Concentration and Potential Ecological Risk of PAHs in Different Layers of Soil in the Petroleum-Contaminated Areas of the Loess Plateau, China

The three most representative areas of petroleum pollution on the Loess Plateau are the research subjects of this study. In this study, 16 priority polycyclic aromatic hydrocarbons (PAHs) were determined by the QuEChERS method combined with gas chromatography-tandem mass spectrometry (GC-MS/MS). The total concentrations of ∑16PAHs in top layer soils (0–10 cm), middle layer soils (10–30 cm), and bottom layer soils (30–50 cm) ranged from 1010.67 to 18,068.80, 495.85 to 9868.56 and 213.16 to 12,552.53 μg/kg, with an average of 5502.44, 2296.94 and 2203.88 μg/kg, respectively. The 3-ring and 4-ring PAHs were the most prominent components in all soil samples. Meanwhile, the average value of ∑16PAHs decreased with the depth, from 5502.44 μg/kg (0–10 cm) to 2203.88 μg/kg (30–50 cm). The PAHs levels in the studied soils were heavily polluted (over 1000 μg/kg) according to the Soils Quality Guidelines and 95% of PAHs come from petroleum sources. Moreover, the total of PAHs in petroleum-contaminated soils was assigned a high ecological risk level. Toxic equivalency quantities (TEQs) indicated that PAHs in petroleum-contaminated soils presented relatively high toxicity.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a class of diverse organic compounds containing two or more fused aromatic rings made up of carbon and hydrogen atoms [1]. Generally, they are produced from incomplete combustion of organic materials, fossil fuels, petroleum product spillage and various domestic and industrial activities [2,3]. Once emitted, PAHs can be widely dispersed in air, water, soil and sediment. Due to the hydrophobicity and lipophilicity of PAHs, soil is the most important sink for PAHs in natural environment [4,5]. It has been reported that soil can store approximately 90% of PAHs [6]. PAHs in soils can be carried into surface/ground water through precipitation and surface runoff, emitted into atmosphere by volatilization, and transported into crops from polluted soil and air via root and leaf adsorption, which may further accumulate in human and other organisms via food chains [7]. Thus, monitoring the concentration of PAHs in soils is important for understanding its environmental fate.
(GPS) was used to accurately provide the location of each sampling point as shown in Figure 1. The basic information of the sampling sites in details is given in Table 1. After transport to the laboratory, the soil samples were air dried, ground, passed through a 60-mesh screen, homogenized, and stored at 4 • C until analysis. to the laboratory, the soil samples were air dried, ground, passed through a 60-mesh screen, homogenized, and stored at 4 °C until analysis.

Sample Extraction
In the laboratory, the samples were air-dried at room temperature and stones, roots and other debris were removed. The samples were then ground and sieved through a 60-mesh screen. Soil samples (2.0 g) were mixed with anhydrous sodium sulfate (3.0 g), and extracted with dichloromethane (20 mL) for 30 min under ultrasoound. After centrifuging the tubes at 9500 r/min for 10 min, a 2-mL supernatant sample was transferred to a single-use centrifuge tube containing 150 mg of PSA, 50 mg of C18, and 900 mg of anhydrous Na 2 SO 4 . The mixtures were shaken vigorously for 1 min using a vortex mixer to ensure that the solvent contacted the entire sample. Subsequently, the samples were centrifuged at approximately 9500 r/min for 10 min. Then, the upper layer of the prepared sample was filtered through a 0.22 µm syringe filter and transferred to an autosampler vial for injection.

Instrumental Analysis
The determination of PAHs was performed on GCMS-TQ8040 (Shimadzu (China) Co., Ltd., Xi'an, China) with splitless injection, MRM acquisition mode. The capillary column Rxi-5Sil Ms (30 m × 0.25 mm × 0.25 µm) was used for separations. Helium (99.999%) was used as the carrier gas. The oven temperature program was as follows: initial temperature of 50 • C was held for 2 min, then increased to 250 • C at a rate of 20 • C/min and held for 3 min, and finally increased to 300 • C at a rate of 5 • C/min and held for 5 min.

