Herbicide toxicity in Eisenia fetida: Ecotoxicological insights for sustainable agriculture

Introduction

Earthworms are widely recognized as keystone species in terrestrial ecosystems due to their profound influence on soil structure, fertility and biological functioning. Their burrowing and feeding activities enhance soil aeration, water infiltration, and nutrient cycling, thereby sustaining agricultural productivity and ecological resilience. Recent studies emphasize that earthworms not only recycle organic matter but also regulate microbial communities, contributing to soil carbon sequestration and long term fertility (den Toonder et al., 2026; Murali et al., 2026) [1,5] . Their ecological services pedogenesis, decomposition, and bioremediation make them indispensable for sustainable agriculture, particularly in regions facing soil degradation and intensive chemical inputs (Saharan et al., 2025) [7] . The ecological importance of earthworms has led to their designation as "farmer's friends," yet their populations are increasingly threatened by anthropogenic pressures, especially the in discriminate use of agrochemicals. Herbicides, which account for nearly half of global pesticide consumption, are extensively applied in modern agriculture to suppress weeds and enhance crop yields (Parven et al., 2024) [6] . While herbicides such as Pretilachlor and Bispyribac-Sodium are effective in rice cultivation, their persistence in soil and potential to affect non-target organisms raise serious ecological concerns. Herbicide residues can alter soil physicochemical properties, disrupt microbial diversity, and impair soil fauna, including earthworms, which are highly sensitive to chemical contaminants (Meftaul et al., 2024) [6] . The reliance on herbicides has intensified due to labor shortages, rising production demands, and the need for rapid weed control. However, repeated and prolonged applications have contributed to herbicide-resistant weed populations, biodiversity loss, and contamination of soil and water resources (Sharma et al., 2019a; Cobb, 2022). Recent reviews highlight that although herbicide usage trends show a decline in overall volume, there is a shift toward systemic and pre-emergence formulations, which may pose greater risks to soil organisms due to their persistence and bioavailability (Parven et al., 2024) [6] . This underscores the necessity of evaluating herbicide impacts beyond crop productivity, focusing on soil health and ecological sustainability. Ecotoxicological evaluation of herbicides on earthworms has emerged as a critical research area. Earthworms, particularly Eisenia fetida are widely used as bioindicators in toxicity assays due to their sensitivity, ease of culturing, and ecological relevance. Studies reveal that herbicide exposure can impair growth, reproduction, and biochemical functions in earthworms, with variations depending on herbicide type, concentration, and exposure duration (Dixit & Bardiya, 2024) [2] . For instance, glyphosate and atrazine have been shown to induce oxidative stress, reduce cocoon production, and alter enzymatic activity in Eisenia species (Rahman et al., 2020; Lammertyn et al., 2021). Similarly, Pretilachlor and Bispyribac Sodium have demonstrated concentration dependent toxicity, affecting survival rates and metabolic processes (Fouad, 2021) [3] . Recent biomarker based studies provide deeper insights into sub-lethal effects, including disruptions in oxidative stress enzymes, lipid metabolism, and protein synthesis (Yatoo et al., 2021; Dixit & Bardiya, 2024) [13,2] . These biochemical responses serve as early warning indicators of ecological stress, complementing traditional mortality-based assays. Moreover, environmental factors such as temperature and soil properties modulate herbicide toxicity, influencing earthworm survival and reproduction under varying agro climatic conditions (Tsukamoto & Watanabe, 1977; Sanchez-Hernandez et al., 2019) [9] . The need for eco toxicological evaluation is further reinforced by the role of earthworms in mitigating pesticide mobility and persistence. By metabolizing herbicide residues in their gut and transporting them deeper into the soil, earthworms influence the fate of these chemicals in agro ecosystems (Sanchez-Hernandez et al., 2019) [10] . However, when herbicide concentrations exceed tolerance thresholds, earthworm populations decline, jeopardizing soil fertility and ecosystem services. This dual role as both regulators and victims of chemical contamination positions earthworms at the center of risk assessment frameworks.

