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Research Article
Life Sciences

Impact of application of insecticide quercetin/azadirachtin and chlorpyrifos on earthworm activities in experimental soils in Uttar Pradesh India

Pradeep Kumar1, Tunira Bhadauria2, Jagadish Mishra1


Intensive agriculture is practiced in Uttar Pradesh and wide varieties of insecticides are used on cropland. Insecticides pose serious threats to the natural ecosystem and non-target (earthworms) animals. Therefore, in the present study earthworms (Eutyphoeus orientalis) were used to assess if the insecticide (quercetin/azadirachtin) application is conducive to their survivability, reproductive behavior, and casting activity, and this was compared to the ecotoxicological effect of chlorpyrifos (organophosphate (OP) pesticide) and to the control untreated soils. For determination of earthworm’s toxicity test forty five earthen pots were used and endemic earthworm E. orientalis, were exposed to varying concentrations (9.3, 7.1 and 4.7 ml kg-1 of soil) of insecticide (quercetin/azadirachtin) and to (3.2, 2.6 and 1.6 ml kg-1 of soil) chlorpyrifos concentrations. The study carried over a period of 42 days showed that E. orientalis in insecticide (quercetin/azadirachtin) treated soil was positively impacted and showed higher survivability rate and biomass, higher fecundity and hatching pattern and increased casting activity and improved C:N ratio, whereas the chlorpyrifos spiked soils were more toxic to the earthworms as measured through their higher mortality rate, reduced reproductive potential, and lower cast production. Therefore, from this study it can be concluded that quercetin/azadirachtin insecticide can be a safe and efficient insecticide which improved the activity of E. orientalis and is non toxic to it, and also is environmentally less harmful as it is easily biodegradable.

Keywordsneem, organophosphate, native earthworms, Eutyphoeus orientalis, survivability, reproduction test, cast, carbon, nitrogen, C:N ratio

Author and Article Information

1 Department of Zoology, MD PG College, India
2 Department of Zoology, Feroze Gandhi College, India

RecievedOct 16 2014 Accepted Jan 9 2015 Published Feb 12 2015

CitationKumar P, Bhadauria T, Mishra J (2015) Impact of application of insecticide quercetin/azadirachtin and chlorpyrifos on earthworm activities in experimental soils in Uttar Pradesh India. Science Postprint 1(2): e00044. doi:10.14340/spp.2015.02A0001.

Copyright ©2014 The Authors. Science Postprint published by General Healthcare Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 2.1 Japan (CC BY-NC-ND 2.1 JP) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Funding Pradeep Kumar was financially supported by the University Grants Commission (UGC), New Delhi under the scheme Rajiv Gandhi National Fellowship for the promotion of higher education for Schedule cast/Schedule tribe candidates as a Junior Research Fellow at Dr. RML University Faizabad. The financial support is duly acknowledged.

Competing interest No relevant competing interest weather financial, professional, or personal were disclosed between the authors and with any other parties involved.

Ethics statement No human participants were involved in our experiments, and all studies involving earthworms were conducted according to national and international guidelines for carrying out our research work.
Corresponding authorTunira Bhadauria

Address Feroze Gandhi College Rae Bareli- 229 001, UP, India

E-mail tunira@gmail.com

Peer reviewers Andressa Cristhy Buch1 and Reviewer B
1. Affiliation: University Federal Fluminense


Soil is a dynamic and complex system functioning as habitat for microorganisms, plants, animals and humans 1. Nowadays, insecticides are being used extensively in the control of crop pests, therefore production and consumption of insecticides has greatly increased in recent years, but synthetic insecticides have also caused unprecedented ecological damage, soil and also induced serious health hazard among workers during manufacture, formulation and field application 2. Chlorpyrifos is an organophosphate insecticide, which is acetylcholinesterase (AChE) enzyme inhibitor and is thus directly toxic to the nervous system of organism 3. In order to mitigate the problem arising out of the indiscriminate use of chemical insecticides, biological insecticides derived from the plants can be utilized as alternative control measures 4. These natural insecticides are renewable and preparations as well as application of botanicals by the farmers and the use of plant derivatives for pest control were a common practice in the tropics before the advent of synthetic insecticides 5.