Ecological Risk of PAHs in Soils
PAHs accumulated in soils may enter water bodies and plants, posing a potential ecological risk. A risk quotient (RQ) was used to assess ecological risk of some organic substances. The negligible concentrations (NCs) and the maximum permissible concentrations (MPCs) of PAHs in soils were used as the quality values in the medium [16]. Therefore, RQ (NCs) and RQ (MPCs) were defined as follows: where C QV(NCs) was the quality values of the NCs of PAHs in the medium and C QV(MPCs) was the quality values of the MPCs of PAHs in the medium. The RQ ∑PAHs , RQ ∑PAHs(NCs) and RQ ∑PAHs(MPCs) were defined as follows: Based on the ecosystem risk assessment of 16 individual PAHs, RQ (NCs) and RQ (MPCs) of individual PAHs which were not less than 1 were added to calculate the RQ ∑PAHs(NCs) and RQ ∑PAHs(MPCs) of ∑PAHs. RQ (NCs) < 1.0 indicated that the single PAHs might be of negligible concern, RQ (MPCs) > 1.0 would indicate that the contamination of the single PAHs posed high risk, and RQ (NCs) > 1.0 and RQ (MPCs) < 1.0 indicated that the contamination of the single PAHs was of moderate risk.

Toxicity Assessment of PAHs in Soils
PAHs can be absorbed by humans through the skin and respiratory tract, and they may cause skin cancer, lung cancer and other diseases. Exposure to PAHs in the environment for a long time may cause chronic poisoning. Toxic equivalency factors (TEFs) were used to estimate the exposure risks posed by individual and total PAHs to human health. The toxicities of PAHs in sampling sites were evaluated BaP equivalent concentration (BaPeq). The TEFs for the 16 PAHs were calculated according to USEPA and Nisbet and LaGoy [10,17]. The total toxicity equivalency concentrations (BaPeq) were calculated using the following equation: where C i is the concentration of individual PAHs and TEF i is the corresponding toxic equivalency factor.

Properties Analysis
Soil pH was measured (soil: water 1:2.5 w/v) by using a pH-meter (pHS-3B, Leici, Shanghai, China) and the soil organic carbon contents were determined by the Walkey-Black method [18].

Characteristics of PAHs Concentrations in Soils
As shown in Table 2, all 16 priority PAHs were detected in petroleum-contaminated soils, indicating that PAHs were ubiquitous pollutants in the tested soil environment. The total concentrations of ∑16PAHs in top layer soils (0-10 cm), middle layer soils (10-30 cm), and bottom layer soils (30-50 cm) ranged from 1010.67 to 18,068.80, 495.85 to 9868.56 and 213.16 to 12,552.53 µg/kg, with an average of 5502.44, 2296.94 and 2203.88 µg/kg, respectively. Moreover, the human carcinogen compounds (BaA, CHR, BbF, BkF, BaP, DBA and InP) were also investigated in petroleum-contaminated soils, and the results are presented in Table 2. The highest total carcinogenic PAHs (∑7PAHs) were distributed in top layer soils with a range of 223.97-4642.40 µg/kg (mean: 1832.55 µg/kg), followed by the bottom layer soils (range: 23.89-6588.26 µg/kg, mean: 1039.09 µg/kg) and the middle layer soils (range: 85.71-3466.19 µg/kg, mean: 921.45 µg/kg). Among these human carcinogen compounds, BaP is a typical PAH which is of greatest interest in terms of potential cancer hazard [19].  (Table 2).
According to the European classification system of soil contamination [20], the PAHs pollution in soils was divided into four grades. A ∑16PAHs soil concentration below 200 µg/kg indicates no polluted, a concentration of 200-600 µg/kg represents lightly polluted, and a soil concentration of 600-1000 µg/kg represents moderately polluted. Concentrations over 1000 µg/kg would be indicative of heavy pollution. According to this classification standard, it is worthwhile to note that ∑16PAHs in petroleum-contaminated soils were 2.2-5.5 times higher than the standard level (1000 µg/kg) of heavy polluted. It indicated that the petroleum-contaminated soils stored great amount of PAHs and regulatory measures are needed to prevent the areas from turning into pollution sources, which would transfer PAHs into the air or groundwater in the region.
In addition, a comparison of ∑PAHs concentrations in soils from different cities worldwide is given in Table 3, where it can be seen that the mean concentration of ∑16PAHs in petroleum-contaminated soils was much lower than that in urban soil from London (UK) and garden soil from New York (USA). However, it was higher than that in different types of soil from some Chinese region such as Xianyang, Shanghai, Nanjing, Tianjin, Jilin, Momoge Wetland and Yangtze River Delta, as well as from Dhanbad (India), New Orleans (USA), Ulsan (Korea), Viseu (Portugal) and Isfahan (Iran). The results indicate people should be cautious about the environmental quality of the petroleum-contaminated soils.

