# Evolution of the Residual Pollution in Soils after Bioremediation Treatments
## 3 Results and Discussion
### Pollutant Concentration in Soils
australis, F. angustifolia, and P. alba_ the most effective species for reducing PHEs availability in the soil [40], based on the accumulation of pollutants in the root system and the low transfer of PHEs from roots to leaves. The use of shrubs was also implemented in the bioremediation of the area, being _Myrtus communis, Retama sphaerocarpa, Rosmarinus officinalis_ and _Tamarix gallica_ the most used species; in this case, the transfer from soil to aerial parts of the plants was low and the decrease in the availability of PHEs in soils was promoted in most cases, highlighting the _R. sphaerocarpa_ as the most adequate species for phytostabilize these type of soils [38, 41]. Moreover, the use of shrubs as nurse plants to improve and favor the growth of trees and the phytostabilization of the area under Mediterranean conditions is recommended [39]. Therefore, the implementation of the phytostabilization in the area presented a positive evolution by the increase in the percentage of plant cover and diversity [6]. No previous studies in such a long-term real case of phytostabilization (20 years) are found for this study area in relation to the toxicity assessment. The high concentration of As and Pb in some sectors of the recovered area, together with the extensive growth of the vegetation, require the monitoring of the soil-plant transfer of PHEs and the assessment of the potential toxicity by the use of bioassays. In this sense, we conducted a short-term toxicity bioassay based on the root elongation of lettuce (_Lactuca sativa_ L.) seeds in these soils. This test evaluates the phytotoxicity using the soil solution of potentially polluted soils and the root elongation of the lettuce seeds after five days of germination. Our results (Figure 4) indicated a significant reduction of the root elongation in relation to the control (distilled  
Figure 3: Cumulative frequency-distribution curves of the main PHEs twenty year after the accident and in the three studied depths (**a**) Total Pb; (**b**) Total As; (**c**) Total Zn; (**d**) Total Cu concentrations. water) in around 25% of the soils into the GGC. According to the different soil types, acidic sector (S1) presented higher phytotoxicity, with a root elongation reduction around 60% in the 25% of the soils in this sector and no toxicity was detected in the 10% of the sampled soils. Otherwise, in the neutral-alkaline sector (S2), around 90% of the soils presented no phytotoxicity, and a slight elongation reduction (around 20% in relation the control) was detected in only the 5% the samples in this sector. The correlation analysis between the root elongation and the main properties and pollutants in the soils indicated differences in S1 in relation to S2. The variables with higher influence in the phytotoxicity in the acidic sector (S1) were the pH, electrical conductivity (EC) and total concentration of As, with significant correlation coefficients (\(p\)\(<\)0.05) of 0.790, \(-\)0.663 and \(-\)0.488, respectively. These results indicate that the acidity, the presence of soluble salts coming from the sulphide oxidation and the high concentration in As, were strongly related with the reduction in the root elongation of lettuce seeds. These results agree with previous toxicity bioassays developed in soil of the area, where a potential risk of pollution was reported after root elongation and soil respiration tests [42]. Otherwise, in the neutral-alkaline sector (S2) the low percentage of samples in relation to the total area that showed phytotoxicity hinders the potential relationship among variables, so further studies in the potentially polluted sites of the neutral-alkaline sector are needed. Other studies in the area found a potential ecotoxicity risk by a reduction in the abundance of ectomycorrhizal fungi associated with holm oak [43], or by accumulation of Cd and Zn in leaves of _Populus alba_ growing in these soils [44; 45].