The popularity of the plant products or phytoinsecticides has been increasing day by day because of their biodegradability, least persistence and are expected to be below the toxic level of non-target (earthworms) organisms, as well as being economical and easily available. The suitability of earthworms as bio indicators in soil toxicity is largely due to the fact that they ingest large quantities of the decomposed litter, manure and other organic matter deposited on soil, helping to convert it into rich topsoil 6. Among the natural products, one of the most promising natural chemical compounds is quercetin/azadirachtin, an active compound extracted from the tree Azadirachta indica A. Juss (Meliaceae), popularly known as neem whose antiviral, antifungal, antibacterial, and insecticidal properties have been known for several years 7. India has been traditionally used and now introducing the neem product as a bio insecticide. Earthworms are at the basis of terrestrial food chains. Understanding different mechanism in these organisms is therefore essential for the prediction of potentials food-chain effects of soil contamination. Earthworm toxicity tests are useful tools for terrestrial risk assessment 8, 9 and this study, therefore will provide important ecotoxicological information for native earthworms, as Eutyphoeus orientalis. The earthworms are largest soil fauna, playing an important role in the management of soil fertility, therefore the purpose of this study was to compare the effects of insecticide (quercetin/azadirachtin) on non target organism (earthworms) survivability, reproductive behavior and activity as measured through their cast availability and C:N ratio and compare these effects with those of chemical insecticide (chlorpyrifos) and control untreated soil. The experiment will help us to assess if the insecticide quercetin/azadirachtin actually enhanced the survivability, reproductive behavior and activity of earthworms as measured through their cast availability and C:N ratio.

Materials and Methods

Study Site

Study site is located in the semiarid zone (26°17´37.89˝N–81°12´18.20˝S) in district Raebareli of Uttar Pradesh, India, at an altitude between 8–10 m. The climate is hot and dry, summer extends from May to June (max temp. 42.2ºC and minimum temp. 38ºC) with severe winter extending from November to February (max temp. 25.2ºC and minimum temp. 7.8ºC), rainy season extends from last week of June to October with an average annual rainfall of 60–100 cm 10.

Test materials


The soil (24.1% clay, 20.5% silt, 55.4% sand, pH = 8.7 (1:5 soil:water) was collected from a depth of a top 0–15 cm. From agriculture field in May 2012 before the cropping season to ensure that there was no recent input of pesticides. The soil so collected was air dried, homogenized and sieved (2 mm-mesh). It was stored in plastic bags for further use.

Test animal

Native endogeic species E. orientalis was used for evaluating the ecotoxicological studies. Total 450 mature clitellate earthworms were collected from the natural ecosystem. Average weight of adult earthworms varied between 1.2–1.5 g.

Preparation of neem leaf extract

Quercetin/azadirachtin (neem extract) was prepared according to the protocol of 11. To prepare the extract the known quantity of fresh green neem leaves were taken in a plastic tub and left to soak at room temperature for 24 hours. After 24 hours the soaked leaves were ground and made into a fine paste using the grinder. The grounded leaves were then mixed thoroughly with distilled water in 1:5 leaves-water ratio in a mixer for 10 minutes. The extract was then filtered using filter paper. The extractant so collected was now ready for use. The advantage of using neem leaf extract is that it is available throughout the year. There is no need to boil the extract since boiling reduces the quercetin and azadirachtin content. Therefore the cold extract was used as it is more effective.

Preparation of test

Based on the methodology used by Field Pest Management, Department of State Uttar Pradesh (INDIA) and also by the village farmers who commonly use the said doses in their agriculture fields to control the crop pests three bio and chemical insecticides concentrations were evaluated. The concentration/grades of quercetin/azadirachtin thus used were B1: 9.3 ml kg-1 soil, B2: 7.1 ml kg-1 soil, B3: 4.7 ml kg-1 soil. The insecticide (chlorpyrifos 20% EC) was purchased from RaeBareli, India (M/S JU pesticides and chemicals (P) LTD, Janak Puri New Delhi) and three different concentration/grades of chemical insecticide were C1: 3.2 ml kg-1 soil, C2: 2.6 ml kg-1 soil, and C3: 1.6 ml kg-1 soil, with respect to the control treatment. LC50 (median lethal concentration) was estimated using probit analysis 12.

