Acute (14 DOE) and chronic toxicity (56 DOE) experiments were conducted to compare the lethal and sub-lethal effects of chlorpyrifos and dimethoate on earthworms (Eisenia fetida). Earthworms were cultured in test soil (agricultural soils + cow dung) spiked with various concentrations of these two organophosphorus pesticides of different half-life. The determined effects were avoidance response, biomass change, growth inhibition, mortality, biomass gain per unit feed, cocoon and hatchling production. The resulted order of effects for acute and chronic toxicity exposure periods for E. fetida was dimethoate > chlorpyrifos. The reported median lethal concentration (LC50) in present study was 17.45 and 228.06 mg kg-1 dry test soil for dimethoate and chlorpyrifos, respectively. Growth inhibition was more critically influenced in dimethoate as compare to chlorpyrifos. Rather than lesser growth inhibition in chlorpyrifos, cocoon and hatchling production is influenced greatly at higher concentrations. The cocoon and hatchling production is significantly affected at lower concentrations of dimethoate. All the tested parameters were found to be dose dependent. The present results showed that even a small concentration of dimethoate is potentially dangerous to earthworms than higher concentrations of chlorpyrifos. In comparison the chlorpyrifos was found safer than dimethoate at sub-lethal concentrations for the development and reproduction of E. fetida. Our study provides a clear insight of different antagonistic effects of two organophosphate pesticides with different half-life.
Research Article
Delineating the ecotoxic effects of Chlorpyrifos and Dimethoate on Eisenia fetida
https://doi.org/10.21203/rs.3.rs-1516313/v1
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Eisenia fetida
Dimethoate
Chlorpyrifos
Avoidance response
LC50
Acute and Chronic toxicity
In the present scenario land use paradigms are shifting drastically worldwide, which is one of the major concerns followed by the burgeoning population. The availability of arable land resources is becoming scarce due to some of the burning environmental issues such as climate change, environmental pollution, globalization, industrialization, unsustainable development etc. (Suhani et al. 2020). Some of the major factors governing land resource deterioration includes, soil erosion, natural and man-made disasters, soil pollution (fertilizers, pesticides, heavy metals, etc.), and irrelevant extensive farming techniques. According to projections by 2050, the global population count will cross 9 billion and there will be demand to increase food supply by 70% to fulfill the global food demands (Rattan 2015). The projections raise a justified concern to the future food security. In agroecosystems, the growing crops needs a healthy soil-plant environment to attain its maximum growth and produce optimal yield. But the presence of plethora of environmental issues (biotic and abiotic stresses) diminishes the crop growth and yield (Sahab et al. 2020). Biotic stresses that include plant pathogens, diseases, pests and weeds, that are responsible for reducing 20–40% crop production and it cost the economy nearly US$ 290 billion annually on a global scale (Savary et al. 2019; Gautham and Bhardwaj 2020). Regarding the global crop production losses due to insects and pests, the use of pesticides is imperative to combat the rising food demand (Dasgupta et al. 2012).
In India, nearly 15–20% yield of principal food and cash crops lost to production apparently due to pests (Rathee and Dalal 2018; Kumar et al. 2021). Dealing with variety of pest and weed infestations, there are number of solutions available which majorly include chemical agents, popularly known as pesticides, insecticides, weedicides, herbicides, etc. Pesticides are the chemical compounds or mixture of chemicals or agents (either biological or man-made) which are mainly used for pest eradication purposes (Lackmann et al. 2021). Organophosphate pesticides (OPPs) are widely used because of their broad spectrum of activity and short residual time in comparison with persistent organochlorine pesticides (Aswathi and Sukumaran 2019; Omwenga et al. 2020). OPPs are well known acetylcholinesterase (AChE) inhibitors, it disrupts normal functions of nervous system (Omwenga et al. 2020). AChE is a cholinergic enzyme especially found in nerves and muscles, its inhibition can cause neurotoxicity, developmental impairments and alterations from lower to higher biological organization levels (Gravato et al. 2021). OPPs are broad spectrum pesticide which are used to control a variety of boring, chewing and sucking insects, spider, mites, aphides and pest that attacks crops like cotton, sugarcane, vegetables and fruits (Dong et al. 2020). Unfortunately, the extensive and unmannered use of OPPs is found hazardous to many non-target organisms residing in soil biota including earthworms (Dasgupta et al. 2012; Sahab et al. 2021). Earthworms live in a proximate contact with soil and constitute up to 90% of the invertebrate biomass depending on different soil conditions (Gupta et al. 2011; Kumar et al. 2020). Due to the remarkable abundance and vital contributions of earthworm in ecologically complex processes in soil biota (i.e., nutrient cycling, debris decomposition, soil formation), they can be well defined as an ecological engineer (Dasgupta et al. 2012; Sahab et al. 2021; Srivastava et al. 2021). Because of their higher proportion out of total invertebrate biomass they become victims of predation by organisms of different trophic level such as, mammals, reptiles, birds and amphibians (Gupta et al. 2011; Martinuzzi et al. 2020). Since many literatures reported about the ability of earthworms to accumulate pesticides and other contaminants, it renders a possible risk of contaminant transmission in higher trophic levels of ecosystems (Singh et al. 2020; Bošković et al. 2021).
In many literatures, the exposure of pesticides has been found to cause alteration in biomarkers at several levels of organizations in earthworm such as, cellular, biochemical, physiological and behavioral (Sanchez-Hernandez 2006; Reinecke and Reinecke 2007; Morcillo et al. 2013; Tiwari et al. 2016). Pesticide exposures are known to alter enzymatic functions and metabolism, reduce biomass, inhibit growth, affect reproduction, increase mortality and change behavioral responses in different species of earthworms (Rico et al. 2016; García-Gómez et al. 2019) (Fig. 1). There are numerous studies revealed reduction in gut enzymatic activities, imbalance in antioxidant enzyme levels, inhibition of AChE, carboxylesterase and other cholinesterase activities in earthworms due to pesticide exposure (Table 1). Histopathological studies revealed that acute and chronic exposures of pesticides produce morphological deformities such as damage to longitudinal and circular muscle layers, epidermal cells, cuticular membrane in earthworms (Saxena et al. 2014; Solé 2021).