Materials and Methods

Study Area and Test Organism

The experiment was carried out under controlled laboratory conditions using Eisenia fetida, a widely recognized test organism in ecotoxicological research. Adult worms exhibiting a well-developed clitellum, measuring approximately 5-7 cm in length and 3-5 mm in diameter, were selected for the assays. This species was chosen due to its rapid growth rate, high reproductive capacity, and resilience to environmental variability, characteristics that make it particularly suitable for standardized toxicity evaluations.

Mortality and Biomass

After 28 days of exposure, adult Eisenia fetida were carefully removed from the soil by hand sorting. Mortality was recorded, and the biomass of surviving individuals was measured to determine the direct toxic effects of herbicide treatments under varying temperature conditions.

Cocoon Development

After removal of adults, the soil was returned to the experimental containers and incubated for an additional 28 days at the respective temperatures. This incubation period facilitated cocoon deposition and development, thereby providing insights into reproductive performance under herbicide-induced stress.

Carbohydrate Estimation

The total carbohydrate content of Eisenia fetida tissues was determined using the phenol sulfuric acid method described by Masuk et al. (2005). After 60 days of exposure, tissues were collected and homogenized in 20% cold trichloroacetic acid (TCA) at a ratio of 1 ml TCA per 100 mg tissue. The homogenate was incubated at 70 °C for 20 minutes to facilitate protein precipitation and carbohydrate release, followed by centrifugation to obtain a clear supernatant. For analysis, 1 ml of supernatant was mixed with 1 ml of 5% phenol solution, and 5 ml of concentrated H₂SO₄ was rapidly added. The mixture was allowed to stand for 30 minutes, during which a yellow orange color developed. Absorbance was measured at 490 nm using a UV-Vis spectrophotometer. A standard curve was prepared using glucose solutions (10-100 µg/ml), treated identically to the samples, and carbohydrate concentrations were expressed as mg carbohydrate per g tissue.

Lipid Estimation

Total lipid content was quantified using the Soxhlet extraction method (Soxhlet, 1879). After 60 days of exposure, tissues of Eisenia fetida were collected, homogenized, and dried to constant weight. Approximately 2-5 g of dried tissue was placed in a cellulose thimble and inserted into a Soxhlet extractor. Petroleum ether (boiling range 40-60 °C) was used as the solvent, and extraction was carried out continuously for 6-8 hours. The lipid-containing solvent was collected in the boiling flask, evaporated under reduced pressure, and the lipid residue was weighed gravimetrically. Results were expressed as mg lipid per g tissue.

Protein Estimation

Protein concentration was measured using the Lowry method (Lowry et al., 1951). After 60 days of exposure, tissues were homogenized in 20% cold TCA and incubated at 70 °C for 20 minutes. The supernatant was collected, and 1 ml was transferred into a test tube. Alkaline copper sulfate reagent was added, and the mixture was incubated for 10 minutes at room temperature. Subsequently, 0.5 ml of Folin Ciocalteu reagent was added, mixed thoroughly, and incubated for 30 minutes, resulting in the development of a blue color. Absorbance was measured at 750 nm using a spectrophotometer. A standard curve was prepared using bovine serum albumin (BSA) solutions (10-100 µg/ml), and protein concentrations were expressed as mg protein per g tissue.

Results

Mortality and LC₅₀ in Eisenia fetida

Bioassays with Eisenia fetida showed distinct mortality responses to the two herbicides. No mortality occurred in controls. Pretilachlor caused progressively higher mortality with concentration and time: 5% LC₅₀ led to 5-20% mortality (12-48 h), 10% LC₅₀ reached 40% at 48 h, and 15% LC₅₀ rose to 60% at 48 h. The estimated LC₅₀ was 75-95 mg/kg, indicating higher acute toxicity (Figure 1). The Bispyribac Sodium produced lower mortality: 5% LC₅₀ caused 3-18% mortality, 10% LC₅₀ reached 35%, and 15% LC₅₀ reached 50% by 48 h. Its LC₅₀ ranged from 90-110 mg/kg, suggesting comparatively lower toxicity than Pretilachlor (Table 1

