Assessing Cocoon Production by Earthworm in Response to Soil Pesticide Contamination

Introduction

Rapid urbanization and industrialization have led to enormous solid waste generation. This includes domestic, industrial, and agricultural waste, which is escalating at an alarming rate. Failure to address this issue seriously could lead to ecological imbalance ^1,2. Municipal solid waste generated annually in the world is around 1.3 billion metric tons and it is expected to rise by 2.2 billion metric tons by 2025 ³. One of the well-known reasons for environmental degradation is the pollution of land, water, and air through different toxic chemical substances ^4,5.

Pollutants in the soil cause both quantitative (decrease in biomass, affects reproduction) and qualitative (depletes natural fertile capacity of soil) changes in fauna. Soil microfauna is a good indicator of toxicity in soil. Protozoans, Nematodes and mesofauna (oligochaete annelids) are used for ecotoxicological studies. The soil macrofauna (snails and terrestrial gastropod molluscs) is utilized for bioaccumulation studies ⁶.

Technologies such as vermiremediation are economical and undemanding to maintain and apply. Earthworms contribute positively to soil productivity and fertility without disturbing the top layer of the soil, thereby causing minimal environmental impact. The coelomic fluid of earthworms has antibacterial properties hence, minimises the amount of pathogens in the soil. Earthworms are good organisms for bioremediation but only in low or moderately-polluted soils as earthworms’ growth, reproduction and physiology will be affected in contaminated soil ⁷. Assessing the response of earthworms is crucial in determining the soil ecological toxicity in contaminated soil ⁸.

Eisenia fetida, an epigeic earthworm can be used for soil bioremediation. They have been shown to survive in soil contaminated with oil (Petroleum and Diesel) (3500mg/kg). Moreover, the survival rate depends on exposure and metabolism. It was also found that even polycyclic aromatic hydrocarbon concentration decreased upon bioremediation with Eisenia fetida and Lumbricus rubellus ⁹.

Toxic pesticides such as aluminium phosphide and organophosphates like dichlorvos (esters of phosphoric acid) and their residue have contaminated the air, water, and soil and thus they are a threat to the co-existence of different plant and animal species ¹⁰. A plentiful amount of applied pesticides (15%- 40%) is dispersed in the air through volatilization ¹¹. Pesticides can be harmful even after many years of application because of their characteristics such as bioaccumulation, lipophilicity, and long half-life ¹⁰.

The first step towards sustainable management is soil protection. Invertebrates like earthworms help modify the soil environment by accumulating cast, pellets, and galleries (biogenic structures). Earthworms also play a major role in the carbon and nitrogen cycle. Urine, mucus and fresh earthworm casts contain more N, P, K and Ca than dry casts. When casts dry, the breakdown of solid organic matter ceases for several months ^12,13. However, all these services earthworms provide are at risk due to the excessive use of pesticides. The most lethal toxic class of pesticides is insecticides, which also pose a risk to the non-target soil fauna ¹⁴.

Bioremediation is an inexpensive, effective and eco-friendly technology to clean up hazardous materials from the soil ¹⁵. This technique includes the breakdown of organic materials by fungi, bacteria and earthworms (soil organisms) ¹⁶. In-situ bioremediation involves bioaugmentation (degrading unwanted compounds by adding pre-grown microbes) which enhances the degradation rate ^17,18. Earthworms are tolerant to heavy metals, pesticides and other organic pollutants. This tolerance capacity of earthworms makes them convert even unstable solid organic wastes such as sewage sludge, industrial waste and animal waste into compost (vermicompost) which has high NPK (nitrogen, phosphorous and potassium) content. An Increased amount of Ca (calcium), and Mg (magnesium) is also found in such composts ¹⁹. This vermicompost can be used as organic fertilizer in crops, it increases the leaf number and leaf area, root length, and number. It also improves stem height and seed germination ^20,21. The study investigates how sub-lethal doses of pesticides AlP and DDVP affect the reproductive capacity of earthworms. In agriculture, even small amounts of pesticide residues can adversely affect the reproductive health of earthworms. Therefore, it is essential to prioritize techniques such as bioaugmentation using substances like cow dung and biochar to protect populations of soil fauna.

