of H2O2 [51] and the extinction of ascorbate was measured at 290 nm for 1 min. Catalase (CAT) activity (EC 1.11.1.6) was measured employing hydrogen peroxide mode [52]. A 0.1 ml sample was mixed with 100 mM phosphate buffer (pH 7.2, 1 mM EDTA). The addition of 10 mM H2O2 commenced the reaction. Glutathione-S-transferases (GST) activity (EC 2.5.1.18) was measured by measuring the coupling rate of glutathione (GSH) and 1-chloro-2, 4-dinitro-benzene (CDNB). The appearance of the reduced glutathione ion GSH-CDNB complex was monitored at 340 nm.

Measurement of lipid peroxidation

Malondialdehyde (MDA) content was evaluated spectrophotometrically [53]. Enzymatic fluid was mixed with a reaction mixture (acetic acid (20%), thiobarbituric acid (TBA 1%), and deionized water), and then the contents were incubated for 1 h in a boiling water bath. The MDA levels in the supernatant were determined at 532 nm.

Statistical analysis

All the experimentation was executed in triplicate, and outcomes were expressed as Mean ± Standard Deviation (SD) following statistical data analysis using MS Excel, Office 16 from Microsoft; GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. All data collected of the mean value of 3 replicates of control, and all the treatment groups exposed worms, were subjected as mean ± SD for analysis by “Two-Way ANOVA followed by Tukey’s multiple comparisons test was performed using GraphPad Prism. The mean and standard deviation were also calculated using MS Excel. The significance of utilizing ANOVA single factor as the statistical tool for analysis of the results was to compare the mean value of more than two groups, control, and two different amendments. Two-way ANOVA with correction was invoked to evaluate the relation among various treatment groups keeping a significance level equivalent to P˂0.05).

Data are presented as-ns(P > 0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001) and **** (P < 0.0001). Two hypotheses were considered in the ANOVA analysis. A significance level of 0.05 indicates a 5% risk of concluding that a difference exists where there is no actual difference. So, a P-value less than 0.05 indicates a significant result, and a P-value of more than 0.05 states that the results are insignificant; in that case, the null hypothesis is not rejected.

Results & discussion

The SEM pictures were analysed for morphology [54, 55]. The findings proved the amorphous, porous structure of RSB had disintegrated and uneven plates with more active sites for adsorption. The amorphous nature implies an increased surface area for absorption activity and contains various functional groups, such as hydroxyl (-OH) and carboxyl (-COOH) groups, which catalyze antioxidant activities by scavenging free radicals [56,57,58,59]. A porous structure enhances the capturing and retaining ability [60,61,62]. Fly ash is known for its pozzolanic properties, which complement the adsorption capabilities. Fly ash, may facilitate ion exchange processes, allowing the materials to interact with ions involved in antioxidant reactions. Each component plays a distinct role, such as the release of phenolic compounds in rice straws and the coordination of metal ions in fly ash, which together increase the overall antioxidant activity.

The earthworms typically discharge the coelomic fluid to maintain moisture levels and their body functions like breathing and tunnelling. Watery fluid, the plasma, and many coelomocytes, a vital part of the innate immunity of earthworms, compose coelomic fluid. Based on different shapes and sizes, immune cells are categorized into four types, i.e., amoebocytes, mucocytes, circular cells, and chloragogen cells [63].

Analysis with coelomic fluid of worm (total phenolic content, total antioxidant capacity & total proline quantum)

Quantifying polyphenols showed a significant concentration believed to deliver an exceptional equivalence with antioxidant activity [64, 65]. The polyphenols protect cells from ROS generated under stress and strengthen the antioxidant system [66]. The TPC/TAC can be withdrawn using a highly polar solvent, and a water and methanol mixture is a potent candidate for extraction. All the phenolic content, total antioxidant capacity, and proline content of earthworms are cited in Figs. 1(a-c) and 2. The total phenolic content of worms was seen at its highest in week 1, and after that, as the stress increased, its decrease continued until week 3, when it again showed an upsurge in content, marking the adaptation of worms in stressful conditions. Worms followed a similar trend in proline content as they did in phenolic content. As a conclusive remark, the key factor responsible for the antioxidant and anti-inflammatory properties of worms is their polyphenolic content. This study is supported by Song et al., (2009), whose results state that earthworm paste possesses a remarkable quantity of phenolic compounds with significant antioxidants, impacting antioxidative enzymes and damaging DNA in E. fetida as a result of exposure to herbicide atrazine.