| Pesticide and their group | Doses | Earthworm Species | Experimental duration | Tested biochemical and other activities | Importance of tested activities | Responses | Reference |
|---|---|---|---|---|---|---|---|
| Chlorpyrifos and azinphos methyl (OPP) | Chlorpyrifos- 0 to 15µgkg− 1; azinphos methyl- 0 to 2.5 µgkg− 1 | Aporrectodea caliginosa | Chronic (6 months) | Cholinesterase activity | - | Cholinesterase inhibition | Reinecke and Reinecke 2007 |
| Lindane (organochlorine) and deltamethrin (pyrethroids) | Lindane- 20, 50, 80, 120, 150 mgkg− 1; Deltamethrin- 5, 25, 50, 100, 150 mgkg− 1 dry soil | E. fetida | Acute (14 day) and subchronic (42 day) | Cellulase activity | Cellulase plays important role in litter and other cellulosic plant material degradation | Lindane was found to increase cellulase activity in earthworms at both acute and subchronic test, while deltamethrin exerted effect only at acute toxicity bioassay. Deltamethrin decreased cellulase activity. | Shi et al. 2007 |
| Methomyl (Carbamate) | 0.86, 1.37, 2.19, 3.51, 5.62, 8.98, 14.4, 23 mgkg− 1 | E. andrei | Acute (48 h) | AChE activity | - | The inhibition of AChE activity is shown by Eisenia andrei in reponse to increasing concentration of methomyl. | Pereira et al. 2010 |
| Chlorpyrifos and fenvalerate (pyrethroid) | 10, 20, and 40 mgkg− 1 dry soil | E. fetida | Acute (7 day) | Enzyme activities (cellulase, SOD, CAT) | Cellulase is one of the digestive enzymes which is apparently important in organic matter degradation. SOD is an important component of the earthworms antioxidative-defense system. | The cellulase activity decreased with increasing chlorpyrifos and fenvalerate concentrations. SOD activity significantly inhibited in initial 3 days of experiment in case of both pesticides, while it gets stimulated with increasing exposure period. The CAT activity increased with increasing concentration and exposure period in case of fenvalerate. In case of chlorpyrifos, CAT activity increased in initial 3 days and decreased with increasing exposure period. | Wang et al. 2012 a |
| Carbaryl (carbamate), chlorpyrifos, and endosulfan (organochlorine) | Carbaryl- 0, 0.75, 1.51, 3.03 mgkg− 1; chlorpyrifos- 0, 0.91, 1.82, 3.65 mgkg− 1 , and endosulfan- 0, 3.75, 7.50, 15.0 µgkg− 1 | Perionyx excavatus | Acute (28 day) and subchronic (56 day) | Rate of respiration | Respiration is an important indicator of metabolism and growth of earthworms. | CO2 evolution is found to be increasing with increasing concentrations of pesticides. Highest CO2 production is reported in chlorpyrifos and lowest in endosulfan bioassay. The increased respiration rate revealed the increased stress due to pesticide exposure. | Dasgupta et al. 2012 |
| Dimethoate (OPP) | 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 25, 50 µg cm− 2 | Octolasion lacteum and E. andrei | Acute (24 h) | AChE activity, carboxylesterase activity, efflux pump activity, CAT activity | Carboxylesterase is a hydrolytic enzyme which metabolizes xenobiotics (that contains thioester, ester, amide bonds). Its inhibition is considered to be a stoichiometric mechanism for reducing pesticide concentration at target sites. Efflux pump activity play a major role in removal of toxicants from cells. | AChE activity significantly inhibited in O. lacteum at lower concentration, while increased in E. andrei. Carboxylesterase activity was found to be significantly inhibited in E. andrei, while in O. lacteum a slight inhibition is seen in last two concentrations. Efflux pump activity was majorly affected in E. andrei at higher concentration of dimethoate. CAT activity is inhibited significantly in both species of earthworms. | Velki and Hackenberger 2012 |
| Chlorpyrifos (OPP) | 0.6, 3, 15 mgkg− 1 dry wt. | Lumbricus terrestris | Acute (72 h) | Esterase activities | - | ChE activity was significantly inhibited (upto 87%) at 48 h in all the concentrations used. AChE activity is not significantly inhibited. | Morcillo et al. 2013 |
| Dimethoate, and trichlorfon (OPP) | Dimethoate- 5, 11, 25, 57, 128 mgkg− 1; Trichlorfon- 33, 50, 75, 113, 169, 253 mgkg− 1 | E. fetida | Acute (14 day) | Histopathological examinations and biomarker analysis- cholinesterase (ChE), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activities. | Cholinesterase is an enzyme which is required in functioning of nervous system, it break down acetylcholine. LDH is a glycolytic enzyme found in nearly all tissues of earthworm. | The ChE and LDH activities are significantly inhibited by pesticides exposure at lower and higher concentrations. Both pesticides resulted in significant decrease in ALP activity in E. fetida. Histopathological assessment revealed that higher concentrations of both pesticides seriously damaged the longitudinal and circular muscular layers and affected epidermis. | Rico et al. 2016 |
| Dinotefuran (neonicotinoid) | Acute- 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 mgkg− 1; Subchronic- 0.1, 0.5, 1 and 2 mgkg-1 | E. fetida | Acute (14 day) and subchronic (28 day) | Antioxidant enzymes- superoxide dismutase (SOD), catalase (CAT), ROS (OH− radical), lipid peroxidation, DNA damage, Gene expression | OH− radical (ROS) play a major role in mediating O2 toxicity (in vivo). SOD is an effective O2− radicle remover. CAT enzyme is an important H2O2 scavenger. Lipid peroxidation is a process which disrupts cellular functions and was considered as a cell damaging process. | SOD activity increased with increasing pesticide concentrations. Higher concentrations enhanced oxidative stress inside E. fetida cell, as a result CAT activity also enhanced with time. The higher doses of dinotefuran induced lipid peroxidation in E. fetida. At 14th and 28th day 8-OHdG content has increased indicating that higher concentrations induced DNA damage. | Liu et al. 2018 |
| Chlorpyrifos (OPP) | 0, 20, 40, 80, 160, 320 mgkg− 1 | E. andrei | Subchronic (28 day) | Acetyl cholinesterase activity (AChE), Glutathione-S-transferase (GST), CAT activity, and MDA content | Alteration in MDA content and antioxidant enzyme activity show the degree of cell injury inside earthworms. | There is a significant reduction in AChE activity caused by chlorpyrifos exposure, which implicates the neurotoxicity of the pesticide. MDA content, CAT, and GST activities are decreased with increasing chlorpyrifos concentrations. | García-Gómez et al. 2019 |
In last few decades, chlorpyrifos (O, O -diethyl O-3, 5, 6-trichloro-2-pyridyl phosphorothioate) and dimethoate (O, O-dimethyl S-methylcarbamoylmethyl phosphorodithioate) insecticides are massively used in agriculture (Anwar et al. 2009; Dasgupta et al. 2012; Dong et al. 2020; Martinuzzi et al. 2020). Both OPP insecticides are non-persistent in nature and possess a lower half-life in environment. The half-life of dimethoate ranges from 4 to 16 days and that of chlorpyrifos ranges from 7 to 120 days in environment (Anwar et al. 2009; Srivastava et al. 2010; Yadav et al. 2020). In most of the European countries as well as in USA, chlorpyrifos and dimethoate pesticides are banned from the past few years (López-Dávila et al. 2021; Yadav et al. 2021).