Cocoon Development

Control worms produced the highest number of cocoons (120 at day 60). Pretilachlor exposure reduced cocoon output in a concentration-dependent manner, with values declining from 100 (5% LC₅₀) to 70 (15% LC₅₀) (Figure 2). Bispyribac Sodium showed comparatively less reduction, yielding 110 (5% LC₅₀) to 78 (15% LC₅₀) cocoons by day 60. Overall, Pretilachlor exerted stronger inhibitory effects on reproduction than Bispyribac-Sodium (Table 2).

Biochemical Estimation

Exposure to both herbicides reduced carbohydrate, lipid, and protein levels in Eisenia fetida compared to controls.

Pretilachlor caused a stronger decline, with values dropping to 9.0 mg/g carbohydrates, 4.5 mg/g lipids, and 14.0 mg/g proteins at 15% LC₅₀ (Figure 4). Bispyribac Sodium showed comparatively milder reductions, with 10.0 mg/g carbohydrates, 5.0 mg/g lipids, and 14.8 mg/g proteins at the same concentration. The Pretilachlor exerted greater biochemical suppression than Bispyribac Sodium (Table 3).

Mortality and LC₅₀ in Eisenia fetida

The acute toxicity assays demonstrated that Eisenia fetida responded differently to Pretilachlor and Bispyribac Sodium. Control groups showed negligible mortality, confirming the absence of solvent effects. Pretilachlor produced a clear concentration-and time-dependent increase in mortality, with LC₅₀ values between 75-95 mg/kg, indicating higher acute toxicity. In contrast, Bispyribac Sodium induced comparatively lower mortality at equivalent concentrations and exposure intervals, with LC₅₀ values ranging from 90-110 mg/kg. These findings suggest that Pretilachlor exerts stronger toxic stress on earthworms, while Bispyribac Sodium is relatively less harmful under similar conditions.

Cocoon Development

Cocoon production in Eisenia fetida was clearly affected by herbicide exposure. Control worms showed the highest reproductive output, confirming normal conditions. Pretilachlor caused a concentration-dependent decline, with cocoon numbers reduced from 100 at 5% LC₅₀ to 70 at 15% LC₅₀ by day 60. Bispyribac Sodium also reduced cocoon production but to a lesser extent, yielding 110 at 5% LC₅₀ and 78 at 15% LC₅₀. These results indicate that Pretilachlor exerts stronger inhibitory effects on reproduction compared to Bispyribac Sodium, highlighting its greater ecological risk to soil invertebrates.

Biochemical Estimation

Biochemical assays revealed that both herbicides suppressed carbohydrate, lipid and protein levels in Eisenia fetida compared to controls. The reduction was concentration dependent, with Pretilachlor exerting stronger effects, lowering values to 9.0 mg/g carbohydrates, 4.5 mg/g lipids, and 14.0 mg/g proteins at 15% LC₅₀. Bispyribac Sodium caused comparatively milder declines, with 10.0 mg/g carbohydrates, 5.0 mg/g lipids, and 14.8 mg/g proteins at the same concentration. These results indicate that Pretilachlor imposes greater biochemical stress, reflecting higher toxicity potential relative to Bispyribac Sodium.

Conclusion and Future Prospective

The findings of this study clearly demonstrate that Eisenia fetida serves as a reliable bioindicator for evaluating herbicide toxicity in soil ecosystems. Pretilachlor consistently exhibited stronger adverse effects, including higher mortality, reduced cocoon production, and greater suppression of carbohydrate, lipid, and protein levels, compared to Bispyribac-Sodium. Although Bispyribac-Sodium was comparatively less toxic, it still induced measurable stress, confirming that both herbicides pose ecological risks to soil invertebrates. These results highlight the importance of assessing chemical impacts not only on crop productivity but also on non-target soil organisms that play vital roles in maintaining soil health and fertility.