Materials and Methods

Rearing of Earthworms (Eisenia fetida)

 Rearing of worms was done according to OECD guidelines no. 207 (1984), ISO (1993,1998), Tropical Artificial soil was utilized as a medium.

Plastic trays, Size 50 cm X 25 cm X 22 cm for culture and rearing – For Tropical Artificial soil (adapted from Garcia, 2004 and De Silva and Van Gestel, 2009) – 10 % Non-decomposed Coconut coir dust/ coconut peat, decomposed Coconut coir dust/ coco peat was used as alternative for sphagnum peat along with rice husk dust and saw dust moistened to 50 % of its water holding capacity, 20 % kaolin clay, 70% fine industrial sand/silica. Calcium carbonate was added, and pH was adjusted to 6 ± 0.5. Nutritive medium – 30 days-old cattle manure was finely ground and sieved through a 500μm sieve. The nutritive medium was placed in a plastic tray and stabilized for 24 hours. Muslin cloth was used to cover the culture trays and to maintain proper aeration. The temperature of the medium was maintained at 25 ± 20 C and relative humidity at 80 %. Scraps of paper and cardboard were used as the bedding material.

For testing acute toxicity, six concentrations in increasing geometric value were chosen. Aluminium phosphide powder is a dry test substance so it was mixed with artificial soil for exposure, but for Dichlorvos, the concentrations were mixed with distilled water and then evenly sprayed in artificial soil. Ventilation of test medium was done before use. Test was conducted in replicates of four with control. Adult clitellated worms were picked from culture medium, washed in distilled water, dried and introduced in the test medium. Any behavioural and physiological change observed was noted. Mortality was plotted versus Log concentration and LC₅₀ was calculated by probit analysis. Moisture and pH were assessed at the start and end of the test period. 

Experimental Tests

Earthen pots were used for exposure to sublethal doses of AlP and dichlorvos. The exposure to sub-lethal dose was done in artificial soil, finely ground, and sieved cattle manure was given weekly as food. Post-exposure to two sub-lethal dose (1/2 and 1/3 of LC₅₀ for both pesticides) constant monitoring was done. To check the combined effects of both pesticides, combined exposure was also done, along with control. All the tests were performed in replicates of four. For analysis of reproduction in earthworms (cocoon production), the test period was 8 weeks. After the end of each week, the earthen pot was emptied to check cocoon production. Every week the test medium was augmented with cattle manure, finely sieved, and ground. Any physical and behavioural changes were also observed in earthworms.

All the data collected of mean weight of 4 replicates of control, AlP, DDVP exposed worms as well as combined exposure of both pesticides, was subjected to analysis by two way ANOVA. The standard deviation and standard errors were also calculated. After statistical analysis using ANOVA, Bonferroni correction (multi comparison correction) was also done. The results were subjected to Tukey- Kramer multiple comparison post hoc T- test. 

Results

For analysis of cocoon production, the test media were emptied every week and the artificial soil medium was sieved and searched for any cocoons laid by the worms. The cocoon production was assessed for 8 weeks.

As compared to the control, there was a significant decline in the cocoon production in both AlP and DDVP exposed worms as well as combined exposure. In the control, average cocoon production per 10 worms per week reached close to 2 in week 5 and remained 1.3 to 1.8 throughout the test period of 8 weeks.

For worms exposed to sublethal dose of aluminium phosphide powder residue, cocoon production was significantly low throughout the test period, p-value was less than 0.05. There were fluctuations observed in the number of cocoons laid throughout the test period. The highest cocoon production was observed in the 7^th week which was 0.7 ± 0.08 for 1/2 LC₅₀ AlP and 0.75 ± 0.10 for 1/3 LC₅₀ AlP (Table 1).

Table 1: Mean cocoon production over eight weeks in Control and AlP exposed worms, significant difference observed from control, p <<0.05. No Significant difference between AlP 1/2 LC₅₀ and AlP 1/3 LC₅₀, p >0.05.