Fig. 1
figure 1

Graphical presentation of a) TPC; b) TAC; c) Proline and d) ROS Content of worms. A significant difference was observed between the control and treated groups, P˂ ˂ 0.05, except the ones marked ns

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Fig. 2
figure 2

Graphical presentation of a) SOD; b) GST; c) APX and d) MDA Content of worms. A significant difference was observed between the control and treated groups, P˂ ˂ 0.05. Lines representations from a-c marked ns and in d marked significant differences

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This finding suggests that worms can adjust their antioxidant defences in response to environmental stress, which is consistent with previous studies that have shown that worms can produce and accumulate phenolic compounds in response to stress. The study also found that worms followed a similar trend in proline content as they did in phenolic content. Proline is an amino acid that plays an essential role in plant and animal stress responses, and its accumulation has been associated with increased tolerance to abiotic stressors. The study’s results suggest that worms can use proline as a stress response mechanism, which may help them cope with pesticide-induced stress. Worms can accumulate polyphenolic compounds in response to stress is noteworthy, as it suggests that worms may have the potential to serve as a source of natural antioxidants and anti-inflammatory compounds.

ROS content analysis

ROS production was induced when E. fetida met pollutants, affecting the earthworm’s antioxidant indexes. A wide variety of ions are included in ROS as superoxide anion, hydroxyl, hydrogen peroxide, peroxyl, singlet oxygen, alpha oxygen, and many more that enhance the risks of oxidative stress, lipid peroxidation, proteotoxicity, mimicry and amplification of growth factor, and DNA damage. The effect of treatments on the ROS content of earthworms is shown in Fig. 1d. Results confirmed an upsurge in ROS level in all weeks and as compared to control, with an almost negligible increase in the content when we compared groups week-wise because the antioxidant enzymes performed ROS elimination. For 28 days, the organisms were exposed to different doses, and after that, tissue homogenate of organisms, taken from each application to estimate the protein content was prepared. The protein content is one of the sensitive biomarkers to study the toxic effects. Protein, carbohydrates, lipids, and DNA are the vital molecules that were damaged by excessive ROS production in the earthworm body (Fig. 3c). The reduction was observed in the protein content of worms as ROS production increased [67].

Fig. 3
figure 3

Graphical presentation of a) POD; b) CAT and c) protein content of worms. No significant difference was observed between the control and treated groups, P > 0.05 for POD and CAT but all the values were significantly different in the control and treated groups, P < 0.05 for protein

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Antioxidative enzyme assay

In the present work on E. fetida, pesticide toxicity was determined by analyzing their effects on protein concentration and antioxidant enzymes like superoxide dismutase. E. fetida activates antioxidant defence and detoxification system against excessive ROS by catalyzing superoxide radicals to peroxides by SOD and then maintaining homeostasis by scavenging it by CAT and POD [68]. SOD maintains the electron transport chain in living organisms. Only SOD, a massive scavenger of oxygen free radicals, effectively catalysed the dismutation reaction of O2−. The monitoring of oxidative stress in the biological system was reflected by a level change in SOD and CAT, which were antioxidants that limit oxidative stress [69, 70]. Figures (SOD 2a; APX 2c; GST 2b; MDA 2d; POD 3a; CAT 3b) summarize the antioxidant enzyme assays of treated and control groups.