Characteristics of the PAHs Distribution in Soils
PAHs represent complex chemicals which consist of multiple aromatic rings. Based on the number of aromatic rings, the 16 PAHs are divided into five groups: 2-ring, 3-ring, 4-ring, 5-ring, 6-ring PAHs. The distribution pattern of the 16 PAHs is shown in Figure 2a. It can be seen that the sequence of the PAHs proportion in top layer soils (0-10 cm) was detected as 3-ring (49.88%) > 4-ring (35.73%) > 5-ring (7.39%) > 6-ring (5.04%) > 2-ring (1.96%). Correspondingly, the sequence of the PAHs proportion in middle layer soils (10-30 cm) was detected as 4-ring (41.46%) > 3-ring (39.82%) > 5-ring (8.52%) > 6-ring (5.81%) > 2-ring (4.39%). In addition, the distribution pattern of PAHs in bottom layer soils and middle layer soils is same. It is obvious that the soil samples in different layers had the same PAHs compositions in terms of the number of aromatic rings. The 3-ring and 4-ring PAHs were analyzed as the most prominent components in all soil samples. Moreover, due to their high volatility, 2-ring PAHs was lower in the top and bottom layer soils, but higher in middle layer soils. The 5-ring and 6-ring PAHs levels increased with the increasing depth, the reason being that they have high hydrophobicity and molecular mass, so they can accumulate more easily by adsorption on soil organic matter.
The vertical distribution of PAHs in petroleum-contaminated soils was assessed from the soil samples collected from vertical sections at three depths in the sampling areas. The results of the vertical distribution profile of PAHs component are shown in Figure 2b. It is expected that ∑16PAHs would gradually decrease with the increasing depth, from the top layer (0-10 cm) to the bottom layer (30-50 cm), resulting in decreasing ∑16PAHs from 5502.44 µg/kg to 2203.88 µg/kg. Compared to the top layer soils, the ∑16PAHs in 30-50 cm depth decreased by 59.95% in the sample area. The vertical distribution profile of ∑7PAHs is similar to that of ∑16PAHs. It is also found that ∑7PAHs would gradually decrease with the increasing depth, and the concentration decreased from 1832.55 µg/kg (0-10 cm) to 1039.09 µg/kg (30-50 cm).
What's more, the results of the individual PAHs concentration in different vertical sections are also shown in Figure 2b. The vertical distribution characteristics of individual PAHs appeared to be different. The highest concentration of Nap, FLU, PHE, ANT, FLA, PYR, CHR, BbF and BgP were obtained in the top layer soils (0-10 cm). More accurately, FLU, PHE and CHR were found to be the most prominent compounds in all soil samples.

Correlation Analysis
The relationships between ∑16PAHs, soil organic matter (SOM) and pH were investigated in the present study (Table 4). Soil pH can affect the residual of PAHs in soils [33]. However, no significant correlation relationships between soil pH and PAHs were found in the present study, implying soil pH was not a key factor in the soil PAHs levels. SOM is considered to be key factor influencing the concentration of PAHs in soils. Nam et al. [34] reported that, in an environment where there is continuous input of fresh PAHs, a lack of correlation should be expected, at least until equilibrium is reached. In this study, good correlation existed between SOM and the concentration of 16 PAHs was found, suggesting that soil PAHs were close to steady state and in equilibrium with SOM.