Looking ahead, future research should emphasize long-term and chronic exposure studies to better understand cumulative effects on earthworm populations and soil ecosystems. Field-based validation of laboratory findings will be essential to capture real world variability in soil conditions. Mechanistic investigations at the molecular and enzymatic levels could provide deeper insights into pathways of herbicide-induced stress. Comparative risk assessments across diverse soil fauna would broaden ecological relevance, while exploration of sustainable alternatives such as bio-herbicides and integrated weed management strategies could help mitigate ecological damage.

Credit authorship contribution statement

Kajal:Writing original draft, Validation, Funding acquisition, Formal analysis,

Acknowledgement

AcknowledgementThe authors are thankful to Department of Zoology, Baba Matnath University, AsthalBoahr, Rohtak, and Haryana, India for providing infrastructural facility to conduct the research.Data availability: All the data is provided with in the manuscript.

References

1. den Toonder J, Angst Š, Angst G. Earthworms promote soil organic carbon formation across management histories. Biology and Fertility of Soils. 2026.
2. Dixit S, Bardiya S. A review on effect of different herbicides on growth, reproduction and biochemical markers of earthworm. World Journal of Advanced Engineering Technology and Sciences. 2024;13(2):655662. https://doi.org/10.30574/wjaets.2024.13.2.0643
3. Fouad MR. Toxic effects of Bispyribac-Sodium on earthworms (Aporrectodeacaliginosa) by filter paper and soil mixing method. Ecotoxicology and Environmental Safety. 2021;220:112356. https://doi.org/10.1016/j.ecoenv.2021.112356
4. Murali JS, Kumar R, Ashwin M. Earthworm and earthworm cast-associated microbes for sustainable agriculture. In: Harnessing Microbial Resources for Sustainable Agriculture. Springer; 2026.
5. Parven A, Meftaul IM, Venkateswarlu K, Megharaj M. Herbicides in modern sustainable agriculture: Environmental fate, ecological implications, and human health concerns. Environmental Science and Pollution Research. 2024. https://doi.org/10.1007/s13762-02405818-y
6. Saharan BS, Lata P, Duhan JS. Insights of agricultural sustainability by the use of earthworms. Discover Agriculture. 2025.
7. Sanchez-Hernandez JC, Capowiez Y, Jänsch S. Earthworm biomarkers in ecological risk assessment: Review of current research and future perspectives. Applied Soil Ecology. 2019;134:57-68. https://doi.org/10.1016/j.apsoil.2018.10.015
8. Sanchez-Hernandez JC, Cares XA, Pérez MA, Del Pino JN. Biochar increases pesticide-detoxifying carboxylesterases along earthworm burrows. Science of the Total Environment. 2019;667:761-768.
9. Wang H, Ren W, Xu Y, Wang X, Ma J, Sun Y, Hua W, Chen S, Dai S, Song J, Jia J, Teng Y. Long-term herbicide residues affect soil multifunctionality and the soil microbial community. Ecotoxicology and Environmental Safety. 2024;283:116783.
10. Xuzhi L, et al. Combined toxicity of Siduron and Cadmium on earthworm (Eisenia fetida) using biomarker response index. Ecotoxicology and Environmental Safety. 2019;171:324-332.
11. Yatoo AM, Ali MN, Zaheen Z, Baba ZA, Sheikh TA, Gupta PK. Assessment of pesticide toxicity on earthworms using multiple biomarkers. Environmental Chemistry Letters. 2021.
12. Yatoo MI, Saxena A, Sharma S. Impact of glyphosate on soil fauna and microbial communities: A review. Environmental Monitoring and Assessment. 2021;193(12):789. https://doi.org/10.1007/s10661-02109542-1
13. Zhang Y, et al. Acute toxicity of seventeen herbicides commonly used to earthworm (Eisenia fetida). Journal of Ecology and Environment. 2023; 32(4):455-463.