Acute Test

Guidelines proposed by OECD 13. In experiment 45 earthen pots were used and for the experimental studies 2 kg treated soil was taken in each of the 45 earthen pots. The experiment was divided into three sets with each set having five replicates. First set was used as control treatment, i.e. without any insecticide either quercetin/azadirachtin or chlorpyrifos added to it, second set was for quercetin/azadirachtin treatment and the third one was for chemical treatment. Control had three treatments C without any insecticides added and with five replicates each. Quercetin/azadirachtin set had three treatments B1, B2 and B3 and each treatment had five replicate. Similarly chemical chlorpyrifos set also had three treatments with five replicates in each of the three treatments, C1, C2 and C3. Before introduction the worms were incubated for 24 hours on wet filter paper to empty their gut contents, after incubation they were washed, dried and then weighed (15.6 g).

The test containers were placed in the room at the room temperature. Soil water content was adjusted to 40% of the water holding capacity with distilled water. Soil pH and soil moisture percentage was checked at regular intervals during the experiments. Ten adult clitellates worms were then introduced into each of the experimental pots which were covered with transparent and perforated cotton cloth. The experiment was checked at regular weekly intervals for 42 days as E. orientalis under goes diapause during winter months and therefore experiment was confined to 42 days only. At 7 days interval earthworms were sorted out from one of the replicated experimental pots of each treatment of the test substrate, they were counted and their biomass recorded to determine their weight loss/gain and survivability.

Reproduction test

A second set of experiments was set up similar to that discussed above and at weekly interval over a period of 42 days; two earthworms were collected from one of the replicates and weighed. After a period of 15 days from the start of the experiment, cocoons emerged, they were harvested at weekly interval and were placed on wet filter paper soaked in distilled water in petri dishes and incubated in dark at room temperature. As per the guidelines 14 the wet filter paper in the petri dishes was changed every second day and the percentage of hatching success assessed.

Method to study cast production and collection

A third set of experimental set up was done similar to the manner discussed earlier to observe the role of earthworms on cast production and on carbon and nitrogen concentration in casts produced under different treatments. The cast were collected two days after the introduction of earthworms in the experimental plots at an interval of twenty four hours. They were weighed and dried, ground to fine powder and stored for carbon and nitrogen analysis. The evaluation of the amount of nitrogen, carbon and C:N ratio in earthworm tissue, soil and cast was done as per standard methodologies. Thus nitrogen was estimated in freshly frozen earthworm tissue, by semi micro-Kjeldahl method 15, organic carbon in soil and earthworm cast was determined following Walkey Black method using wet oxidation, total nitrogen in earthworm casts as well in soils was estimated following micro-Kjeldahl method 16.

Statistical Analysis

Statistical analysis was done using XL Stat computer software package (version 2009). Significant differences (p ˂0.05) in the population and biomass of earthworms across different treatments (Within same treatment of (quercetin/azadirachtin and chlorpyrifos insecticide) and in between different treatment of (quercetin/azadirachtin and chlorpyrifos insecticide), were tested using one-way ANOVA (F-test), using the probability level p ˂0.05 accepted for statistical significance of the differences, and Student-Newman-Keuls test (SNK) multiple range test. The sample standard error was calculated as the standard error of the mean (± SE).