As per our extensive literature review, we found that there is plenty of research works which comprises of avoidance response, acute and chronic toxicity of OPP pesticides on earthworms. In this study we have tried for a wholesome approach to provide a comparative study about the short and long term effects of two organophosphorus (OPP) insecticides of different persistence (half-lives) time on earthworms.
2.1. Procurement of earthworms
Earthworms were collected from vermicomposting unit of Institute of Environmental and Sustainable Development, Banaras Hindu University, India. The earthworm species, Eisenia fetida was chosen for the study because it was robust, easy to control and easily available. The specimens were brought to the laboratory 2 weeks prior to experiment in culture pots supplemented with finely ground soil and cow-dung (preconditioned) mixed in the ratio of 1:1 (Dasgupta et al. 2012). The culture pots were covered with lid (fine meshed iron net) and kept inside the BOD incubators at 28 ± 1°C and an optimal moisture content (50–60% moisture level) was maintained by adding double distilled water (DDW) into the culture medium (Dasgupta et al. 2012). Only the adult (having clitellum) and healthy earthworms of weight ranging between 400–500 mg were selected for the toxicity tests. Before the commencement of experiments, the earthworms were removed carefully from the culture, and rinsed with DDW, after that placed on blotting sheets to remove soil and litter particles. After surface cleaning the worms then placed in moistened filter paper for next 12 hours (h) to clear the food content present in gut of earthworms.
2.2. Procurement of chemicals and experimental containers
The insecticides used in the experiments were OPP insecticides, i.e. chlorpyrifos and dimethoate. Insecticides and acetone were of analytical reagent grade (98% purity) was ordered from Sigma-Aldrich, India. Plastic containers of two different size selected for the experiments, big containers (dimensions are 25cm × 17cm × 8cm) are subjected to avoidance response, acute and chronic toxicity assay and small containers (dimensions are 8cm × 4cm × 3cm) are selected for cocoon and hatchling study.
2.3. Test soil preparation and pesticide spiking
The upper layer soil (5cm) with no pesticide application history was used in experiments which were collected from the field of Institute of Environmental and Sustainable Development, Banaras Hindu University, India. 25 days preconditioned cow dung (CD) was procured from agricultural farm, Institute of Agricultural Sciences (IAS), BHU. The collected soil and CD were screened manually and air dried under shadow. After drying passed through a 10-mesh (2mm) sieve and stored for further use. The test soil (TS) was prepared by mixing sieved soil and CD in 3:1 ratio (Kavitha et al. 2020). General physico-chemical properties of prepared TS, was measured, pH and EC (electrical conductivity) was analyzed using water parametric analysis kit (Model 371, systronics, India), percent nitrogen using kjeldahl method (Khjeltron, tulin, India), and organic carbon was done by Walkley and Black method (Walkley and Black 1934). The physico-chemical characteristics of TS used in this study were mentioned in Table 2.
For desired concentrations, a particular amount of insecticides (active ingredient, a.i.) was dissolved in 10 ml of acetone (solvent). The dissolved solution was spiked and mixed thoroughly in 100 gm of dry TS. The spiked TS was placed overnight (12 h) under an exhaust hood to evaporate the solvent and then mixed meticulously with the desired amount of TS according to the different experimental assay. Similarly, the control TS was prepared by mixing only 10 ml acetone without insecticides.
|
Characteristic |
Value |
|---|---|
|
pH |
7.4 ± 0.2 |
|
EC (mS cm− 1) |
1.27 ± 0.23 |
|
Organic carbon (%) |
1.82 ± 0.30 |
|
Organic matter (%) |
3.13 ± 0.52 |
|
Nitrogen (%) |
0.38 ± 0.10 |
|
Soil texture |
Sandy loam (sand 57%, silt 20%, and clay 20%) |
2.4. Avoidance response test
Avoidance response test was designed following the recommendations of the ISO protocol (ISO 17512-1, 2008). The concentrations of insecticides used in the avoidance response test are mentioned in Table 3. Rectangular plastic containers (25cm × 17cm × 8cm) were divided into two equal sections with the help of a removable plexiglass divider. One half of the container was filled with 0.5 kg of insecticide spiked TS and another half was filled with 0.5 kg control TS (Bernardino et al. 2021). Then the plexiglass was removed, and 10 healthy earthworms with well-developed clitellum (weighing between 400 to 500 mg) were placed on the separating line of each test containers. To prevent the earthworms escaping from containers, it was covered with perforated plastic lid which was permeable to light and air. Then, the test containers were incubated for 48 h at 23 ± 2°C under continuous light exposure (Light intensity: 700–800 lux).
|
Active ingredient |
Tested concentrations (mg kg− 1 dry soil of a.i.) |
||
|---|---|---|---|
|
Avoidance response test |
Acute test |
Chronic toxicity test |
|
|
Dimethoate |
1; 5; 10; 25; 50 |
1; 5; 10; 15; 20; 50; 100 |
1; 5; 10; 15; 20 |
|
Chlorpyrifos |
5; 10; 25; 50; 100 |
5; 10; 25; 50; 100; 175; 200; 300 |
5; 10; 25; 50; 100 |
After fulfillment of incubation period, the control and spiked TS were separated by introducing plexiglass divider and the number of alive earthworms was counted throughout each section. Earthworms which are getting divided due to introduction of plexiglass divider were counted as 0.5 independently to the remaining body length. Each insecticide was tested at varying concentrations with four replicates. To guarantee that the earthworms were distributed randomly throughout the container, a double-control (control-control) test was carried out in which control TS was placed in both compartments, in four replicates (Bernardino et al. 2021). The simplified experimental set up is explained diagrammatically in Fig. 2.
