# Bioremediation of Hydrocarbon-Polluted Soil: Evaluation of Different Operative Parameters
## 2 Materials and Methods
### Response Variable
#### 2.4.2 Determination of the TPH Concentration in Test B  
The response variable was measured with samples collected from each biopile. Regarding this issue, a procedure was established to standardize the sample collection.  
The biopiles were appropriately homogenized before the collection, destroying their initial geometry design (truncated pyramid), and intensively mixing all the mass, emphasizing the destruction of lumps; 5 samples of mass around 100 g, were taken from the four corners of the basin and the centre, alternating between bottom and surface samples.  
At last, a quartering was done, and 100 g was taken for the determination of the TPH concentration. The samples were uniformly dried and grounded as part of the pretreatment  
\begin{table}
\begin{tabular}{c c c} \hline
**Operative Conditions** & **Microcosms (Test M)** & **Biopiles (Test B)** \\ \hline Response variable & TPH & TPH \\ \hline  & M1 (8 WC\%-120 C/N) & B1 (sugarcane bagasse) \\  & M2 (8 WC\%-180 C/N) & B2 (sugarcane filter cake) \\ Factor 1-Treatments & M3 (12 WC\%-120 C/N) & B3 (sawdust) \\ (6 categorical levels) & M4 (12 WC\%-180 C/N) & B4 (coffee pulp) \\  & M5 (15 WC\%-120 C/N) & B5 (beef manure) \\  & M6 (15 WC\%-180 C/N) & B6 (_Thalassia testadinium_) \\ \hline  & 0 & 0 \\ Factor 2-Time & 15 days & 60 days \\ (5 levels) & 70 days & 90 days \\  & 112 days & 150 days \\  & 131 days & 240 days \\ \hline Mass & 200 g & 38 kg \\ \hline Pollutant & Diesel oil & Oily Sludge \\ \hline  & 8\% & \\ Water content (WC\%) & 12\% & 20\% \\  & 15\% & \\ \hline  & 60 & \\ Carbon to nitrogen ratio (C/N) & 120 & 10 \\  & 180 & 300 \\ \hline Kind of soil & Sandy soil & Sandy soil \\ \hline pH & 6–8 & 6–8 \\ \hline \end{tabular}
\end{table}
Table 1: Operative conditions for the studied tests.  
to increase homogeneity. Then, they were spread on an aluminum foil inside a room at a controlled temperature of 30 \({}^{\circ}\)C for 48 h.  
The TPH concentration was determined by adapting the EPA Method 1664 to the biopile system. The analysis is based on the TPH extraction from solid by an organic solvent and evaluation of the TPH mass by gravimetry. This method is reliable when the solid sample is over 5 g and the TPH concentration is over 0.3% by weight.  
The applied method consists of the extraction of a dry soil sample with \(n\)-hexane in a microwave at 120 \({}^{\circ}\)C for 20 min. Then, the extract (\(n\)-hexane + TPHs) is filtered through a funnel containing anhydrous Na\({}_{2}\)SO\({}_{4}\). After, the solution is distilled at 85 \({}^{\circ}\)C in a water bath. The residual mass is weighted to estimate the TPH quantity. The TPH concentration is achieved by the ratio of TPH mass to the dry sample soil mass.  
The monitoring of the TPH concentration was done at 0, 60, 90, 150, 240 days, corresponding with the levels of the factor \"Time\".  
Test B has combined all the levels of the factors (6 \(\times\) 5). There was a total of 120 runs due to four replicas of the experimental array. This ensured 90 degrees of freedom, which were considered statistically robust, compensating for the weakness of the gravimetric determination method if compared to the gas chromatographic one.