Earthworm’s population and biomass

Under B2 treatment, no mortality occurred in earthworms and the biomass improved 7 days after treatment which was significantly higher than the control. The increase in biomass and abundance was significantly (F = 68.02, p <0.05; q = 9.6, p <0.05) higher in B2 treatment as compared to B1 and B3 treatment up till first 21 days of the experiment compared to control and declined subsequently towards the end of the experiment. In C1 and C2 treatments within the first week of application earthworms had 100% mortality rate. The lower biomass of earthworms at mortality under C1 and C2 treatment indicate that they had stopped feeding. The result under C2 treatment was similar to C1 treatment. Under C3 treatment the mortality rate was 50%, up till 21 days and declined their after. The increase in earthworm biomass was significantly higher in B3 treatment as compared to control treatment and C3 treatment under both the7 days and 14 days. Weight loss of earthworms was significantly (F = 9.94, p <0.05) higher at mortality under C3 treatment when compare to the B3 and control treatment (Table 1).

Table 1 Abundance (numbers/pot) and biomass (g) of E. orientalis under different concentrations of insecticide quercetin/azadirachtin and chlorpyrifos (N = 5, ± SE)

B = Bio insecticide; B1: 9.3 ml kg-1 soil; B2: 7.1 ml kg-1 soil; B3: 4.7 ml kg-1 soil
C = Chemical insecticide; C1: 3.2 ml kg-1 soil; C2: 2.6 ml kg-1 soil; C3: 1.6 ml kg-1 soil

Reproduction test

Higher cocoon production occurred in B2 and B3 treatments when compared to the control and B1 treatments. However the cocoon production occurred after 21 days in control treatment, whereas it took 28 days in B1, B2 and B3 treatments. Due to adult mortality no cocoons were obtained in C1 and C2 treatments and even though adult were present in C3 treatment, but no cocoon production occurred (Figure 1a). Under B2 and B3 treatments 50% of hatching occurred however the hatching of cocoons was delayed in these two treatments (after 42 days) and occurred earlier that is after 35 days in control and B1 treatments. The cocoons and juvenile were higher in the insecticide quercetin/azadirachtin treatment when compared to control/chemical soil (Figure 1b).

Figure 1 (a) Number of cocoons produced by earthworms under two treatments; (b)The hatching patterns of cocoons and emergence of juveniles under two treatments

Cast production

Total quantity of cast produced was significantly higher (F = 19.20, p <0.05) in B1 treatment when compared to control and chemical treatments (Figure 2). The quantity of cast produced was higher in B1, B2 and B3 treatment as compared to control and C1, C2 treatments. Cast production increased after 14 days in the control treatment. Under C1 and C2 treatments, there was no cast production due to the higher mortality rate of earthworms but in C3 the cast, production occurred up till 14 days subsequent to the treatment after which it declined. Maximum cast was produced in B1 and B2 treatments 14 days after the start of the experiment compared to B3 treatment, with a decline in production subsequently. B3 had lower cast production as compare to control 7 days subsequent to the treatment, but it improved significantly (F = 2.39, p <0.05) after 14 days and then declined after 20 days (Table 2).

Figure 2 Total amount of cast produced under insecticide quercetin/azadirachtin and chlorpyrifos treatments

Table 2 Comparison of different concentration of insecticide quercetin/azadirachtin and chlorpyrifos (between different treatments) (N = 5, ± SE) on cast production by E. orientalis

B = Bio insecticide; B1: 9.3 ml kg-1 soil; B2: 7.1 ml kg-1 soil; B3: 4.7 ml kg-1 soil
C = Chemical insecticide; C1: 3.2 ml kg-1 soil; C2: 2.6 ml kg-1 soil; C3: 1.6 ml kg-1 soil

Amount of nitrogen percentage in tissue

Nitrogen assimilation tissues were significantly (F = 46.44, p <0.05) higher in C1, C2 and C3 when compared to control, B2 and B3 fed earthworms also had higher tissue nitrogen assimilation when compared to control but these values were lower than the C1, C2 and C3 treatments (Figure 3a). Tissue nitrogen assimilation was significantly (F = 56.46, p <0.05) higher in control, B1 and C1 treatments when compare to control, B2, C2 and control, B3 and C3 treatments. Nitrogen assimilation was lower in B3 treatment as compared to B1 and B2. Tissue nitrogen was higher in C3 when compared to C1 and C2 fed earthworms (Figure 3b).