After fulfillment of 48 h of incubation period the avoidance response (%) was calculated following Rico et al. (2016), using the following equation:
Avoidance response (%) = [ \(\frac{(\mathbf{C}-\mathbf{T})}{\mathbf{N}}\times 100\)]
Where, C is the numbers of earthworm present in the control TS containing section, T is the number of earthworm present in spiked TS containing section, and N is the total number of earthworms introduced in the test container. A positive (+ ve) value indicates avoidance of earthworms to spiked TS, a zero (0) represents no avoidance, and a negative (-ve) value indicates an attraction of earthworms towards spiked TS (Amorim et al. 2005).
2.5. Acute toxicity
The acute toxicity test was designed by following the protocol of ISO 11268-1 (ISO, 2012a). The TS selected for the acute toxicity test consists of sieved soil and cow dung in 3:1 ratio, for maintaining the same condition as agricultural fields (Kavitha et al., 2020). The information about the selected concentrations of insecticides for acute toxicity assay were detailed in Table 3. Rectangular plastic containers (25cm × 17cm × 8cm) were used for the acute toxicity test. Each container contains 0.5kg spiked TS (60% soil moisture) and 10 adult earthworms (with well-developed clitellum ranging between 400–500 mg weights). Additionally, a control with no pesticide spike was carried out. To prevent the worms from escaping, containers were covered with perforated plastic lid. The containers were kept in an incubation chamber (23 ± 1°C, 70–90% relative humidity with continuous illumination of 500–700 lux) throughout the 14 days of exposure (14 DOE). Three replicates were used for each dose and control.
After 14th DOE the number of dead earthworms is counted in each experimental container and mortality at various concentrations were determined. Earthworms were considered as dead if they don’t move and did not respond to any external stimulus. Because earthworms disintegrate very fast after death, they were considered dead if missing (Yang et al. 2015). The LC50 values were calculated in the study to compare the acute lethal effects of chlorpyrifos and dimethoate separately on earthworms. For the calculation of LC50 probit regression modeling was used (Shi et al. 2007; ISO 2012a). The test endpoint was mortality.
2.6. Chronic toxicity
2.6.1. Determination of growth inhibition (GIt) and net biomass gain per gram feed (NBGPGFT)
For determining the chronic effects of chlorpyrifos and dimethoate on growth attributes of E. fetida, bioassays were installed same as in the acute toxicity test, differed only in the experimental duration, concentrations used and assessment methods. According to acute toxicity test results, 5 sub-lethal concentrations of chlorpyrifos and dimethoate were selected for chronic toxicity test (Table 3). Ten adult earthworms (with developed clitellum) weighing between 400–500 mg were taken from the cultured earthworm stock. For voiding the gut content earthworms were placed on damp filter paper inside petri dishes and put in the dark at 23 ± 1°C. Before introduction, the earthworms were rinsed with DDW, blotted dry on filter paper and biomass of 10 earthworms was determined by weighing. Then they were released onto the soil surface of each experimental container containing 0.5kg of TS either spiked or control (feed matter). The test containers were maintained at 23 ± 1°C inside an incubator and to prevent earthworms from starvation a total of 5 g (dry weight) of finely sieved cow dung (moistened to 60% w/w) was added each week on the TS upper surface. Moisture loss from the test soil was checked by weighing the test containers at the weekly intervals and replenished by adding DDW if needed. Bioassay run for 56 DOE with three replicates used for each dose and control groups. The earthworms were weighed on the 0th, 7th, 14th, 21st, 28th, 35th, 42nd, 49th and 56th DOE to determine the change in biomass and weekly growth rates.
Mosleh et al. (2003) found growth rates as important biomarkers of toxicity of pesticides (endosulfan and aldicarb) to Lumbricus terrestris. The GIt was analyzed according to the increase or decrease in biomass of earthworms in each dose group at different exposure periods. The GIt was calculated following Shi et al., (2007), using this equation:
Where GIt is the growth inhibition percentage for the dose or control groups at tth DOE, B0 is the mean initial biomass of earthworms at start of the experiment and Wt is the mean biomass of earthworms at time, t (DOE). Positive value of GIn indicates growth inhibition of earthworms due to experimental exposure and negative value represents increase in growth of earthworms.
For calculation of earthworm net biomass gain per gram feed (NBGPGFT) at time (T), a formula was designed which is as follows:
Where, BT is mean biomass of individuals at a time (T) (in mg), Bi is mean initial biomass of individuals (in mg), W is the total weight of feed mixture (in gram) and N is the total number of individuals. Using this formula, the net biomass gain of earthworms per gram feed at 28th DOE (NBGPGF28 DOE) and 56th DOE (NBGPGF56 DOE) were calculated.
2.6.2. Determination of cocoon and hatchling production
Reproduction tests with E. fetida were carried out in accordance to the ISO 11268-2 guideline (ISO 2012b). The cocoon production determines the reproductive success of test specimens. For determination of cocoon and hatchling production, the spiked and control TS were examined carefully under a magnifying glass, every week throughout 56 DOE of chronic toxicity experiment. After every week cocoons and hatchlings were hand sorted and transferred in small experimental containers (8cm × 4cm × 3cm) containing same TS. At the last day of experiment, the survived hatchlings were rinsed with DDW, blotted with blotting paper, then weighted and biomass per hatchling were calculated.
2.7. Statistical significance
Statistical analysis was performed using the SPSS (version 16) software for windows program. Treatments were compared using analysis of variance (ANOVA) and Duncan’s multiple ranged tests (DMRT) to test the significance of difference (at P < 0.05) between the treatments. The graphs were drawn using Sigma Plot version 10 software.
2.8. Chemicals and quality control
All the chemicals used in this study were of analytical grades and used without further purification. The analytical quality control was guaranteed through the use of laboratory quality assurance and quality control protocols and standards. Standard operating procedures were followed.
