## 3 Results and Discussion
### Overview of Metabolites Discovered in Filed Soil (Contaminated Site)  
The metabolites in the soil near the accident site were excavated to trace the ecological effects and find the biomarker metabolites having a trace of toluene fire accident by identifying metabolites formed in toluene-affected soil (field soil) for three years after the chemical accident had occurred. PLS-DA was applied to assess the propensity of the metabolites. PLS-DA analyses enable highlighting differences between control and field (contaminated soil) (Figure 3). PLS-DA results showed a clear separation of contaminated soils located within 500 m and control soil located at 1 km away from the chemical accident site. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Field** & \multicolumn{3}{c}{**Texture (\%)**} & \multirow{2}{*}{**pH**} & **GRP** & **CEC** & **TOC** & \multirow{2}{*}{**Mineral**} \\
**Soil** & **Sand** & & **Silt** & & & **(mV)** & **(cmol/kg)** & **(\%)** \\ \hline
100 m & 68.57 & 15.42 & 16.01 & 7.27 & 113.4 & 21.64 & 1.05 & \\
200 m & 69.42 & 15.24 & 15.34 & 6.67 & 137.8 & 29.87 & 1.05 & Kaolinite \\
300 m & 69.56 & 13.95 & 16.49 & 6.14 & 179.0 & 23.40 & 0.39 & Halloysite \\
400 m & 57.78 & 28.54 & 13.68 & 6.97 & 106.9 & 20.76 & 0.62 & Anorthite \\
500 m & 76.36 & 11.00 & 12.64 & 8.29 & 44.1 & 23.43 & 1.25 & Quartz \\ \hline \hline \end{tabular}  
* ORP: Oxidation-reduction potential (mV); CEC: Cation exchangeable capacity (cmol/kg); TOC: Total organic carbon (%).  
\end{table} 
Table 2: Soil characterization of field soil (contaminated soil). Figure 3 shows that field soil does not currently recover metabolically comparing control soil (not contaminated soil) because the \"field\" group is not close to the \"control\" group on the PLS-DA score plot, which means components of metabolites (variables) in two soil groups are quite different. Each group was tightly clustered. The heat map shown in Figure 4 displays different levels among the two groups and metabolites with similar variation trends that were clustered together. Overall, 114 metabolites (variables) less than 0.05 \(p\)-value were shown in the heat map analysis. The distribution of the metabolites group can be divided into two groups of A and B (Figure 4). The black vertical line distinguishes the group control and field groups, while the black horizontal line divides metabolite categories such as up- and downregulation of metabolites in field soil. The metabolites in group A are considered inhibited and downregulated metabolites in field soil and the metabolites in group B are upregulated in field soil (Figure 4). The metabolic result of the field group soil in the heat map still presents several metabolites derived from toluene or its combustion products (Figure 4 and Table 4). The metabolic pathway analysis result shows the major metabolism in the chemical accident site (Figure 5 and Table 3). The metabolisms of alanine, aspartate, and glutamate satisfied the cut-off standard of raw \(p\)-value less than 0.05, which means significant and major metabolism in field soil (Figure 5). The oxaloacetate and carbamoyl phosphate discovered in field soil joined this metabolism. According to [23], upregulation of oxaloacetate can lead to the generation of nicotinamide adenine dinucleotide phosphate (NADPH) during oxidative stress by promoting the conversion of NADH (Nicotinamide adenine dinucleotide). The reduced NADPH is significant for the cellular anti-oxidative protection. Oxaloacetate is evidence that indicates microbes struggling to overcome chemical stress. Moreover, there are several biomarker metabolites such as naphthyl-2-methyl-succinic acid involved in naphthalene degradation, streptomycin 6-phosphate in streptomycin biosynthesis, malonyl-[acyl-carrier protein] in fatty acid biosynthesis, and 3-nitroacrylate. On the other hand, down-regulated metabolites also can be a significant biomarker.