In forensic science, soil is an important form of trace evidence and can be used to test for potential links between soil on a questioned item and a potential source location. Since traces of soil can readily become trapped on the underside of footwear and on vehicle tires, soil has long been considered to be a potential source of forensic information ^{[1][2][3][4][5][6]}[1,2,3,4,5,6]. Provided that a well-tested and robust method (with chemical, physical, mineralogical and organic data) for the characterisation of the samples is available and used appropriately, a potential link can be tested and established between a suspect and a crime scene ^[3][4][3,4].

Inorganic soil analyses have been successfully used in forensic investigations ^{[3][4][7][8][9][10][11]}[3,4,7,8,9,10,11], and organic approaches have less frequently been employed. Many soils have less than 5% (w/w) of organic matter content, particularly tropical and subtropical soils, and this has expressive effects on the biological, chemical and physical characteristics of the soil, even if it is present at reduced contents. Furthermore, this material can be used successfully in forensic studies, as organic matter is an inherent part of the soil, that is, for a given material to be considered as soil, it must have a minimum level of organic material, in addition to containing the inorganic mineral fractions (sand, silt and clay) in different proportions. As the concentration of organic matter varies with the soil concentration, dilutions of the organic matter content depend on the amount of soil sampled at the crime scene, such as the amount found on the sole of shoes and on the vehicle tires ^{[3][4][12][13]}[3,4,12,13].

Traditional forensic approaches in Brazil have focused on the determination of the total elemental content and the physical fractionation of the soil [14]. In an alternative forensic approach, the composition of soil organic matter is determined by pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) [15]. Py–GC/MS analyses allow a qualitative investigation of the nature of organic materials and are efficient in identifying different compounds at the molecular level [16]. Soil organic biomarkers have received limited attention in forensic soil science, with only a few studies worldwide ^{[17][18][19][20]}[17,18,19,20]. The relative abundance of organic compounds can vary considerably between soil samples only a short distance apart ^[13][21][13,21]. In most soils, especially in the topsoil, the organic compounds which have originated from the surface wax of plants are abundant. These compounds enter into the soil as fallen plant leaves and plant litter such as seeds, flowers and stems. Since plant roots have low concentrations of plant-wax compounds, they generally make a small contribution to the total content of these compounds in soils ^[4][18][4,18]. The most common and widely studied wax-marker compound class is the hydrocarbons, including n-alkanes, and the free and esterified long-chain fatty alcohols (high molecular-weight, straight-chain primary alcohols, derived from natural fats and oils) and fatty acids (carboxylic acids with a long aliphatic chain, containing a hydrocarbon chain and a terminal carboxyl group) ^{[17][22][23][24]}[17,22,23,24] can also be used. 

Forensic studies using plant-wax compounds have been tested mainly under temperate conditions, over a wide range of both rural and urban areas ^[17][20][17,20]. Under tropical and subtropical conditions, the potential of the use of plant-wax biomarkers in a forensic context is still unknown. The soil has a unique and ubiquitous nature given its physical, chemical, biological and mineralogical characteristics, which allow it to be differentiated between the most varied environments. The uniqueness of the soil in regard to its characteristics makes it a powerful source of information in forensic investigations, because, due to its variability, it is possible to discriminate and associate it with a specific individual or object related to a crime scene. In tropical and subtropical conditions, it is observed that inorganic mineral fractions are more frequently used to investigate traces of soil sampled at crime scenes, possibly due to several factors, such as the heterogeneous nature of soils under different climatic conditions which leads, for example, to the wide variations of the organic matter decomposition rates in relation to soils from temperate regions. 

2. Soil Sites and Sampling

Two areas from the Curitiba municipality and two areas from the Colombo municipality, Paraná State, in the South of Brazil, were selected: the Santa Cândida and Boa Vista neighbourhoods of Curitiba and the Guarani and Guaraituba neighbourhoods of Colombo, located in the Metropolitan Region of Curitiba (Figure 1 and Figure 2). All soils were classified as inceptisol according to U.S. Soil Taxonomy ^[25][34] and as cambissolo háplico according to the Brazilian Soil Classification System ^[26][35].

The samples collected in the Guarani neighbourhood (Table 1) presented the lowest values for the E4/E6 ratio (Figure 3a). The E4/E6 ratio is related to the condensation degree of the aromaticity or aliphaticity and the molecular weight of organic compounds ^[27][28][29][39,42,43]. Higher E4/E6 ratios (Boa Vista neighbourhood) are generally associated with more aliphatic and low-molecular-weight structures ^[30][44], with branched and straight-chain carbons ^[31][32][45,46]. The greater presence of vegetation with aliphatic groups (less humified) in Boa Vista could also be observed in the greater reflectance at 725–720 nm (red region) (Figure 3b), typical of long aliphatic chains with C–C and C–H bonds ^[31][45]. The RGB reflectance of the visible spectrum (500 to 720 nm) may be attributed to less aromatic chemical structures. These relationships obtained through colour spectroscopy and the qualitative aspects of the organic matter may be related to the nature of the residue of plants present in the soil ^[30][33][44,47]. At all the sites there was a predominance of odd long-chain alkanes (C29, C31 and C33); on the other hand, the values of C35 were low (Figure 4a). The hydrocarbons, including n-alkanes, are the most common and widely studied wax compounds ^[22][34][35][22,26,48]. The provenance of these compounds is mainly from the wax of the vegetation that enters into the soil as fallen leaves and other plant litter such as seeds, flowers and stems [12]. The n-alkanes, with odd-numbered carbon chains, are the predominant markers found in cuticular wax, especially the C27, C29, C31 and C33 alkanes ^[24][36][33][24,27,47], with C29 and C31 being the dominant compounds (Figure 4a).