3.1. Avoidance response test
Avoidance response test is relatively a recent technique in terrestrial eco-toxicological assessments, offering rapid and cost-effective analysis of pesticide contamination in soil (Garcia et al. 2008; Bernardino et al. 2021). The data generated from avoidance response tests are helpful in defining acceptable concentration of pesticides in soil (Nunes and Espíndola 2012; Bernardino et al. 2021). In our study, the results of the test performed with the double control TS (control–control) showed that E. fetida were randomly distributed among both soil compartments. A significant avoidance response showed by E. fetida in TS spiked with higher concentrations of insecticides, except first two low concentrations. At first two low concentrations of insecticides, more earthworms were found in compartment containing spiked TS (Figs. 3 and 4). Apparently, E. fetida preferred the first two low concentrations of insecticides chlorpyrifos and dimethoate.
On the highest concentration (50 mg kg− 1 dry TS) of dimethoate, an avoidance of 93.33 ± 3.33% was observed. According to ISO guidelines (ISO, 2008), the concentrations that reported avoidance higher than 80% can be concluded as the pesticide concentration which caused reductions in habitat functions in the soil.
In the case of chlorpyrifos a significantly low avoidance response was reported as compared to dimethoate. At the highest concentration (100 mg kg− 1 dry TS) of chlorpyrifos, an avoidance of 60% was reported. Besides the higher concentrations (upto 100 mg kg− 1 dry TS) of chlorpyrifos used in the avoidance response study, E. fetida reacted more sensitively in dimethoate spiked TS than in chlorpyrifos spiked TS. The avoidance response for different earthworms varies with the differences in type and composition of feed matter and pesticide (Table 4).
| Test medium | Pesticide used | Concentrations | Earthworm Species | Avoidance response | Reference |
|---|---|---|---|---|---|
| OECD artificial soil | Chlorpyrifos (OPP) | 0, 5, 10, 20, 40, 60 mgkg− 1 | E. fetida | A significant avoidance response by E. fetida was found in last two higher concentrations. | Zhou et al. 2007 |
| OECD artificial soil | Dimethoate (OPP) | 1, 3, 10, 100, 300, 900 mgkg− 1 dry soil | E. andrei | There was no significant avoidance was shown in first two concentrations. At 30 mgkg− 1 dimethoate there is 35% avoidance was shown and it increased to 85% at 100 mgkg− 1. | De Silva and Amarasinghe 2008 |
| OECD artificial soil | Lambda-cyhalothrin (pyrethroid) | 0.32, 1, 3.16, 10, 31.6, 100 mgkg− 1 dry soil | E. fetida | E. fetida started avoiding Lambda-cyhalothrin from 3.16 mgkg− 1 concentration and showed 100% avoidance at pesticide concentration 10 mgkg− 1 dry OECD artificial soil. | Garcia et al. 2008 |
| Tropical artificial soil (TAS) | Lambda-cyhalothrin | 0.32, 1, 3.16, 10, 31.6, 100 mgkg− 1 dry soil | E. fetida | In TAS E. fetida started avoidance from the first lowest concentration. More than 60% avoidance reported at 0.32 mgkg− 1 concentration and it increased to 80% at 3.16 mgkg− 1. An avoidance of 100% reported at 31.6 mgkg− 1. | Garcia et al. 2008 |
| The European natural field soil (LUFA 2.2) | Lambda-cyhalothrin | 0.32, 1, 3.16, 10, 31.6, 100 mgkg− 1 dry soil | E. fetida | In LUFA soil, an avoidance of 40% is reported at lowest concentration (1 mgkg− 1), which increased to 70% at 1 mgkg− 1, 80% at 10 mgkg− 1, 90% at 31.6 mgkg− 1 and more than 90% at 100 mgkg− 1. | Garcia et al. 2008 |
| LUFA 2.2 | Methomyl (99.5% purity) | 0.86, 1.37, 2.19, 3.51, 5.62, 8.98, 14.4, 23 mgkg− 1 dry soil | E. andrei | An increasing net avoidance response was showed by E. andrei in response to increasing concentration of methomyl. | Pereira et al. 2010 |
| TAS | Imidacloprid (Gaucho® 600 FS), fipnoril (Standak® 250 SC), thiamtoxam (Cruiser® 350 FS), captan (Captan® 480 SC), carboxim + thiram (Vitavax® 200 SC) | Imidacloprid- 0.125, 0.25, 0.5, 1, 2 mgkg− 1; fipnoril- 0.1, 1.25, 2.5, 5, 10 mgkg− 1; thiamtoxam- 1.25, 2.5, 5, 10, 20 mgkg− 1; captan- 0.625, 1.87, 5.62, 16.87, 50.62 mgkg− 1; carboxim + thiram- 0.313, 1.25, 5, 20, 80 mgkg− 1 dry soil | E. andrei | There is no sign of avoidance shown towards fipnoril concentrations used in study, as earthworms are attracted towards this pesticide. Significantly higher avoidance was shown at lower concentrations of Imidacloprid, 100% at 1 mgkg− 1. An avoidance of 80% was shown at 50.62 mgkg− 1 for captan, 60% at 80 mgkg− 1 carboxim + thiram, and 20% at 10 mgkg− 1 thiamtoxam concentration. | Alves et al. 2013 |
| Agricultural soil | Chlorpyrifos | 0.6, 3, 15 mgkg− 1 dry soil | Lumbricus terrestris | There is no significant avoidance response was observed in L. terrestris at any concentration of chlorpyrifos used in study. | Morcillo et al. 2013 |
3.2. Acute toxicity test
Acute toxicity test has been widely used to evaluate the short-term toxicity of pesticides to earthworms (Zhou et al. 2007; Cang et al. 2017; García-Gómez et al. 2019). Earthworm’s mortality can be used as a predictable indicator of environmental pollution (Shi et al. 2007). The most probable end point measured when evaluating acute toxicity of chemicals to earthworms was LC50 (the concentration that is lethal to 50% of individuals of a population). The endpoint “LC50” shows effects at relatively high pesticide concentrations and indicates the maximum casualty likely to earthworms. According to Mosleh (2009), those pesticides which have LC50 value higher than 1000 mg kg− 1 were safe and harmless to earthworms in the agricultural field.
