**3. Results and discussion**

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

The soil profile and laboratory results confirmed that both soils are Gleysols, with the same pedogenetic origin [43] (Table 1), but the contamination had abnormalities in their chemical characteristics (Table 2), the which have been reported in other studies undertaken in the South East (Table 3).

**Figure 1.** Study zone

25 years). Both soils were characterized as Gleysols, with the same pedogenetic origin, as

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

*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

*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

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].

The soil profile and laboratory results confirmed that both soils are Gleysols, with the same pedogenetic origin [43] (Table 1), but the contamination had abnormalities in their chemical characteristics (Table 2), the which have been reported in other studies undertaken in the South

mortality, height, root length and dry matter accumulation aerial and root.

analyzed with SAS software version 9.1, using PROC GLM.

**3.1. Effect on the physical and chemical properties of the soil**

**2.3. Effect on soil microorganisms**

**3. Results and discussion**

East (Table 3).

described in the previous section.

90 Environmental Risk Assessment of Soil Contamination

symptoms of stress in the plant.


\* pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos).

† Control soil

†† Soil with weathered oil (OM without previous removal of TPH)

**Table 1.** Chemical properties and concentration of TPH in the soils studied.


†† Soil with weathered oil (OM without previous removal of TPH)

**Table 2.** Interpretation of the characteristics of the soils studied. The interpretation was based on the ranges indicated in NOM-021-2000-RECNAT. Alterations Effects High ratios of C / N and C / P Unfavorable microbial growth [44]

Table 3. Alterations reported in soils contaminated with TPH, in southeastern Mexico.


**Table 3.** Alterations reported in soils contaminated with TPH, in southeastern Mexico. cations Ca and K and K [32]

Also, many physical properties of soil are altered, such as water retention capacity; this is because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49]. On the other hand, increasing the moisture content in the soil reduces the adsorp‐ tion of liquid hydrocarbons in the organic matter and clay and the filling of the pores and capillaries (Figure 2) [50]. Also, many physical properties of soil are altered, such as water retention capacity; this is because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49]. On the other hand, increasing the moisture content in the soil reduces the adsorption of liquid hydrocarbons in the organic matter and clay and the filling of the pores and capillaries (Figure 2) [50].

Figure 2. Alteration in the retaining moisture of soil. **Figure 2.** Alteration in the retaining moisture of soil.

The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die. The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die.

7

**Figure 3.** Formation of macroaggregates in soil with 12,155 mg.kg-1 TPH weathered.

#### **3.2. Effects on plants**

**Sampling**

in NOM-021-2000-RECNAT.

up to 5.6 times

cations Ca and K

capillaries (Figure 2) [50].

capillaries (Figure 2) [50].

**0-30cm pH**

92 Environmental Risk Assessment of Soil Contamination

†† Soil with weathered oil (OM without previous removal of TPH)

**Alterations Effects**

Alterations Effects

The electrical conductivity can be increased up to 5.6 times Salinity [48]

Figure 2. Alteration in the retaining moisture of soil.

**Figure 2.** Alteration in the retaining moisture of soil.

The electrical conductivity can be increased

Interruption in the interaction between

**Table 3.** Alterations reported in soils contaminated with TPH, in southeastern Mexico.

High ratios of C / N and C / P Unfavorable microbial growth [44] Retaining TPH fractions in organic matter Alter the solubility of phosphorus [45]

Increasing Na Limitations in the production of plants [11, 46]

Decreasing pH Decrease microbionas populations [47]

Interruption in the interaction between cations Ca and K Reduction in capacity of soil to retain Ca and K [32]

Also, many physical properties of soil are altered, such as water retention capacity; this is because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49]. On the other hand, increasing the moisture content in the soil reduces the adsorp‐ tion of liquid hydrocarbons in the organic matter and clay and the filling of the pores and

Also, many physical properties of soil are altered, such as water retention capacity; this is because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49]. On the other hand, increasing the moisture content in the soil reduces the adsorption of liquid hydrocarbons in the organic matter and clay and the filling of the pores and

The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long

The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period

period of contact [51], so that the plants may suffer water stress and die.

of contact [51], so that the plants may suffer water stress and die.

Decreasing pH Decrease microbionas populations [47]

Control soil Moderately acid Medium High Medium Very High Polluted soil †† Strongly acidic Very high Low Medium Very High

Table 3. Alterations reported in soils contaminated with TPH, in southeastern Mexico.

