**2. Materials and methods**

The experimental protocol was installed in the field to the Agricultural Experiment Station of Oued Souhil - Nabeul, situated about 60 kilometers from Tunis and belonging to the National Institute for Research in Rural Engineering Water and Forest.

The urban mud used in this study is taken from the wastewater treatment plant in Korba with a treatment system at low load activated sludge followed by maturation. Sludge from this station underwent a stabilization in aerobic followed by drying on beds. The dry sludge is removed from the drying bed.

The industrial mud is provided from wastewater treatment plant Bou Argoub which hosts two big companies, the Tunisian beverage manufacturing company (SFBT) specialized in the food industry, and Assad company specialized in the electrical industry. Sludge from this station underwent a stabilization in aerobic followed by drying on beds. This sludge is loaded with heavy metals especially lead and chromium.

The plant materials that were used in this experiment are the rapeseed (*Brassica napus*), which is an annual plant with yellow flowers of the family Brassicaceae and durum wheat (*Triticum turgidum*) that can be defined as a species of wheat characterized by its hard and glassy kernel. Rapeseed was chosen for its ability to accumulate metals and also because it is one of the three main sources of edible vegetable oil with sunflower and olive.

Experimentation was carried out on two juxtaposed plots reserved for each crop (wheat or rapeseed). For each type of sludge, four doses (5, 25, 50 and 100 t ha−1) were used. Results were compared to a control soil without any treatment.

Sludges were manually dug into the soil. Before utilization, the sludge was analyzed.

The soil was analyzed before the application of sludge and after the harvest. Sampling was conducted between the lines using an auger at four depths (0–10, 10–20, 20–40 and 40–60 cm).

In the laboratory, soil samples were dried in open air and sieved to 2 mm or 0.2 mm depending on the type of analysis required. The main measured parameters

### *Impact of the Spreading of Sludge from Wastewater Treatment Plants on the Transfer… DOI: http://dx.doi.org/10.5772/intechopen.103745*

were particle size, total calcium, conductivity, carbon, organic matter, total nitrogen and heavy metals concentration. For the particle size, we used the method of the International pipette Robinson, which is essentially based on the destruction of organic matter in the soil using H2O2 and the dispersion of clays by sodium hexametaphosphate. Clays and silts are measured in the suspension of land following the decay time that depends on particle diameter (NF X 31–107). The settling velocity was measured by the formula of Stokes. The Mud and soil samples were analyzed by XRF (X-Ray Fluorescence) and ICP-AES (Inductive Coupled Plasma Atomic Emission Spectrometry Activa–Horiba Jobin Yvon Spectrometer) in the Geosciences and environment Department of National School of Mines in Saint Etienne. The Soil pH was measured by using a 1:2 soil to water ratio. Plant samples were washed with tap water and rinsed three times with distilled water, then separated into leaves, stems and roots, dried at 40°C to constant weight, crushed and sieved at 2 mm. Moreover, the digestion of plant samples was performed using nitric concentrated acid, according to [5–8]. The plant extracts were analyzed by ICP-AES.

The sowings were performed with 50 seeds m2–1 for rapeseed and 350 seedsm2–1 for wheat. The rapeseed harvest was performed after the formation of slices. We weighed the aerial part and the root. The same work was done to wheat. The samples were subsequently dried and crushed ore to determine the mix of metals in different parts of the plant. The different parts of the plant were dried at 80°C to constant weight and then crushed to a fine powder using a porcelain mortar to prevent metal contamination. Digestion is done at high temperature (70°C) with aqua regia. For histological analysis, preparing the samples carefully for transmission microscopy was essential for obtaining reliable results. Therefore, samples were set at 4°C with a solution of 20.5% glutaraldehyde, pH was maintained at 7.4 with a solution of sodium cacodylate (0.1 M). The samples were then washed with sodium cacodylate buffer (0.1 M) and post-fixed in a solution of 1% osmium tetroxide in veronal buffered (0.1 M) [9]. After several washes in distilled water, the samples were dehydrated with a graded ethanol series of increasing concentrations going from 30 to 100%. The final inclusions were made from a mixture of resin [10]. Only the sections with interference colors are gray or silver, that is to say (thickness of 600 to 900A° (1A° = 0.1 nm)) were collected and deposited on a copper grid with 3 mm diameter. The ultrathin sections were mixed using an alcoholic solution of uranyl acetate and 7 by 1% lead citrate [11]. On top of that, observations were made using a Hitatchi H800 electron microscope.

The data were subjected to analysis of variance. The comparison of means at 5% level of significance was performed by the Newman–Keuls test using the Statistica 7 software.

The amount of heavy metal in sludge is not a good indicator for metal availability for *T. turgidum* plant uptake; accumulation factors were calculated based on metal availability and its uptake by a particular plant. A calculation of biological concentration factor (BCF) was as in Eq. 1, biological accumulation factor (BAF) as in Eq. 2, and transfer factor (TF) as equation

BCF = Metal content (mg kg−1) in root/metal content (mg kg−1) in sludge (1)

BAF = Mean metal content (mg kg−1) in shoot (root+straw+spike)/metal content (mg kg−1) in sludge (2)

TF = Mean metal Content (mg kg−1) in shoot (root+straw+spike) /metal content (mg kg−1) in root (3)