| Country | Test medium | Pesticide used | Concentrations | Earthworm Species | LC50 value | Response | Reference |
|---|---|---|---|---|---|---|---|
| The Netherlands | OECD artificial soil (characteristics- 70% fine sand, 20% kaolinite clay and 10% ground sphagnum peat), Clayey soil, Humus soil | Dimethoate (97–99% purity) | 0, 3, 9, 27, 81, and 243 mgkg− 1 | Aporrectodea caliginosa tuberculata | In OECD artificial soil- 56 mgkg− 1; Clayey soil- 40 mgkg− 1, and Humus soil- 65 mgkg− 1 | The biomass changes and LC50 values are found different in different soil type. The soil CO2 production was found increased with increasing dimethoate concentration. | Martikainen 1996 |
| India | Black soil and Cow dung (3:1) | Dimethoate (Rogor 30 EC) | 0.4, 1.6 mgkg− 1 dry soil | E. fetida | - | Application of dimethoate showed negative impact on biomass and reproductive output of earthworms. | Yasmin and D’Souza. 2007 |
| China | OECD artificial soil | Lindane and deltamethrin | Lindane- 20, 50, 80, 120, 150 mgkg− 1; Deltamethrin- 5, 25, 50, 100, 150 mgkg− 1 dry soil | E. fetida | Lindane- 162.1 mgkg− 1; Deltamethrin- 432.9 mgkg− 1 (14 day) | Both the pesticides exerted significant effects on cellulase activity and growth of E. fetida. | Shi et al. 2007 |
| China | OECD artificial soil | Chlorpyrifos | 75, 90, 100, 125, 150, 200 mgkg− 1 dry soil | E. fetida | At 14 day- 118.5 mgkg− 1, and 21 day- 91.78 mgkg− 1 | The biomass of E. fetida was significantly decreased with increasing concentration of chlorpyrifos. The decrease in biomass, cocoon number, and cocoon viability is seen to be dose dependent. | Zhou et al. 2007 |
| Germany | OECD artificial soil, Tropical artificial soil (TAS), The European natural field soil (LUFA 2.2) | Lambda-cyhalothrin | 0.32, 1, 3.16,10, 31.6, 100 mgkg− 1 dry soil | E. fetida | In OECD soil- 99.8 mgkg− 1, TAS- 23.9 mgkg− 1, and LUFA- 139.9 mgkg− 1. | Different soil composition resulted in different LC50 values. | Garcia et al. 2008 |
| Germany | OECD artificial soil, TAS, LUFA 2.2 | Carbendazim | 1, 3.2, 10, 31.6, 100, 316, 1000 mgkg− 1 dry soil | E. fetida | In OECD soil- 5.8 mgkg− 1, TAS- >1000 mgkg− 1, and LUFA- 4.1 mgkg− 1. | Different soil composition resulted in different LC50 values. | Garcia et al. 2008 |
| Germany | OECD artificial soil, TAS, LUFA 2.2 | Benomyl | 1, 3.2, 10, 31.6, 100, 316, 1000 mgkg− 1 dry soil | E. fetida | In OECD soil- 22 mgkg− 1, TAS- 633 mgkg− 1, and LUFA- 14.6 mgkg− 1. | Different values of LC50 in different soil type resulted due to different composition and characteristics of soil. | Garcia et al. 2008 |
| India | Grassland soil and FYM (1:1) | Chlorpyrifos (20 EC) | 5.75, 11.5, 17.25, 23 mgkg− 1 dry soil | E. fetida | 28.58 mg kg− 1 (96 h) | Chlorpyrifos found moderately toxic to E. fetida and environmentally less hazardous than other insecticides such as, endosulfan, carbamates, and cypermethrin. | Gupta et al. 2011 |
| India | Grassland soil and FYM (1:1) | Chlorpyrifos (20 EC) | 0.91, 1.82, 3.65 mgkg− 1 dry soil | Perionyx excavats (Perrier) | 7.3 mgkg− 1 | Highest concentration of chlorpyrifos rendered 24.84% hatching reduction. A slight decrease in cocoon number, hatching success and body weight reported with no mortality of earthworms. | Dasgupta et al. 2012 |
| China | Filter paper test | Chlorpyrifos (97% pure) | 0.1, 1.0, 10, 100, 1000 µg cm− 2 | E. fetida | 14.19 µg cm− 2 (48 h) | - | Wang et al. 2012 b |
| China | OECD artificial soil | Chlorpyrifos (97% pure) | 0.1, 1.0, 10, 100, 1000 mgkg− 1 dry soil | E. fetida | 7-day (421.3 mg kg− 1) and 14-day (384.9 mg kg− 1) | The LC50 value of chlorpyrifos showed that it is less toxic to the earthworms in soil as compared to other pesticides. | Wang et al. 2012 b |
| Croatia | Filter paper test | Dimethoate (Chromgor 40) | 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 25, 50 µg cm− 2 | Octolasion lacteum and E. andrei | O. lacteum (1.98 ± 0.25 µg cm− 2) and E. Andrei (10.36 ± 1.39 µg cm− 2) | - | Velki and Hackenberger 2012 |
| Brazil | TAS | Imidacloprid (Gaucho® 600 FS), fipnoril (Standak® 250 SC), thiamtoxam (Cruiser® 350 FS), captan (Captan® 480 SC), carboxim + thiram (Vitavax® 200 SC) | Imidacloprid- 6.25, 12.5, 25, 50, 100 mgkg− 1; fipnoril-62.5,125,250, 500, 1000 mgkg− 1; thiamtoxam- 62.5,125,250, 500, 1000 mgkg− 1; captan- 62.5,125,250, 500, 1000 mgkg− 1; carboxim + thiram- 62.5,125,250, 500, 1000 mgkg− 1 | E. andrei | Imidacloprid- 25.53 mgkg− 1, fipnoril- >1000 mgkg− 1, thiamtoxam- >1000 mgkg− 1, captan- >1000 mgkg− 1, carboxim + thiram- >1000 mgkg− 1 | At tested concentrations only imidacloprid caused mortality in earthworms. Except fipronil, all other pesticides induced negative effect on E. andrei in chronic toxicity and avoidance test. | Alves et al. 2013 |
| India | Laterite type soil and Cow dung (9:1) | Dimethoate (Rogor 30% E.C.) | 1,2,3,5,7,9,11,13,15,17 mgkg− 1 dry soil | Drawida wilsi (Michaelsen) | LC50 for adult, immature and juvenile worms were 9.0, 6.7, and 5.5 mgkg− 1 (96 h) | Mortality started at 3 mgkg− 1 and 100% mortality was achieved at 17 mgkg− 1 dry soil. | Bhattacharya and Sahu. 2013 |
| Spain | OECD artificial soil | Dimethoate (Citan 40 EC) | 5, 11, 25, 57, 128 mgkg− 1 dry soil | E. fetida | 28 mg kg− 1 (14 day) | The insecticide dimethoate showed higher toxicity than other tested insecticides such as, prochloraz, trichlorfon, and tebuconazole. | Rico et al. 2016 |
| China | Filter paper contact test | Chlorpyrifos (96% technical grade) | - | E. fetida | LC50 at 24 h and 48 h were 5654 mg a.i. L− 1 and 1015 mg a.i. L− 1 respectively | Based on 24 h LC50 results authors conclude that chlorpyrifos was 968.2 times less toxic than insecticide imidacloprid. | Cang et al. 2017 |