Figure 3 Effect of application of insecticide quercetin/azadirachtin and chlorpyrifos on N (%) in E. orientalis tissue (a) between different treatments (b) within the same treatments

Carbon content in soil and cast

B3 and C3 treated soil had lower carbon % than the control soil, however, the carbon % was higher in the casts than the normal soil, though these values did not vary significantly between control, B3, and C3 treatments (Table 3). Carbon % in the cast was higher under B2 as compared to B1 and did not vary between B2 and B3.

Table 3 Effect of different treatments on carbon (%), nitrogen (%) and C:N ratio in normal soil and in cast produced by earthworms

B = Bio insecticide; B1: 9.3 ml kg-1soil; B2: 7.1 ml kg-1 soil; B3: 4.7 ml kg-1 soil
C = Chemical insecticide; C1: 3.2 ml kg-1 soil; C2: 2.6 ml kg-1 soil; C3: 1.6 ml kg-1 soil

Nitrogen content in soil and cast

Nitrogen (%) was higher in B1 soil as compare to control and C1, B1 had significantly (F = 7.06, p <0.01) higher nitrogen % in cast as compared to control, similar results were obtained for B2 and C2 soils and cast nitrogen. But in B3 and C3 treatments nitrogen % of soil did not vary significantly as compared to control values, however C3 has higher nitrogen % in cast as compared to B3 and control treatments (Table 3). Cast nitrogen was lower in B3 treatment when compared to B1 and B2 treatments.

Estimation of C:N ratio in soil and cast

Control soil and cast had higher C:N ratio when compared to the treated soils (B1, B2, and B3), however, under all the treatments these values were higher in cast than the soil. C:N ratio was significantly (F = 9.0, p >0.05) lower in cast soils in B1 and B2 as compared to control cast. But it did not vary significantly between B3 and C3 cast when compared to control cast (Table 3). C:N ratio was lower in B1 cast when compared to B2 and B3 cast.


Earthworm’s population and biomass

The study has highlighted that insecticide quercetin/azadirachtin is nontoxic to the native earthworm E. orientalis as shown through their increased body weight, abundance, survivability and faster growth rate, this agrees with the work of Rossner and Zebitz 17, who reported that incorporation of neem products in 10 cm soil, had a positive impact on weight gain and survival of seven species of earthworms, this was also observed in our studies and was more prominent under B2 treatment. Chemical insecticide on other hand negatively impacted the same parameter because of transformation of chlorpyrifos to chlorpyrifos-oxon inside animals 3, which is about 3000 times as potent against the nervous system as chlorpyrifos itself 18, 19. This probably directly influences the nervous system of earthworm causing higher mortality rate. Tang et al. 20 have also shown insecticide quercetin/azadirachtin is a favored food for earthworm species showing a positive effect on their growth. This however is contradictory to the reports of Mikunthan and Piratheban 21, who observed weight loss in Eisenia foeitida fed on neem plus cow dung mixture, thus it can be presumed that different earthworm species respond differently to biopesticide but this needs further study. Insecticide quercetin/azadirachtin needs to be used in optimum quantity as per Muangphra and Gooneratne 22, because using it in higher concentration can cause chronic toxicity to non targeted (earthworms) organism. This was also observed in our study where earthworms showed improved biomass under B1 and B2 treatment as compared to B3 treatment.

Reproduction test

Insecticide quercetin/azadirachtin favored the production and hatching of cocoons when compared to chemical insecticide. This was also reported by Sivakumar et al. 23, who reported normal hatching success in earthworms in neem treated feed showing its non toxic nature. The fecundity rate was higher with improved hatching percentage under B2 treatment when compared to B1 and B3 and this could be due to the dose dependence of E. orientalis to the bio insecticides as has been highlighted by Bakthavathsalam 24, who through his experiment showed that the cocoon production and hatching ability of earthworms are dose dependent and increased dose has negative impact on both, this was also observed in our study where. Chemical insecticide also influenced the above factors of worms in a dose- dependent manner, with greater impact at higher concentration of chemical 25 as has also been observed in our study. Zhou et al. 26, showed chlorpyrifos adversely effected fecundity in earthworm when exposed to 5mg kg-1 chlorpyrifos after eight weeks. In our study the adverse effect on E. orientalis exposed to (C1: 3.2 ml kg-1 soil) was observed within a week of its application therefore it can probably be said that the tolerance level of species to insecticide is also species specific.