At 60% and 80% LC50 POD and APX isozyme, CAT activity showed a cycle of increasing as time passed. Compared with the controls, the higher doses of pesticide increased and stimulated POD, and APX isozyme, CAT activity in earthworms and, significant differences were found in exposed duration. Peroxidase activity is a potent biomarker for the sublethal toxicity of xenobiotics to worms for oxidative stress conditions [47]. GST is involved in the cellular detoxifying of a wide range of xenobiotics [71]. GSH, as long as three amino acids long peptide and a free radical scavenger, converts harmful poisons into harmless products to be excreted out of the body. A significant change in GSH content demonstrated that the treatment could up-regulate GSH content in worm bodies. SOD and GST activities showed a significant difference between treated and amended groups, and the results were in synchronization with Song et al., 2009. While APX, CAT, and POD showed a continuous upregulation in their levels, contrary to their findings. Possible reasons for this that can be explained are that treated groups showed mild stress for worms as compared to atrazine causing severe stress.

Specifically, the study found that at 60% and 80% LC50, POD and APX isozyme, and CAT activity showed a cycle of increasing as time passed. These findings are consistent with previous studies that have shown that antioxidant enzymes, such as CAT, POD, and APX, play a critical role in defending organisms against oxidative stress induced by pesticides. The study also found that exposure to higher doses of pesticides led to significant differences in the levels of antioxidant enzymes in earthworms. For instance, the higher doses of pesticide increased and stimulated POD and APX isozyme, and CAT activity in earthworms. Additionally, there was a significant change in GSH content, demonstrating that the treatment could up-regulate GSH content in worm bodies. These findings suggest that earthworms can respond to pesticide exposure by upregulating their antioxidant defences, which can help mitigate the harmful effects of the pesticide.

The study also found that SOD and GST activities showed a significant difference between treated and amended groups. These results are consistent with previous studies that have shown that SOD and GST activities can serve as useful biomarkers of pesticide toxicity. However, the study’s results differed from Song et al. (2009), who found that APX, CAT, and POD showed continuous upregulation in their levels. The possible reason for this discrepancy may be that the treated groups in this study showed milder stress for worms than atrazine, which causes severe stress. Overall, the study’s results suggest that exposure to higher doses of pesticides can induce oxidative stress in earthworms, which can trigger the upregulation of antioxidant defences. The findings highlight the importance of monitoring antioxidant enzyme activity in earthworms and other organisms exposed to pesticides to better understand the potential risks associated with pesticide use in agriculture. Additionally, the study’s results can provide valuable information for the development of new strategies for the remediation of pesticide-contaminated soils.

MDA acts as index senescence physiology, a byproduct of the cell membrane’s lipid peroxidation. MDA levels aggravate lipid damage and explain the degree of lipid peroxidation. The treatment increased MDA content in worm bodies, which had previously been shown to result in significantly altered MDA content [72]. Lipid peroxidation is a result of the presence of oxygen-free radicals. Lipid peroxidation was measured in terms of malondialdehyde content expressed in µM/ml. Malondialdehyde is a lipid peroxidation product, which, in combination with thiobarbituric acid, gives a pinkish hue. At the end of Week 1, there was a significant increase in MDA content between control and treatment-exposed worms. Increased MDA content meant that lipid peroxidation was high under pesticide exposure. There was a significant increase in MDA content in exposed worms from week 2 to week 4. There wasn’t much fluctuation observed in the MDA content of the control worms. But in the case of group 4 exposed worms, the most fluctuations were seen. MDA content initially increased, but by the end of 4th week, it had declined.

Lipid peroxidation is a process that occurs when free radicals attack lipids in cell membranes, resulting in the formation of MDA as a byproduct. The increase in MDA content in the treatment-exposed worms at the end of week 1 indicates that pesticide exposure triggered lipid peroxidation in earthworms. This result is consistent with previous studies that have shown that pesticides can induce oxidative stress in earthworms and other organisms. The continuous increase in MDA content in exposed worms from week 2 to week 4 suggests that the exposure to pesticides was persistent and that the earthworms were unable to cope with the oxidative stress caused by the exposure. The lack of fluctuation in MDA content in control worms indicates that there was no significant impact of environmental factors on lipid peroxidation in these worms.

