The study was carried out in Badougou Djoliba (Figure 1Figure 1) in collaboration with the Institut Polytechnique Rurale de Formation et de Recherche Appliquée de Katibougou, the Laboratoire d’Entomologie et Parasitologie at USTTB-FST, and the École Doctorale des Sciences et Technologies du Mali.

Figure 1.Map of the study area.

Biological and technical materials were used to conduct this research. The experimental site was surveyed to identify areas for earthworm collection, which was facilitated by the presence of casts on the soil surface.

The biological materials included earthworms, indigenous epigeic worms, and manure worms.

Earthworms were identified and characterized through the morphological observation of their external features. For this, we used binocular magnifying glasses, tweezers, a precision electronic scale, a ruler, alcohol, a camera, Petri dishes, data sheets, and pens.

Earthworm sampling primarily uses chemical methods for anecic species and physical methods for endogeic and epigeic species.

The sampling took place at the experimental site during the rainy season, from June to July 2024. Earthworms were collected following the methods outlined by [10], which involved digging at the sampling point, manually sorting through manure piles, or examining the surface layers of the soil (up to 15 cm deep) in areas known to harbor earthworms. This was done to minimize biotype variation within the same plot of land.

The earthworms were collected three weeks before the experiment began (Figure 2Figure 2). After collection, the samples were stored in a cool place in containers filled with moist soil from the sampling site, taking into account the soil’s physicochemical properties.

Figure 2. Sampling and collection of earthworms on site.

Pre-composting is an initial stage in the accelerated decomposition of organic matter that occurs before final composting or vermicomposting. This process can reduce waste volume by nearly 60%, minimize odors, and sanitize the material.

In this study, traditional pre-composting was essential before introducing earthworms (Figure 3Figure 3). This phase, characterized by high heat generation, achieved sufficient temperatures to eliminate most pathogens, prevent harmful fermentation that could impede worm development [11]. And sanitize the material.

Figure 3.Different types of pre-compost.

A test was conducted to evaluate the viability of earthworms based on the ratio of pre-compost to soil. This test utilized the optimal compost-to-soil ratio for promoting earthworm development, focusing on their survival, growth, and reproduction across various substrates, including soil, compost, and manure.

The viability of the earthworms in these different substrates was assessed using a specific formula.

Three types of pre-compost manure were utilized. For each type of manure, we prepared four different percentages, varying from raw manure to a mixture of one part pre-compost and four parts soil (Table 1).

Table 1.Substrate report for the viability test.

Each container was filled with substrate (pre-compost/soil from the experimental site) and three earthworms.

Two types of studies were conducted: one in a natural environment and the other under controlled conditions.

3.4.1. Field Tests of Vermicomposting

Vermicomposting is an eco-friendly method that breaks down organic waste into compost using macro- and microorganisms, such as bacteria and worms. The experiments were conducted in rural areas with animal waste to determine the environmental conditions conducive to vermicomposting.

Experimental Setup:

The experimental setup consisted of specific materials, experimental units, and organizational arrangements. Trenches measuring 40 cm wide, 50 cm long, and 10 cm deep were dug. The spacing between the trenches was 60 cm in width and 1 m in length (Figure 4Figure 4).

A total of 9 pits were filled with substrates and arranged in a completely randomized block design. Each experimental unit was covered with a mosquito net featuring a 2 mm square mesh to prevent earthworms from escaping and to exclude other macroinvertebrates from entering.

Indigenous earthworms (Eudriluseugeniae) and exotic earthworms (Eiseniafoetida) were introduced at a density of 3 individuals per unit, based on their weight. The pits were filled with substrate, either containing or lacking earthworms, arranged in a completely randomized block design with 9 treatments, each replicated 3 times.

Figure 4. Experimental block design in a natural environment.

3.4.2. Tests Conducted under Controlled Conditions in Vermiculture

The controlled-condition vermiculture test aims to optimize the decomposition of organic matter using epigeic worms. This study also measures the degradation rate, worm reproduction rate, and the quality of the compost produced. The research was conducted in a controlled environment utilizing animal waste, municipal waste, and green waste at varying incorporation rates. The objective was to determine the pre-compost/soil ratio that is most favorable for earthworm survival and to assess the effect of earthworms on the substrate.

Setting up and monitoring the experiments:

The experiments were carried out in plastic bins (45 at each site) arranged horizontally as compost bins. Each bin had dimensions of 45 × 45 × 20 cm and contained three earthworms. To maintain moisture for the worms, the bins were covered with straw. Following Chaoui’s method [11], the total duration of the experiment was 40 days. This period included not only the active decomposition phase (spanning from days 22 to 32) but also a necessary maturation period for the organic waste to transform into compost. In this experiment, two types of waste were utilized: plant waste and municipal waste, which were prepared into the following substrate types: (i) T Du: municipal waste (100%) (ii) T fv: manure poultry (5%) + plant waste (95%) (iii) T v + u: plant waste (2/3) + municipal waste (1/3) (iv) T u + v: plant waste (1/3) + municipal waste (2/3) (v) T n: bare soil.

Experimental design: The study tested two categories of earthworms (native and exotic), five types of substrates, and conducted three replicates for each substrate, along with a control (Figure 5Figure 5). The fifteen treatments were arranged in a full factorial design (Figure 6Figure 6).

3.4.3. Method for Identifying Earthworms

The two categories of earthworms were identified at the Laboratoire d’Entomologie et de Parasitologie at the Faculté des Sciences et Technique of the Université des Sciences Techniques et Technologies de Bamako.

Figure 5.Block design in a controlled environment.

Figure 6. Installation of experimental setups in a controlled environment.

Figure 7. Different stages of earthworm identification.

The collected earthworms were identified using the identification key from [8].