Correlation Analysis
The relationships between ∑16PAHs, soil organic matter (SOM) and pH were investigated in the present study (Table 4). Soil pH can affect the residual of PAHs in soils [33]. However, no significant correlation relationships between soil pH and PAHs were found in the present study, implying soil pH was not a key factor in the soil PAHs levels. SOM is considered to be key factor influencing the concentration of PAHs in soils. Nam et al. [34] reported that, in an environment where there is continuous input of fresh PAHs, a lack of correlation should be expected, at least until equilibrium is reached. In this study, good correlation existed between SOM and the concentration of 16 PAHs was found, suggesting that soil PAHs were close to steady state and in equilibrium with SOM. * Correlation is significant at p < 0.05 (two-tailed); ** Correlation is significant at p < 0.01 (two-tailed).

Source Identification of PAHs in Soils
Understanding the sources of PAHs is crucial to determine how PAHs are carried into the environment. Generally, the diagnostic ratios method was the most widely used to distinguish between the sources of PAHs in the soil ecosystem. Ratios such as low molecular weight (2-3 rings, LMW)/high molecular weight (≥4 rings, HMW), FLA/(FLA + PYR), BaA/(BaA + CHR) and ANT/(ANT + PHE) have been reported in many studies. For example, the ratio of LMW/HMW < 1 indicates pyrogenic source, while the ratio >1 indicates petrogenic source [35]. A ratio of FLA/(FLA + PYR) < 0.4 indicates a petroleum source, a ratio between 0.4-0.5 indicates a fossil fuel combustion source, and a ratio >0.5 indicates coal/wood/grass combustion source [36]. For BaA/(BaA + CHR), the ratio < 0.2 indicates a petroleum source, the ratio between 0.2-0.35 indicates a mixed source, and the ratio >0.35 indicates a combustion source [37]. Values of ANT/(ANT + PHE) ratio are <0.1 and >0.1 indicative of petroleum and combustion sources, respectively [38].
In this study, the diagnostic ratios of FLA/(FLA + PYR) and BaA/(BaA + CHR) were used to distinguish the possible PAHs origins in petroleum-contaminated soils. The ratios for BaA/(BaA + CHR) versus FLA/(FLA + PYR) are shown in Figure 3, where the BaA/(BaA + CHR) values for 95% of the samples are <0.2, while the FLA/(FLA + PYR) values for 75% of the samples are <0.5. This suggests that the PAHs in soil samples come from petroleum sources and only a small quantity of them comes from combustion sources.

Source Identification of PAHs in Soils
Understanding the sources of PAHs is crucial to determine how PAHs are carried into the environment. Generally, the diagnostic ratios method was the most widely used to distinguish between the sources of PAHs in the soil ecosystem. Ratios such as low molecular weight (2-3 rings, LMW)/high molecular weight (≥4 rings, HMW), FLA/(FLA + PYR), BaA/(BaA + CHR) and ANT/(ANT + PHE) have been reported in many studies. For example, the ratio of LMW/HMW < 1 indicates pyrogenic source, while the ratio >1 indicates petrogenic source [35]. A ratio of FLA/(FLA + PYR) < 0.4 indicates a petroleum source, a ratio between 0.4-0.5 indicates a fossil fuel combustion source, and a ratio >0.5 indicates coal/wood/grass combustion source [36]. For BaA/(BaA + CHR), the ratio < 0.2 indicates a petroleum source, the ratio between 0.2-0.35 indicates a mixed source, and the ratio >0.35 indicates a combustion source [37]. Values of ANT/(ANT + PHE) ratio are <0.1 and >0.1 indicative of petroleum and combustion sources, respectively [38].
In this study, the diagnostic ratios of FLA/(FLA + PYR) and BaA/(BaA + CHR) were used to distinguish the possible PAHs origins in petroleum-contaminated soils. The ratios for BaA/(BaA + CHR) versus FLA/(FLA + PYR) are shown in Figure 3, where the BaA/(BaA + CHR) values for 95% of the samples are <0.2, while the FLA/(FLA + PYR) values for 75% of the samples are <0.5. This suggests that the PAHs in soil samples come from petroleum sources and only a small quantity of them comes from combustion sources.