| China | OECD artificial soil | Chlorpyrifos (96% technical grade) | - | E. fetida | At 7-day (421.3 mgkg− 1) and 14-day (384.9 mgkg− 1) | Based on 14 day LC50 results authors conclude that chlorpyrifos was 137.5 times less toxic than imidacloprid. | Cang et al. 2017 |
| Spain | Agricultural soil | Chlorpyrifos (FOSTAN, a commercial product containing 480 gL− 1) | 20, 40, 80, 160 or 320 mgkg− 1 soil | E. fetida | 148 mgkg− 1 dry soil | Chlorpyrifos resulted in reduction of acetylcholinesterase (AChE) activity, which revealed the neurotoxicity of this insecticide. | García-Gómez et al. 2019 |
In present study, the toxicity of dimethoate on E. fetida after 14 DOE was higher than chlorpyrifos. The LC50 (14 DOE) of dimethoate and chlorpyrifos were 17.45 and 228.06 mg kg− 1 dry TS, respectively. Dimethoate caused physiological and morphological alterations in earthworms at higher concentrations (15 and 20 mg kg− 1 dry TS), surviving worms had ruptured skin surface, deformed body and decreased body weight. Rico et al. (2016), found that high concentration of dimethoate and trichlorfon insecticides resulted in internal impairment and some serious damage to longitudinal and circular muscle-layers of E. fetida. Saxena et al. (2014), reported the disintegration of muscle layers, epidermal cells, cuticular membrane thus leading to bleeding, fragmentation and loss in typical architecture of body wall in earthworms Metaphire posthuma and E. fetida exposed to higher concentration of pesticides, carbofuran, carbaryl, fenvalerate and cypermethrin. The calculated LC50 values in this acute toxicity test were not similar to those reported elsewhere in the literature. As Rico et al. (2016), reported LC50 value of 28 mg kg− 1 after 14 DOE to dimethoate in OECD soil. Wang et al. (2012 b), reported LC50 (14 DOE) of 384.9 mg kg− 1, whereas Zhou et al. (2007), reported LC50 (14 DOE) of 118.5 mg kg− 1 for chlorpyrifos using E. fetida in OECD soil. According to De Silva and Amarasinghe (2008), dimethoate became more toxic for earthworms in natural soils rather than OECD soil. In the study by Zhang et al. (1985), feed matter is positively correlated with the LC50 value of pesticides. Previous studies give a clear insight that the LC50 values varies with the type and composition of feed matter, pesticides and earthworm species (Table 5). Difference observed in the LC50 values in our study and previous studies may be because of the difference in composition of TS (feed matter) used.
3.3. Chronic toxicity test
3.3.1. GIt
GIt of earthworms at 7th, 14th, 21st, 28th , 35th, 42nd, 49th and 56th DOE for dimethoate and chlorpyrifos were shown in Figs. 4 and 5. For controls, the GIt value was negative, with mean earthworms biomass increasing throughout the exposure period 0 to 56th DOE. Negative values of GIt indicates the increase in growth of earthworm biomass (Shi et al. 2007).
GIt of the earthworms exposed to dimethoate spiked TS were significantly higher than chlorpyrifos spiked TS as well as controls (Figs. 5 and 6). For concentrations upto 5 mg kg− 1 dry TS of dimethoate, negative value of GIt was observed. This indicates that exposure of concentration level upto 5 mg kg− 1 dry soil of dimethoate in agricultural field soils was safer for the biomass growth of E. fetida. The GIt started at 10 mg kg− 1 dry TS of dimethoate was observed with a significant increase till 35th DOE. A significantly higher GIt was reported at last two concentrations of dimethoate i.e., 15 and 20 mg kg− 1 dry TS (Fig. 5). This sharp decrease in biomass might be due to the higher toxicity, residual nature of pesticides and decrease in feed matter with time. The GIt at 20 mg kg− 1 dry TS of dimethoate found to be highly harmful for the biomass growth, this may be due to its lower LC50 value (17.45 mg kg− 1 dry TS) observed in the study. Reduction in growth of E. fetida at sub-lethal concentrations of insecticides has been observed when exposed to carbendazim, glyphosphate and dimethoate (Yasmin and D’souza. 2007).
In our study, chlorpyrifos was found capable of retarding the growth of E. fetida only at two highest concentrations i.e. 50 and 100 mg kg− 1 dry TS (Fig. 6). GIt at concentrations upto 25 mg kg− 1 dry TS was observed to be negative indicating increase in biomass. The growth was observed reducing manner upto 28th DOE at 50 mg kg− 1 dry TS, and followed an increase from 35th to 56th DOE. Biomass changes can be a good indicator of chemical stress, which can be linked to the chemical effects in energy dynamics (Rico et al. 2016). The slight increase in biomass of control earthworms suggested that the soil nutrients were just sufficient to sustain the survival of earthworms (Fig. 7). Dimethoate produced most severe effects on the growth after exposure to concentration greater than 5 mg kg− 1 dry TS, while earthworms affected least after exposure to 50 mg kg− 1 dry TS of chlorpyrifos. Gupta et al. (2010), also reported that chlorpyrifos and monocrotophos were moderately toxic to E. fetida and environmentally less hazardous than the carbamates, endosulfan and cypermethrin. Reductions in growth of earthworms by sub-lethal concentrations of insecticides have been observed in E. fetida exposed to carbendazim, glyphosphate and dimethoate (Yasmin and D’Souza., 2007). Changes in biomass through progression of experimental duration for both insecticides have shown in Fig. 7.
