**2. Materials and methods**

### **2.1 Soil sampling and analysis**

The metal-contaminated soils were sampled from the abandoned dumping site located in M'Ploussoue Park, Bonoua, Ivory Coast, at latitude 5°16<sup>0</sup> N and longitude 3° 360 W. Soil samples were collected at 18 different points from the surface horizon (0– 30 cm) to cover the entire study area according to the random sampling technique.

The metal-contaminated soil samples were air-dried, sieved to 2 mm, and then were mixed and homogenized to obtain a composite sample. The composite sample was transferred to the laboratory for various analyses and their used for the pot experiment. Some properties of this metal-contaminated soil, which has been previously described by [18], are summarized in **Table 1**. The concentrations of Cd (81 mg kg�<sup>1</sup> ), Cr (130 mg.kg�<sup>1</sup> ), Pb (118 mg kg�<sup>1</sup> ), and Ni (119 mg kg�<sup>1</sup> ) in dumpsite soil are greater than limit values recommended for agricultural soil. However, previous studies performed in pot experiment with polluted soil and *A. mangium*<sup>5</sup> have revealed that only lead, chromium, and nickel concentrations in plant biomass were above the detection limit. Thus, cadmium was not detectable in plant biomass. This stipulates that in the soil, Cd is neither mobile nor exchangeable and is therefore not bioavailable for plants. While for Pb, Ni, and Cr, these metals are bioavailable for plants [5] justifying the choice of the three metals used in this study.

### **2.2 Biological material**

Seeds of *A. mangium* were obtained from the CNRA (Centre National of Research Agronomy) at Oume, Ivory Coast). Seeds were treated with concentrated sulfuric acid (95%) to break hard seed dormancy before germination, as described by [22]. The treated seeds were pregerminated in a Petri dish containing 0.8% water-agar medium (w/v) and sterilized for 30 min at 110°C. Then, the Petri dish was stored at room temperature (30°C) in the dark for 72 hours, after packing with aluminum paper.


### **Table 1.**

*Physicochemical and chemical properties of the contaminated soil used in the pot experiment.* � *standard errors.*

Before germination in the Petri dish, three pregerminated seedlings were transplanted into polyethylene plastic nursery bags (1540150 cm) filled with polluted soil sieved at 2 mm. One month after transplantation in the plastic nursery bags, one seedling of uniform size was transferred into perforated pots filled with 5 kg of dry polluted soil.

*Bradyrhizobium* ORS strains were obtained from the collection of the Laboratory of Soil Microbiology by Institute Research Senegal Agricultural (IRSA), Dakar. *Bradyrhizobium* ORS strains immobilized in an alginate ball were suspended in sterile buffer solution (23 g K2HPO4, 14.6 g KH2PO4, and adjusted at 1liter of distilled water). Five milliliters of *Bradyrhizobium* solution was introduced around the roots of the seedlings after transplantation into plastic pots and then after 1 week. The uninoculated treatment (plant uninoculated with *Bradyrhizobium*) received a similar amount of buffer solution with sterilized inoculum to minimize any possible variation in soil properties.

Earthworms (*P. corethrurus*) were hand-collected from Felix Houphouet-Boigny University, Cocody, Abidjan, Ivory Coast, in not metal-polluted soil. The earthworms were then kept in plastic boxes filled with water for 1 week to monitor their health before starting the experiment. For the treatment with earthworms, five adult earthworms with 5 g as of weight biomass were placed in the perforated pot after transplantation of the seedling.