High ratios of C / N and C / P Unfavorable microbial growth [44]

Retaining TPH fractions in organic matter Alter the solubility of phosphorus [45]

**Table 2.** Interpretation of the characteristics of the soils studied. The interpretation was based on the ranges indicated

Increasing Na Limitations in the production of plants [11,

46]

Salinity [48]

and K [32]

**OM P K CEC (%) mg.kg-1 C mol(+) kg-1**

Reduction in capacity of soil to retain Ca

7

The results indicated that the lower height and root length were found in soil with 79,457 mg.kg-1 of weathered oil, this can be explained because of the limited development of these. Both legumes formed less biomass in soils with higher concentrations of oil, which was associated with lower production of biomass in leaves and stems, as a result of the presence of the contaminant in the soil (Table 4 and 5), which limits the entry of water to the plant. However, the hydrophobic effect also affects the early stages of germination as evidenced with a delay of up to five days in the emergency [52], even in high concentrations of TPH, germi‐ nation may be inhibited completely [53].

Some authors found that exposure to concentrations of 2 791, 9 025 and 79 457 mg.kg-1 of petroleum hydrocarbons in soil inhibited the vegetative growth and reduced plant biomass in seedlings of *Echinochloa polystachya*, *Brachiaria mutica* and *Cyperus sp* [10]. Others establish increased toxicity on the dry weight of rice seedlings (*Oryza sativa*) after 25 days of exposure to 90,000 mg.kg-1 weathered oil [54]. Biomass reduction is possibly due to widespread damage, which begins in the root system, hindering vegetative growth and therefore the accumulation of plant biomass. However, the first studies mentions that at low concentrations, oil could stimulate vegetative growth [55].

*Crotalaria incana* seedlings did not form nodules in soil contaminated with 79,457 mg.kg-1 of weathered oil after 30 days of exposure to oil. These results were similar to those obtained according to [34], who found that nodulation was completely inhibited in *Crotalaria sp*. and *Mimosa pigra* by concentrations above 50,000 mg.kg-1.

In both plant species, growth decreased with higher concentration of hydrocarbons in soil (Table 5 and 6). This response may be related to decreased water absorption through the roots for the presence of the hydrophobic film formed by the oil added to the soil [56]. The lack of water absorbed decreases cell turgor, reduces or inhibits the processes of incorporation of nutrients and also affects vegetative growth [57, 58]. Water stress is related to the water potential gradient (which depends on the conditions present in the soil) and the membrane permeability to water (which depends on the species [59] therefore, *Leucaena leucocephala* shown to be more tolerant to water deficit).


Values with different letter are statistically different (Tukey, p = 0.05)

**Table 4.** Response of *Crotalaria incana* seedlings to 30 days of weathered oil exposure.


Values with different letter are statistically different (Tukey, p = 0.05)

**Table 5.** Response of *Leucaena leucocephala* seedlings to 30 days of weathered oil exposure.

*Crotaria incana* plants showed a greater effect on biomass in response with exposure to the weathered oil (Figure 4), which may be related to increased toxicity of recalcitrants com‐ pounds. There are also some soil properties which allow the adsorption of pollutants [60]. Clay soils with high organic content and low pH may favor the persistence of toxic substances in the soil for a long time after the oil spill occurred [61, 34], due to the adhesiveness of organic matter [62].

Furthermore, fine texture allows the oil form a coarse structure on the outside and around the conglomerate making it waterproof [63], this has effects on root development, growth, and as a result will cause decrease in the accumulation biomass [59]. On the other hand, soil contam‐ ination by hydrocarbons can also modify some characteristics such as texture, bulk density, ratio of the particle size of the soil, reducing aeration and affecting the productive development of plants [64, 65].

11

*Crotaria incana* plants showed a greater effect on biomass in response with exposure to the weathered oil (Figure 4), which may be related to increased toxicity of recalcitrants compounds. There are also some soil properties which allow the adsorption of pollutants [60]. Clay soils with high organic content and low pH may favor the persistence of toxic substances in the soil for a long time after the oil spill occurred [61, 34], due to the

Furthermore, fine texture allows the oil form a coarse structure on the outside and around the conglomerate making it waterproof [63], this has effects on root development, growth, and as a result will cause decrease in the accumulation biomass [59]. On the other hand, soil

Figure 4. Toxicity on *Leucaena leucocephala* seedlings with: a) 150 mg.kg-1 y b) 79,457 mg.kg-1 TPH weathered, and plants with c) 150 mg.kg-1 y d) 79,457 mg.kg-1 TPH weathered. **Figure 4.** Toxicity on *Leucaena leucocephala* seedlings with: a) 150 mg.kg-1 y b) 79,457 mg.kg-1 TPH weathered, and plants with c) 150 mg.kg-1 y d) 79,457 mg.kg-1 TPH weathered.


Values with different letter are statistically different (Tukey, p = 0.05)

adhesiveness of organic matter [62].

development of plants [64, 65].