3.3.2. NBGPGFT
A significant reduction in NBGPGFT was found in earthworms feeding on TS spiked with dimethoate (Fig. 8A). The negative values depict the loss in NBGPGFT due to the exposure to higher concentration of insecticides. A sharp reduction was observed in the last three concentrations i.e. 10, 15 and 20 mg kg− 1 dry TS, with negative values of NBGPGFT. This sharp decrease in NBGPGFT indicate the extent of toxicity of dimethoate to E. fetida.
NBGPGF56 DOE was found greater than NBGPGF28 DOE for concentrations upto 10 mg kg− 1 dry TS and 50 mg kg− 1 dry TS for dimethoate and chlorpyrifos, respectively. A sharp decrease in NBGPGFT was observed in last two sub-lethal concentrations of chlorpyrifos (50 and 100 mg kg− 1 dry TS) (Fig. 8B). The decrease in NBGPGFT is found to be dose dependent for both insecticides. Garg and Gupta (2011) and Daam et al. (2020) reported that the rate of net biomass gain per gram feed in E. fetida was mainly depend on available feed matter (substrate composition and soil quality) and its population density. In the present study it was observed that NBGPGFT of E. fetida was higher in chlorpyrifos in comparison with dimethoate spiked TS.
3.3.3. Cocoon production
The number of cocoons and hatchlings produced in control and different treatments of the two insecticides have been presented in Fig. 9. The cocoon production started from the 14th DOE in both control and dose groups. The number of cocoon at concentration 1 mg kg− 1 dry TS of the dimethoate was approximately equal to that of controls and there was slight increase at 5 mg kg− 1 dry TS as compared to controls. At concentrations above 5 mg kg− 1 dry TS of dimethoate, it was observed that cocoon production was significantly reduced as compared to controls (Fig. 9A). The results indicated that the reproductive performance of E. fetida was significantly affected in concentrations higher than 5 mg kg− 1 dry TS dimethoate. The reduction in cocoon production is found to be dose dependent. The cocoon production was negatively affected in the last two concentrations i.e. 15 and 20 mg kg− 1 dry TS.
In case of chlorpyrifos, there was a reducing trend observed in the cocoon production with increasing concentration (Fig. 9B). The cocoon production started from the 2nd week of DOE as same in dimethoate treatment. The mean number of cocoon produced at 5 mg kg− 1 dry TS was found to be near about the controls. This indicates the concentration below 5 mg kg− 1 of chlorpyrifos was safer for reproductive performance of E. fetida. The cocoon production got severely reduced at highest concentration i.e. 100 mg kg− 1 dry TS of chlorpyrifos. A comparatively lower decrease in cocoon number was found at sub-lethal concentrations of chlorpyrifos than dimethoate. However, the selected concentrations of dimethoate (upto 20 mg kg− 1 dry TS) was lower than the chlorpyrifos (upto 100 mg kg− 1 dry TS), this indicates that dimethoate is more harmful for E. fetida than chlorpyrifos at sub-lethal concentrations.
3.3.4. Hatchling production
Hatchling production is adversely affected with increasing concentrations for both insecticides, while dimethoate produced the most severe effects on the hatchling production of E. fetida. A significant decrease was observed in hatchlings production at the lowest concentration (1 mg kg− 1 dry TS) of dimethoate as compared to controls (Fig. 10A). Average weight of hatchlings was significantly decreased with increasing concentration of dimethoate as compared to control (Fig. 10A). The results suggests that reproductive performance of E. fetida was negatively influenced even at the lowest concentration of dimethoate. In case of chlorpyrifos, there was slight decrease in hatchling production with increase in concentration (Fig. 10B). Major reduction in hatchling production was found at the concentration (100 mg kg− 1 dry TS). At 5 and 10 mg kg− 1 dry TS, the average weight of hatchlings was almost equal to controls. Whereas, at higher concentrations i.e. 25, 50 and 100 mg kg− 1 dry TS, there was slight decrease in the average weight of hatchlings as compared to controls.
The obtained results suggest that dimethoate is more lethal than chlorpyrifos for E. fetida. This might be due to the high stress of dimethoate than chlorpyrifos on reproductive system of E. fetida. Reports from earlier studies have shown effects on reproductive system, histological disorders in spermatheca, alteration of cell proliferation in seminal vesicle and DNA fragmentation in spermatogonia as a result of sub-lethal toxicity of OPP insecticides causing anomalous reproduction in E. fetida (Bustos-Obregón and Goicochea 2002; Espinoza-Navarro and Bustos‐Obregón 2005; Zou et al. 2018; Zhao et al. 2021).
Our study provides a clear insight of different antagonistic effects of two OPP insecticides with different half-life. The avoidance response at highest concentrations of dimethoate (50 mg kg− 1 dry TS) was 93.33 ± 3.33% and for chlorpyrifos (100 mg kg− 1 dry TS) it was 60%. The resulted LC50 values at 14 DOE for dimethoate and chlorpyrifos was 17.45 and 228.06 mg kg− 1 dry TS, respectively. Acute toxicity experiments show the level of toxicity of dimethoate is approximately 13 times higher than that of chlorpyrifos. A significantly higher GIt was reported at 10, 15 and 20 mg kg− 1 dry TS concentration of dimethoate and for chlorpyrifos the growth retarded at two highest concentrations i.e. 50 and 100 mg kg− 1 dry TS. The results of cocoon and hatchlings production strongly suggests that even through the population density not immediately affected on exposures to sub-lethal concentrations but prolonged exposure might result in reproductive changes that ultimately reduces population density of E. fetida. In comparison the chlorpyrifos was safer than dimethoate at sub-lethal concentrations for the development and reproduction of E. fetida.
Acknowledgement
The authors are grateful to the Dean & Head, Department of Environment and Sustainable Development and Director, Institute of Environment and Sustainable Development, Banaras Hindu University, for providing institutional support. Rajeev Pratap Singh is grateful to the authorities of Banaras Hindu University, Varanasi for providing support under the Institute of Excellence (IOE) scheme under Dev scheme No. 6031.
Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical Approval: Not Applicable
Consent to Participate: Not Applicable
Consent to Publish: Not Applicable
Data Availability: All data generated or analyzed during this study are included in this published article.
CRediT roles
Vaibhav Srivastava: Conceptualization, visualization, data analysis, illustrations, writing original draft, review & editing
Sinha Sahab: Visualization, investigation, data analysis, illustrations, writing original draft, review & editing
Rajeev Pratap Singh: Conceptualization, visualization, review & editing, resources, supervision, funding acquisition
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