Interestingly, the group 4 exposed worms showed the most fluctuations in MDA content, with an initial increase followed by a decline by the end of the 4th week. This result could be attributed to the acclimation of the earthworms to the pesticide exposure over time. It is possible that the earthworms in group 4 developed some level of tolerance to the pesticide exposure, leading to a decrease in lipid peroxidation and a subsequent decline in MDA content by the end of week 4. Overall, the MDA analysis results suggest that exposure to pesticides can induce oxidative stress and lipid peroxidation in earthworms. The continuous increase in MDA content over time indicates that the exposure was persistent and potentially harmful to the earthworms. The fluctuations in MDA content in group 4 exposed worms suggest that earthworms can develop some level of tolerance to pesticide exposure over time. These findings highlight the importance of monitoring the impact of pesticides on earthworms and other organisms to better understand the potential risks associated with pesticide use in agriculture.

Coelomic fluid concentrate from E. fetida in our investigation showed a striking measure of phenolic compound. Our study focused on the protein content of earthworms and their defence mechanism against free radicles. Phenolics are secondary metabolites that function as molecules that scavenge free radicals. The present trends were attributed to the adaptation of those earthworms in that condition. The impact of chlorpyrifos on soil invertebrate communities may be underestimated if the ecotoxicological risk was assessed solely based on toxicity data collected from the observations. The second sample’s readings decreased on the 14th day due to stress conditions imposed by agrochemicals, which increased ROS concentrations in earthworms.

On the other hand, antioxidants are crucial in avoiding body cell damage brought on by free radicals, H2O2, and other oxygen ions in the body. Because phenolic compounds possess a hydroxyl group, they are directly responsible for antioxidant action and are essential in scavenging free radicals. It was discovered that when biochar and the earthworms were employed together, the quantity of a variety of potentially harmful substances mobilized by earthworms was reduced, implying that biochar plus earthworms could be a helpful remediation method. However, this will boost biochar’s ability to immobilize the inorganic or organic contaminants. The most basic and crucial defence systems in an organism’s body are antioxidants, which work to repair any harm done by ROS. Free radicals were neutralized by the donating electrons from stable antioxidants.

So, protein content can also act as an important biomarker for the pesticide toxicity study. However, every treatment once checked through the two-way ANOVA showed a significant difference as compared to the control. Fly ash and Biochar have little effect on restoring the protein content but as compared to the control the remediation showed poor outcomes.

The process of drying and restoring the artificial soil from the experiment using a sieve tower and soil testing kits is an essential step in analyzing the impact of the experiment on soil parameters. The removal of moisture content from the soil helps to prevent further microbial growth, which could lead to the alteration of the soil parameters. The use of a sieve tower with different pore sizes helps to separate the soil from any leftover debris, allowing for a more accurate analysis of soil parameters.

Under oxidative stress caused by a chlorpyriphos increase in value, the body is trying to remove these free radicals. However, as the body’s ability to fight pesticides deteriorates with increased exposure and pesticide concentration, the deals begin to dwindle. These ROS irreversibly damage proteins, and these damaged proteins activate signalling pathways and try to adapt to elevated ROS. But cannot do the same for a prolonged period as ROS are highly harmful and result in cell death.

The current investigation shows that the coelomic fluid concentrate of E. fetida contains a noticeable amount of phenolic content and other antioxidant enzymes, which can be extracted using suitable solvents. The concentration of phenols that possess excellent antioxidant properties was enhanced to counterbalance the concentration of free radicals produced. Through the action of antioxidant defence system enzymes, treatment, and chlorpyriphos, the antioxidant capacity in earthworms was increased more than by insecticide alone. As a conclusive remark key factor responsible for the antioxidant and anti-inflammatory properties of worms was their polyphenolic content. This study supported the findings that earthworm paste possessed a remarkable quantity of phenolic compounds with significant antioxidants—impacting antioxidative enzymes and DNA damage in E. fetida induced by the herbicide atrazine [47].