A total of twenty samples were selected, consisting of ten individuals of Eiseniafoetida and ten of Eudriluseugeniae. The worms were rinsed with water in a Petri dish before being fixed in alcohol to immobilize them. This step facilitated the observation of various morphological characteristics under a binocular magnifying glass, including color, number of segments, arrangement of setae, shape of the prostomium, and shape of the tail. Weight and size were measured using a precision balance and a graduated ruler, respectively (Figure 7Figure 7).

Figure 8Figure 8 shows that the observed viability rate was 100% for the proportions FPR-1/1, 1/3, and 1/4. A 67% viability rate was noted for the FPR-1/0 proportion.

The results presented in Figure 9Figure 9 indicate that the FV-1/1 group had a viability rate of 0%, resulting in three recorded deaths. In contrast, the FV-1/3 group exhibited a viability rate of 67%, with one death recorded. The FV-1/4 group achieved a 100% viability rate, with no deaths reported. Similarly, the FV-1/0 group also showed a viability rate of 67%, with one death recorded.

Figure 10Figure 10 displays the following viability rates: 67% for the FB-1/1 ratio (indicating recorded mortality), 100% for the FB-1/3 ratio (indicating no mortality), 100% for the FB-1/4 ratio, and 67% for the FB-1/0 ratio.

Figure 8.Viability of worms according to the proportion rate of FPR substrates + soil.

Figure 9.Viability of worms according to the proportion rate of poultry manure substrates + soil.

Figure 10. Viability of worms according to the proportion rate of cattle manure substrates + soil.

Figure 11Figure 11 indicates a 100% survival rate for the species E.eugeniae and E.foetida when treated in urban waste.

Figure 11. Worm viability in the urban manure (100%).

An analysis of Figure 12Figure 12 indicates a 100% viability rate for both categories of earthworms in the mixture of plant waste (2/3) and municipal waste.

Figure 13Figure 13 presents a survival rate of 100% for Eudriluseugeniae when using a mixture of one-third plant waste and two-thirds municipal waste. In contrast, Eiseniafoetida showed a survival rate of 67% with the same mixture of one-third plant waste and two-thirds municipal waste.

Figure 12. Worm viability in Tv (2/3) + DU (1/3).

Figure 13. Worm viability in T-DU (2/3) + DV (1/3).

Figure 14Figure 14 illustrates a viability rate of 67% for the species Eudriluseugeniae and Eiseniafoetida when using a mixture of 95% plant waste and 5% poultry manure.

Figure 14. Worm viability in the DV (95%) + FV (5%).

Figure 15Figure 15 displayed a mortality rate of 100%, which corresponds to 3 deaths, for the species Eiseniafoetida. In contrast, the survival rate for the species Eudriluseugeniae was 67%, indicating one death.

Figure 15. Viability of worms in bare soil.

In this study, two species of earthworms were identified and described using the identification keys from [8] (Table 2), (Figure 16Figure 16), (Figure 17Figure 17).

Table 2.Comparison of the morphological characteristics of the different collected earthworm species.

Continued

Figure 16.Identification of the species Eiseniafoetida.

Figure 17.Identification of the species Eudriluseugeniae.

Earthworms play a crucial role in the dynamics of soil nutrients by changing the physical, chemical, and biological properties of the soil. They consume decomposing organic matter and soil mixed with organic materials, which enhances soil fertility, recycles organic matter, and enriches the soil with nutrients through their castings.

5.1.1. Morphological Characteristics of the Species Eiseniafoetida

The average size of *E.foetida* recorded in this study is 9.26 cm, which is comparable to the values reported by [12] at 9 cm and [13] at 8.5 cm. It exceeds the value reported by [14], which was 8 cm. The study also found an average weight of 0.79 g, higher than the weights reported by [7] and [15], which were 0.699 g, and [16], which was 0.078 g.

In terms of coloration, the observed red hue aligns with findings from [7], who described it as purplish-red with stripes and dark red, respectively. However, [13] reported brown coloration, while [12] noted yellow-striped patterns.

Additionally, the study revealed that the prostomium type is Epilobic, consistent with observations by [13] and [14], who also reported the same Epilobic shape. In contrast, [7] described the prostomium as zygolobic.

The investigation further indicated that the bristle position observed was of the Distant Pair type. This contrasts with findings from [12] and [14], who reported Germinate and Separate bristle positions, respectively.

The average number of segments noted was 159, which is higher than the counts documented by [13] and [14], at 90 and 101 segments, respectively. However, it is slightly lower than the 162 segments reported by [7].

The tail shape observed was flattened, similar to the cylindrical shape with slight caudal flattening reported by [14] and the cylindrical shape with flattening noted by [13].

Finally, the study identified the clitellum shape as saddle-shaped, which does not align with the annular shape observed by [7].

5.1.2. Morphological Characteristics of the Species Eudriluseugeniae

The average number of segments obtained was 267. This value is higher than those reported by [12]-[15], which were 207, 175, and 207 segments, respectively.

The average size observed was 16.68 cm. This result corroborates that reported by [17], which is 17 cm. It is lower than that reported by [16], which is 11.2 cm. It is higher than those obtained by [7] and [12], who obtained 15 cm.

The shape of the clitellum observed was saddle-shaped. This result contrasts with that obtained by [7], which was annular.

The position of the setae observed was close-set. However, this is contrary to the findings reported by [7] and [12], who found the setae to be separated.

The prostomium shape is zygolobic. This result contradicts that obtained by [7], which is open epilobic.

The observed coloration is black. This result does not corroborate those observed by [7]-[16], who obtained dark pink to violet and dark red, respectively.

The average weight obtained was 2.33 g. This result is similar to that obtained by [17], which is 2 g, and is lower than that obtained by [7], which is 4 g.

The shape of the tail observed was circular.