**2. Materials and methods**

balance, due to bioaccumulation, leaching and extension of contaminants into groundwater with potential effects on all living organisms [8]. In Mexico, current environmental regulations is the NOM-138-SEMARNAT/SS-2003 which establishes the maximum permissible limits of hydrocarbons in soil and specifications for its characterization and remediation [9], although in this standard are omitted criteria for assessing chronic effects on soil microorganisms and plants as a result of weathered oil contamination. However, the contributions of several studies show that pollution and waste generation caused by oil activities have deteriorated soil quality [10,11]; ie, the social responsibility of the industry and the government should not only attend

Crude oil is a complex mixture of thousands of compounds that when released to the envi‐ ronment, is subjected to physical, chemical and biological processes called weatherability [13]. This process includes adsorption, volatilization, dissolution, biodegradation, photolysis, oxidation, and hydrolysis. The effects of weathering are difficult to predict because it depends on many biotic and abiotic factors [14]. Therefore, the mobility of hydrocarbons is also influenced by natural factors [15], which involve: a) chemical processes (hydrolysis, oxidation, reduction, photolysis), b) transport and physical processes (adsorption, advection, dispersion, diffusion, volatilization and dissolution), and c) biological processes (biodegradation, metab‐

When an oil spill occurs, it covers the soil surface, but initially at high viscosity prevents penetration towards the subsoil. The oil is retained in the topsoil, during this phase, the light fraction is photo-oxidized and volatilized through the soil pore space and transported to the atmosphere, in this process involves the first n-alkanes (methane, ethane, propane and butane), which are evaporated in less than 24 hours in tropical climates [18]. The hydrocarbons which are not evaporated are incorporated into the soil to form a waterproofing layer that prevents the normal flow of water. This layer or film affects the structure, porosity, absorption and water

Subsequently, the oil soluble fraction diffuses into the soil solution through infiltration. The behavior of this fraction in the soil depends on the type of texture. The presence of fine texture allows the volatilization of some compounds (C5-C7), but when the texture is coarse, it can leach out and transport themselves to the groundwater, affecting other organisms [20], including the human [21-23]. Otherwise, clay soils rich in organic matter immobilize some compounds, reducing their toxicity and decreasing its spread and leaching through the soil

The most stable fraction of crude oil is composed with more than 18 carbons (Polycyclic Aromatic Hydrocarbons [PAHs] and polar compounds), which are adhered on the soil matrix decreasing the solubility and volatility [26], and increasing the capacity of adsorption on the mineral and organic fractions, owing to the high content of active surface of the soil (clay 2:1) and to the high molecular weight hydrocarbons [27]. At this stage, the development of bacteria and fungi have influence on mineralization as part of natural attenuation process, but it is not

acute environmental emergencies [12].

88 Environmental Risk Assessment of Soil Contamination

olism and toxicity) [16, 17].

penetration into the soil [19].

sufficient to remove all hydrocarbons weathered [28].

profile [24, 25].

In southeastern Mexico, the land has been affected as a result of extraction; handling and transportation of oil, there are also zones of oil discharges, which are deposited in the open, without any environmental protection measure [32]. The bad condition of the pipelines and the dispersion through surface runoff of rainwater, resulting from weather conditions become more complicated to calculate the chronic effects of soil contamination by hydrocar‐ bons [10]. Accordingly, this study was conducted in three stages, which were analyzed the chemical and physical properties of the soil, the toxic effects of weathered hydrocarbons on the growth and development of seedlings and after 150 and 240 days of exposure, and finally was evaluated the behavior of beneficial soil microorganisms in rhizospheric soil.

#### **2.1. Effect on the physical and chemical properties of the soil**

The study was undertaken at the facilities the *Colegio de Postgraduados*, located in Tabasco, Mexico. The soil with weathered oil was collected within 2 km of the Petrochemical "La Venta" (18 ° 04 '54 "N and 94 ° 02' 31" W), Figure 1. The pollution-free soil according to NOM-138- SEMARNAT/SS-2003 was located in the community of Santa Teresa Arroyo Hondo. The objective was to identify the types of soils and their level of similarity.

For both soils were determined the content of organic matter (Walkley and Black), pH (potentiometry), P and K exchangeable (by extraction with 1N ammonium acetate pH 7, quantification by atomic absorption and emission respectively), CEC (extraction 1N ammoni‐ um acetate pH 7, quantification by distillation and titration) and texture (Bouyoucos). The analytical methods used were those indicated in NOM-021-2000-RECNAT [33]. Total Petro‐ leum Hydrocarbons (TPH) were determined by the technique reported in the NOM-138- SEMARNAT/SS-2003 [9].

#### **2.2. Effects on plants**

Bioassays to determine the phytotoxicity of weathered oil on two species of legumes (*Crotalaria incana* and *Leucaena leucocephala*) were carried out in the facilities of the Laboratory of Soil Microbiology, Colegio de Postgraduados, Tabasco, Mexico. Two states were studied phenological (plant and seedling), in two treatments: a) soil with 150 mg.kg-1 TPH (control treatment) and b) soil with 79.457 mg.kg-1 TPH weathered (this has been contaminated by over 25 years). Both soils were characterized as Gleysols, with the same pedogenetic origin, as described in the previous section.

Bioassays were established under a Completely Randomized Design (CRD) with three replications and two legumes, these were selected to be species that grow wild in oiled areas, but the former has tolerance, while the second shows sensitivity to high concentrations of crude oil [34]. In each bioassay was used 208 Protocol of the Organization for Economic Cooperation and Development (OECD) modified according to [35], which allows easily identify the symptoms of stress in the plant.

*Seedling bioassays*: 50 seeds were sown *C. incana* and 25 *L. leucocephala* by repetition, respectively. Glass containers were used (32 x 22 x 5.5 cm). The number of seeds sown per plant species was calculated according to the size of the seed [36], the viability of the species [37] and the area of the container. Seeds were previously scarified to remove impermeable integument, which constitute a barrier for germination [38]. Scarification consisted of immersing the seeds in sulfuric acid for 15 minutes and washed with tap water subsequent to remove all acid residues [39]. Germination tests were performed to determine the initial seed viability, finding viability standard values in both blocks [40]. The test lasted 30 days and the variables evaluated were mortality, height, root length and dry matter accumulation aerial and root.

*Plant bioassays*: Bioassays were established plants seedlings 30 days from uncontaminated soil. Subsequently, two of these were transplanted into containers, 15 days after a plant was removed from each container. Exposure of plants to pollutant lasted 150 days to *C. incana* and 240 days for *L. leucocephala*, because of its tolerance respective. The physiological variables were evaluated: height, root length, biomass (leaves and stems), root biomass and number of nodes, leaves and fruits. During both assays was provided with water to field capacity and were not supplied nutrients (N, P and K) to avoid interference on the growth of the specimens. The material was weighed on an analytical balance to obtain the values of DM. The multiple comparison of means was performed by Tukey test (a = 0.05). The numerical results were analyzed with SAS software version 9.1, using PROC GLM.

#### **2.3. Effect on soil microorganisms**

Quantification of microorganisms was determined by viable count method for serial dilutions [41] for *Rhizobium* extracts in nodules and Free Living Nitrogen Fixing Bacteria (FLNFB) in rhizospheric soil. Culture media were used combined carbon and yeast-mannitol agar [42].