Cast production

Earthworms play important role in improving soil fertility through mineralization of dead plant materials and field straw by cast production. The higher cast production in B1, B2, and B3 treatments when compared to C highlight the importance of quercetin/azadirachtin in enhancing E. orientalis activity and this could be related to the improved metabolic rate of this species in insecticide quercetin/azadirachtin treated soils. Sivakumar et al. 23, also reported the normal casting activity in neem treated soils fed to the earthworms.

Amount of nitrogen percentage in tissue

The lower percentages of nitrogen in earthworm tissue indicate the enhanced efficiency of E. orientalis in mineralization of nitrogen in the presence of insecticide quercetin/azadirachtin when compared to chemical insecticide. Earthworm skin is a significant route of contaminant uptake 27, 28. The presence of a higher tissue nitrogen % in E. orientalis in chemical insecticide treatment compared to the insecticide quercetin/azadirachtin indicates that earthworms assimilate chemical compound in their body during intake of these soils, this gets converted to chlorpyriphos-oxon 29, 30 causing stress condition in them due to toxic affect on the nervous system and subsequently killing them. This also explains their lower weight during mortality 31.

Carbon content in soil and cast

Reduction in organic carbon in cast of B1 treatment when compared to cast of control soil and C1 cast may be attributed to assimilation of carbon during composting as has also been shown through studies of Sivakumar et al. 23.

Nitrogen content in soil and cast

The enzyme nitrogenase activity in cast in greater than the normal soil. The increased nitrogen concentration in the E. orientalis casts under B1 and B2 treatment as compared to control was because probably insecticide quercetin/azadirachtin provides a suitable environment for microbial growth and thereby increases the activity of enzyme nitrogenase 32, 33. This therefore also shows that the activity of E. orientalis is enhanced in the presence of insecticide quercetin/azadirachtin; and it probably improves the mineralization of nitrogen, converting it into plant available form 34, 35.

Estimation of C:N ratio in soil and cast

Earthworms influence the C:N ratio of soil organic matter by feeding selectively on rich neem litter and produce high nitrogen rich cast 32, 36. Lower C:N ratio in E. orientalis cast under B1 and B2 treatment as compared to control soil indicates that the insecticide quercetin/azadirachtin treated soil probably provides favorable environment for micro organisms associated with the casts, they increase combustion of carbon by enhancing total soil respiration rate, this associated with increased rate of nitrogen availability results in decreasing the C:N ratio of the E. orientalis soil 37 and thus improving the fertility of the soil and crop productivity.


From the present study it can be said that insecticide quercetin/azadirachtin has positive effect on E. orientalis as seen through their higher survivability rate, higher fecundity and hatching pattern and increased casting when in low concentrations. Therefore it can be said that insecticide quercetin/azadirachtin could be a safe and efficient option which probably enhances the efficiency of native earthworm (E. orientalis) but this needs further study.


The authors express their sincere thanks to Dr. J.M. Julka (emeritus scientist) of Zoological Survey of India (Himanchal Pradesh) and Dr. Rahul Paliwal (scientist) Zoological Survey of India (Solan Govt. of India) for taxonomic identification of the earthworm species. The authors acknowledge the help extended by the co-ordination unit of TSBF – SARNET based at JNU New Delhi for providing the necessary literature.

Author contributions

Conceived and designed the work: Bhadauria T, Mishra J

Acquired the data: Kumar P

Analyzed and/or interpreted the data: Bhadauria T

Drafted the work: Bhadauria T, Kumar P

Revised and approved the work: Mishra J


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