Ecological Risk of PAHs in Soils
The assessment results of ecological risk of PAHs in petroleum-contaminated soils based on risk quotient are given in Table 5. The mean values of RQ(NCs) for most individual PAHs were found to be greater than 1, with the exception of ACY (0.00), BkF (0.49) and InP (0.67). The mean values of calculated RQ(MPCs) for FLU, PHE and PYR were greater than 1, implying that these three PAHs had high ecological risk to aquatic/soil organisms. The mean value of calculated RQ∑PAHs(NCs) was above 800, while the mean value of calculated RQ∑PAHs(MPCs) was higher than 1, suggesting that the total of PAHs in petroleum-contaminated soils was assigned a high ecological risk level. It is worth noting that though low molecular PAHs are less mutagenic and carcinogenic than high molecular PAHs, it can be seen from Table 5 that ecosystem risk associated with low and molecular PAHs is actually very high. Therefore, control and preventive measures should be implemented to decrease the contamination associated with 2-ring, 3-ring and 4-ring PAHs.

Ecological Risk of PAHs in Soils
The assessment results of ecological risk of PAHs in petroleum-contaminated soils based on risk quotient are given in Table 5. The mean values of RQ (NCs) for most individual PAHs were found to be greater than 1, with the exception of ACY (0.00), BkF (0.49) and InP (0.67). The mean values of calculated RQ (MPCs) for FLU, PHE and PYR were greater than 1, implying that these three PAHs had high ecological risk to aquatic/soil organisms. The mean value of calculated RQ ∑PAHs(NCs) was above 800, while the mean value of calculated RQ ∑PAHs(MPCs) was higher than 1, suggesting that the total of PAHs in petroleum-contaminated soils was assigned a high ecological risk level. It is worth noting that though low molecular PAHs are less mutagenic and carcinogenic than high molecular PAHs, it can be seen from Table 5 that ecosystem risk associated with low and molecular PAHs is actually very high. Therefore, control and preventive measures should be implemented to decrease the contamination associated with 2-ring, 3-ring and 4-ring PAHs.

Toxicity Potential of PAHs in Soils
Toxic equivalency quantities (TEQs) calculated as toxic equivalency factors (TEFs) are given in Table 6. As shown in Table 6, the TEQs of ∑16PAHs in top layer soils (0-10 cm), middle layer soils (10-30 cm), and bottom layer soils (30-50 cm) ranged from 16.59 to 303.50, 2.59 to 165.19 and 0.21 to 1452.16 µg/kg, with an average of 220.31, 106.25 and 292.48 µg/kg, respectively. Meanwhile, the TEQs of ∑7PAHs in soils of 0-10 cm, 10-30 cm and 30-50 cm ranged from 11.90 to 277.19, 3.23 to 277.28 and 0.24 to 1436.87 µg/kg, with an average of 212.13, 103.14 and 288.46 µg/kg, respectively. It is found that the TEQs of ∑7PAHs were very close to that of ∑16PAHs, indicating that the ∑7PAHs were the major carcinogenic contributor to the TEQs of ∑16PAHs. According to the Canadian soil quality guidelines for the protection of environmental and human health, the safe value of the TEQs of ∑7PAHs in soils is 600 µg/kg [39]. All the soil samples in this study were below the safe value. In addition, the TEQs of ∑7PAHs were much higher than that in soils of Hunpu (52.31 µg/kg) [35], Xinzhou (34 µg/kg) [40], Liaohe estuary (30.0 µg/kg) [41] and Yellow River Delta (11.92 µg/kg) [42]; while lower than that in soils of Xi'an (421.05 µg/kg) [43]. These indicated that PAHs in petroleum-contaminated soils presented relatively high toxicity.