In both plant species, growth decreased with higher concentration of hydrocarbons in soil (Table 5 and 6). This response may be related to decreased water absorption through the roots for the presence of the hydrophobic film formed by the oil added to the soil [56]. The lack of water absorbed decreases cell turgor, reduces or inhibits the processes of incorporation of nutrients and also affects vegetative growth [57, 58]. Water stress is related to the water potential gradient (which depends on the conditions present in the soil) and the membrane permeability to water (which depends on the species [59] therefore, *Leucaena leucocephala*

shown to be more tolerant to water deficit).

**Height**

Values with different letter are statistically different (Tukey, p = 0.05)

Values with different letter are statistically different (Tukey, p = 0.05)

**Table 4.** Response of *Crotalaria incana* seedlings to 30 days of weathered oil exposure.

**Root**

**Table 5.** Response of *Leucaena leucocephala* seedlings to 30 days of weathered oil exposure.

**length Mortality**

*Crotalaria* 150 9.8a 9.8a 2.6a 1.6a 1.0a 2.6a 0.6a 3.2a

*Crotaria incana* plants showed a greater effect on biomass in response with exposure to the weathered oil (Figure 4), which may be related to increased toxicity of recalcitrants com‐ pounds. There are also some soil properties which allow the adsorption of pollutants [60]. Clay soils with high organic content and low pH may favor the persistence of toxic substances in the soil for a long time after the oil spill occurred [61, 34], due to the adhesiveness of organic

Furthermore, fine texture allows the oil form a coarse structure on the outside and around the conglomerate making it waterproof [63], this has effects on root development, growth, and as a result will cause decrease in the accumulation biomass [59]. On the other hand, soil contam‐ ination by hydrocarbons can also modify some characteristics such as texture, bulk density, ratio of the particle size of the soil, reducing aeration and affecting the productive development

**Height**

**Root**

**length Mortality**

*Leucaena* 150 7.5a 19.0a 0.0a 3.3a 1.3a 4.6a 1.1a 5.7a

**cm (%) gr**

79,457 5.1b 5.1b 4.2a 1.5b 0.5b 2.0b 0.5b 2.5b

**cm (%) gr**

79,457 2.9b 2.8b 28.6a 1.0a 0.5b 1.5b 0.4b 1.9b

**Biomass Leaves Stems Aerial Root Total**

**Biomass Leaves Stems Aerial Root Total**

**Concentration mg.kg-1**

94 Environmental Risk Assessment of Soil Contamination

**Concentration mg.kg-1**

**Species**

**Species**

matter [62].

of plants [64, 65].

**Table 6.** Response of *Crotalaria incana* to 150 days of weathered oil exposure.


**Table 7.** Response of *Leucaena leucocephala* to 240 days of weathered oil exposure.

#### **3.3. Effect on soil microorganisms**

In plants *Leucaena leucocephala* high concentration of weathered oil did not affect populations of *Rhizobium* into nodules, as happened in the case of *C. incana*, in which both populations were significantly lower in a shorter time exposure (Figure 5). This is because the oil alters the physical and chemical characteristics of the soil, causing the blockage of gas exchange with the atmosphere and affecting microbial populations. Furthermore, the weathered oil is adsorbed in the ground, being less accessible and more difficult to degrade by microorganisms [66, 67]. This may bring a direct impact on rhizobia, because they are aerobic bacteria that remain in the soil as saprophytes, until they infect a radical hair.

Some authors mention that soil conditions have a marked effect on rhizobia, because they can impact the survival and the infectivity of root hairs [68, 69]. However, there are many other factors that influence on effectiveness symbiosis such as specificity and virulence of the bacterium *Rhizobium*, nutrimental factors, soil temperature and pH [70], the latter is of utmost importance because has been reported to decrease significantly in contaminated soil [38]. Added to this there are other factors such as the accumulation of heavy metals and salts that affect soil microbiota [11, 4].

**Figure 5.** Quantification of populations of *Rhizobium* extracts in nodules and Free Living Nitrogen Fixing Bacteria in rhizospheric soil.

*L. leucocephala* almost doubled FLNFB populations with 79,457 mg.kg-1 of weathered oil. This can be explained because in tolerant species, some microorganisms can increase their popu‐ lations in the presence of hydrocarbons [71], allowing support microbial growth. As well as [72] argue that tolerant plants are promising tools to accelerate the removal of PAH in long term polluted soils, due to their ability to thrive in a contaminated site, and its success is probably influenced by the relative amount of exudates and other compounds within the root, that stimulate microbial growth [73]. On the other hand, several authors argue that changing the C: N: P, the water content and the water retention capacity of clay soils are crucial to obtain the highest rate of degradation of TPH [74, 75].