Our data imply that biochars, in addition to causing no mortality by farmland standards, can cause damage at microlevels, such as cellular or biochemical levels Vermitoxicity is considered based on biochar origin, according to trends in phytotoxicity and microbial toxicity. Indeed, numerous studies present both positive and negative impacts of biochar on soil health [73]. Even in the absence of insecticide, a drop in earthworm growth parameters was detected. To check on the bioavailability of contaminants and soil fauna’s health, the fundamental step is to conclude the amount of biochar amendment. However, the concern is on the application risk of biochar as a remediation measure.

Earthworm species have a significant impact on changes in bioavailability as well as the uptake mechanism. Certain species primarily absorb contaminants by cutaneous absorption, but other earthworm species’ biological uptake strategies were significantly influenced by stomach activities [36]. However, biochar amendments increased the buildup of pesticide residues in worms. It’s because biochar can encourage the gizzard’s pulverisation of soil and the release of enhanced contaminants during gut development [34]. Similarly, to this, biochar did not lower the bioavailability of free pesticides in earthworms at very low and very high application rates (less than 1%) because earthworm uptake of pollutants was enhanced through the intestinal system [35]. Comparable to biochar, fly ash consumption and entry into worms’ guts revealed heavier detrimental effects than biochar.

Moreover, in soils with varied textures, the impact of amendments at the same application rate on reducing the bioavailability of organic contaminants varies. The interactions between active surfaces and soil-soluble organic matter, which led to the attenuation of adsorption performance, are the main reasons why the features of soil are significant for the reduction of bioavailability and augmentation of sorption [10]. A soil treated for metal contamination with ashes long ago had shown a noteworthy avoidance by E. fetida after fly ash amendment. Changes in soil texture or an increase in pH are two possible explanations. Conclusively, no soil health recovery was attended to because of the loss of farmers’ friend habitats. Instead, a comparative approach would provide precise information about the earthworm community and soil fauna using those living in ash-treated and untreated areas.

An inverse relationship was observed between biomass and the percentage of fly ash used for amendment. Ramalingam (1997) [74], and Kaushik and Garg (2004) [75], studies back up our findings, as reproduction and worm growth were influenced by food accessibility.

It is extremely difficult to mitigate the harmful effects of chlorpyrifos. There may be intricate relationships between the amendments that call for more research. It was difficult to figure out which amendment ratios work best for reducing the toxicity of chlorpyrifos. There may be unforeseen effects on other soil species or ecosystem activities, and the ideal balance may change depending on soil types, environmental factors, and chlorpyrifos concentrations. Natural ecosystems are complicated systems that can contain extra variables that are difficult to mimic in controlled research. Also, applying research from the lab to actual field situations. The application of amendments showed a noteworthy decrease in the mortality rates of earthworms exposed to chlorpyrifos. Also, demonstrated a direct correlation between the application of amendments and elevated antioxidant activity in earthworms, suggesting a possible strategy for reducing oxidative stress caused by chlorpyrifos. It can help in putting out an integrated plan for pest management that integrates amendments with other ecologically beneficial and sustainable farming practices [76].

The future perspective of extracted phenols and other antioxidant enzymes lies in the fact that they can be used as natural antioxidants to cure various ailments related to inflammation and oxidative stress conditions. These antioxidants have enormous potential to be of immediate use in medicine. This can considerably reduce dependence on artificially synthesized antioxidants by switching to natural antioxidants extracted from these “miracle worms.” Further efforts are required to correctly deliver pesticides so that less or no residue of the same remains in the soil so that it doesn’t harm nontargeted soil fauna. The relationship between worms’ antioxidant indexes and biochar has been testified by three studies to date [77,78,79]. But, surprisingly, none of these was based on animal-origin biochar. Besides, varied biochars have massively diverse features; consequently, additional research on their interactions with earthworm antioxidant indexes is required. The inclusive impacts of explicit biochar on a particular ecosystem of soil or environment must be explored before its actual field use. Grounded on this reflection, developing apt, healthy, and effectual testing approaches will be necessary. Likewise, more focus is needed on afforestation, which benefits the earthworm community.