	CONTROL	AlP 1/2 LC50	AlP 1/3 LC50
WEEK 1	1.325 ± 0.12	0.575 ± 0.09	0.375 ± 0.17
WEEK 2	1.375 ± 0.15	0.5 ± 0.08	0.525 ± 0.05
WEEK 3	1.55 ± 0.30	0.45 ± 0.19	0.525 ± 0.05
WEEK 4	1.35 ± 0.19	0.5 ± 0.08	0.475 ± 0.05
WEEK 5	1.95 ± 0.23	0.575 ± 0.12	0.625 ± 0.05
WEEK 6	1.5 ± 0.24	0.65 ± 0.12	0.75 ± 0.05
WEEK 7	1.4 ± 0.18	0.7 ± 0.08	0.75 ± 0.10
WEEK 8	1.625 ± 0.15	0.6 ± 0.18	0.6 ± 0.14

For worms exposed to a sublethal dose of dichlorvos, cocoon production was significantly lower, there was a significant difference observed between average cocoon production in control and DDVP-exposed worms, after ANOVA analysis. The p-value was << 0.05, pointing towards a statistically significant result. As compared to 1/3 LC₅₀ DDVP, the cocoon production was lower in 1/2 LC₅₀ DDVP, pointing towards the dose-dependent effect of DDVP on the cocoon production. Significant fluctuation was observed throughout the test period with maximum cocoon production in DDVP exposed worms observed in week 8 which was 0.6 ± 0.08 for 1/2 LC₅₀ and 0.675 ± 0.09 for 1/3 LC₅₀ (Table 2).

Table 2: Mean cocoon production over eight weeks in Control and DDVP exposed worms, significant difference observed from control, p <<0.05. No Significant difference between DDVP 1/2 LC₅₀ and DDVP 1/3 LC₅₀, p > 0.05

	CONTROL	DDVP1/2 LC50	DDVP1/3 LC50
WEEK 1	1.325 ± 0.12	0.375 ± 0.05	0.525 ± 0.12
WEEK 2	1.375 ± 0.15	0.175 ± 0.09	0.15 ± 0.05
WEEK 3	1.55 ± 0.30	0.175 ± 0.09	0.275 ± 0.09
WEEK 4	1.35 ± 0.19	0.3 ± 0.14	0.4 ± 0.11
WEEK 5	1.95 ± 0.23	0.35 ± 0.05	0.525 ± 0.05
WEEK 6	1.5 ± 0.24	0.525 ± 0.05	0.6 ± 0.08
WEEK 7	1.4 ± 0.18	0.55 ± 0.05	0.575 ± 0.09
WEEK 8	1.625 ± 0.15	0.6 ± 0.08	0.675 ± 0.09

In combined exposure to both AlP and DDVP, a significant decline from control was seen, after ANOVA analysis, p-value obtained was <<< 0.05 (significantly lower). There were fluctuations throughout the test period of 8 weeks, and the cocoon production in the combined exposure was also significantly lower than in the AlP and DDVP exposed worms (Table 3).

Table 3: Mean cocoon production over eight weeks in Control, combined AlP and DDVP exposed worms, significant difference observed from control, p <<0.05. No Significant difference between combined 1/2 LC₅₀ and combined 1/3 LC₅₀, p >0.05

	CONTROL	COMBINED 1/2 LC50	COMBINED 1/3 LC50
WEEK 1	1.325 ± 0.12	0.175 ± 0.09	0.15 ± 0.05
WEEK 2	1.375 ± 0.15	0.125 ± 0.05	0.15 ± 0.05
WEEK 3	1.55 ± 0.30	0.2 ± 0.08	0.15 ±0.05
WEEK 4	1.35 ± 0.19	0.375 ± 0.05	0.375 ± 0.09
WEEK 5	1.95 ± 0.23	0.4 ±0.08	0.375 ±0.09
WEEK 6	1.5 ± 0.24	0.375 ± 0.09	0.5 ± 0.08
WEEK 7	1.4 ± 0.18	0.525 ± 0.05	0.55 ± 0.12
WEEK 8	1.625 ± 0.15	0.6 ± 0.08	0.65 ± 0.10

The highest cocoon production in combined exposure was 0.65 ± 0.10 for combined 1/3 LC₅₀ at the end of 8^th week. The lowest cocoon production in all the tests conducted was in combined exposure of 1/2 LC₅₀ which was 0.125 ± 0.05 at the end of 2^nd week (Figure 1). It was during these initial two weeks that worms in the test media of combined exposure showed the most behavioural changes of coiling, jumping, excessive mucous secretion and extrusion of the coelomic fluid (Figure 2).

The combined exposure had a significant decline in cocoon production. There was no significant difference in the dose of combined exposure. In fact, at the end of week 1, week 3 and week 5, the cocoon production was lower in 1/3 combined LC₅₀ as compared to 1/2 combined LC₅₀ (Table 3)_.

Figure 1: Graphical representation of changes in cocoon production in control and AlP and DDVP exposed worms over 8 weeks Click here to view Figure

Figure 2: Sections through the body wall of pesticide exposed worms A, B, C & D- the disintegration of the muscle layers and increased number of mucous gland cells in the epidermis can be seen.  Click here to view Figure

Discussion

When earthworms were exposed to sub-lethal doses of AlP, cocoon production reduced in all the weeks from 1 to 8 compared to control. But in both AlP ½ LC₅₀ and AlP 1/3 LC₅₀ in week 8^th cocoon production was slightly increased compared to week 1. Almost similar results were observed when earthworms were exposed to dichlorvos. In the case of a combination of ALP and dichlorvos, the cocoon production by earthworms was significantly reduced in the first four weeks. Exposure to various pesticides causes considerable stress and reduces the capacity for cocoon laying in earthworms due to irregularities in mucus production and changes in subepidermal musculature. Over time, a slight increase was observed, after augmentation of the culture media with cow dung. The cocoons in earthworms are produced by the epidermis of the clitellar region; those exposed to pesticides exhibit an abundance of mucous glands and a modified muscle arrangement that disrupts cocoon formation.

In earthworms, cocoon development, shape, and size greatly vary among different species. In a study more weight and larger size cocoons were found in anecic earthworms whereas low-weight and smaller size cocoons were found in epigeic earthworms ²². The incubation period of a cocoon differs among the species and also depends on the environment. The incubation period is short for epigeic and anecic species whereas for endogeic species it’s long ²³. Further, the rate of cocoon production also depends on the substrate materials. Different species show different growth and reproduction patterns on the same substrate material ²⁴.

The use of earthworms in bioremediation, along with techniques like bioaugmentation and biostimulation, improves soil health and restores soil fertility ^25,26. In addition to bioremediation, phytoremediation (known for its high metal biosorption capacities) is essential for addressing heavily contaminated organic waste from industrial and agricultural sectors. The most commonly studied species of bacteria for bioremediation are Mycobacterium, Pseudomonas and Rhodococcus ²⁷. In addition to bioremediation, adequate supplies of water, oxygen, nitrogen, and phosphorus are essential to enhance degradation rates ¹⁸. In this study, bioaugmentation was performed using cow dung. Cocoon production significantly decreased initially with sublethal doses of AlP and DDVP but showed improvement following bioaugmentation with cow dung.

Conclusion

Maintaining the health of agricultural ecosystems hinges on the preservation of optimum soil fauna populations. Pesticide exposure poses a significant threat to these crucial organisms, necessitating proactive measures to safeguard their survival. Earthworms, integral to soil health, play a pivotal role in bioremediating pesticide-contaminated soils. However, ensuring their vitality requires strategic interventions such as bioaugmentation techniques involving materials like cow dung and biochar. These practices not only support the well-being of earthworms but also contribute to the overall resilience and sustainability of agricultural ecosystems by fostering a balanced soil ecology. By prioritizing the health of soil fauna through thoughtful management practices, we can mitigate the adverse impacts of pesticides and promote sustainable agricultural practices for the future.

Acknowledgment

The authors would like to acknowledge the Department of Zoology, Hansraj College, University of Delhi, and the Department of Zoology, Miranda House, University of Delhi for assisting with the infrastructure needed for conducting the nature walk sessions.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of the article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement       

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Ethics Statement           

This research did not involve human participants, animal, subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Authors’ Contribution

All authors contributed to the study’s conception and design. Material preparation, data collection and analysis were performed by Himanshu Sharma and Yasha Yadav. The first draft of the manuscript was written by all authors. All authors read and approved the final manuscript.

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