Wetlands are one of the most abundant and biodiverse ecological landscapes in the natural world. Wetlands have irreplaceable functions in the conservation of water and soil, water purification, stabilizing the environment, the protection of genetic diversity, and resource utilization among others [1
]. The second national wetland resource survey organized by the State Forestry Administration of China showed that 96% of the available fresh water resources in China were stored in wetlands [2
]. In recent decades, concomitant with the rapid and intensive development of industrialization in China, urbanization and globalization led to increased human disturbances such as sewage discharge, oil exploitation, agricultural irrigation, and smelting. These resulted in wetland reduction and simultaneous pollution of various types and increasing quantities in the wetland soil, water bodies, and sediments [3
]. Because wetlands generally occupy low-lying areas, large amounts of pollutants produced by environmental transition and human activities are collected in wetlands, which have become one of the final destinations for heavy metals, organic pollutants, and other refractory pollutants [5
Polycyclic aromatic hydrocarbons (PAHs) compounds are persistent organic pollutants that exist in the environment [6
]. In 1976, the United States Environmental Protection Agency (US EPA) listed 16 PAHs as toxic organic pollutants [7
]. PAHs in wetland soil and sediments can be transported into the biosphere through animal and plant respiration, absorption, ingestion and some other approaches. Therefore, they can accumulate in living bodies, causing harm to many organisms and entering human bodies through the food chain, resulting in a threat to human health [8
Sustainability of environmental systems largely depends on a sound soil ecosystem. Changes will occur in ecosystems if soils are polluted. PAHs can remain in the environment for years. Even if the sources of PAHs are removed, residual PAHs may also pose long-term risks to the environmental system. Thus, PAHs accumulated in soil have attracted more attention because of the potential risk and adverse impact they cause in soil ecosystems.
In wetland environments, PAHs in wetland soil can be transferred to the atmosphere through particulate matter. Hence, PAHs can easily enter living bodies through ingestion, breathing, skin exposure and other approaches. This can result in the damage of living bodies on a cellular level, such as damaging DNA, which will lead to mutation of microorganisms, animals and plants [9
]. These effects will influence the health of creatures and much of this influence involves teratogenicity, carcinogenicity and mutagenicity. Ma et al. found that the content of PAHs in the Songhua river bottom sludge was greater than their content in river water. Freshwater mussels showed high PAH levels, and the PAH content in catfishes was higher than in mussels (lower in the food chain) [8
]. In addition, materials derived from PAHs can also be harmful pollutants. It is worth mentioning that although some of the PAH derivatives exist only at trace levels in the environment, the toxicity they express is even greater than that of PAHs (already high). Therefore, they become highly toxic organic carcinogens [10
]. As for the Momoge Wetland, existing research has mainly focused on the physical and chemical properties of the soil, ecosystem service functions, tourism development and biodiversity in the Momoge Wetland; moreover, these studies did not involve PAH content or ecological risk assessment. Because of the widespread concern about the environmental issues related to PAHs and the lack of information about PAHs, the Momoge Wetland was selected for this study.
In this study, a method combining source identification, pollution and ecological risk was used to carry out significant research on PAH distribution in the wetland soil and to provide risk assessment. This study was intended to lay a theoretical foundation for developing remediation for soil pollution and fully understanding the degree of environmental pollution in the area. The study also analyzed the extent to which the PAHs in the soil might cause risk to the environment, with the goal of warning relevant departments. There are important theoretical and real consequences of protecting wetland’s creatures and environment.
2. Materials and Methods
2.1. Study Area
The Momoge Nature Reserve (45°45′–46°10′ N, 122°27′–124°04′ E), located in Zhenlai County (west of Jilin Province, China) is a typical wetland protection area and is also the main migration path in the Chinese eastern region of northern migratory waterfowl birds. The total area of Momoge is 14 million hectares, and its natural wetland, which is an inland wetland, accounts for approximately 80% of the total area. The region is flat, and the relative elevation is only 2–10 m [11
]. The dominant types of land-use are cultivated fields, grasslands and other land-use types. The main industry there is petroleum exploitation, which could lead to organic pollution. There are 15 species of cranes in the world, of which 6 are protected in the reserve (Grusleucogeranus
). There are two types of storks in the reserve: Ciconiaboyciana
. Approximately 500–800 Ciconiaboyciana
gather in the Momoge Wetland Reserve every fall, accounting for 1/3 of the world total. It is also a crane stopover. Many international organizations, such as World Wildlife Fund (WWF), Global Environment Facility (GEF) and International Crane Foundation (ICF) are particularly concerned about this area [12
]. The Momoge Wetland provides the necessary breeding and stopover environments for rare birds and waterfowl such as Grusleucogeranus
. The protection zone has remarkable characteristics and a uniquely important value from the perspectives of biodiversity and habitat originality [13
2.2. Collection and Preservation of Soil Samples
A total of 39 topsoil samples from the wetland were collected in November 2014. The sampling sites were roughly evenly distributed to represent the buffer zone and experimental area of the Momoge Wetland considering transport, environmental and security conditions. At each site, five sub-samples (depth of 0–10 cm, within range of 10 × 10 m; per the quincunx layout method) were collected and bulked together to form one composite sample. In the sampling process, a Global Positioning System (GPS) was used to accurately provide the location of each sampling point and the specific locations of the sample sites as shown in Figure 1
Under dry, clean, and ventilated laboratory conditions (approximately 25 °C), the soil samples were put on white paper (2–3 cm thick), allowing them to naturally air dry (~1 week). During the air-drying process, small stones, plant root debris and other inclusions in the soil samples were carefully removed. After grinding the dry samples with a mortar, they were sieved to provide 60-mesh size particles, then sealed in lock bags and conserved in a refrigerator at 4 °C until analysis.
2.3. Laboratory Instruments and Reagents
HPLC-grade solvents (dichloromethane, acetone) were used in sample processing. A composite standard solution of 16 PAHs including naphthalene (Nap), acenaphthylene (Acl), acenaphthene (Ace), fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), pyrene (Pyr), benz(a)]anthracene (BaA), chrysene (Chr), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), indeno(1,2,3-cd)pyrene (Ind), dibenz(a,h)anthracene (Dah) and benzo(g,h,i)-perylene (Bghi) each at a concentration of 2000 mg·L−1. Phenol-d6; nitrobenzene-d5; 2-fluorobiphenyl and 2,4,6-tribromophenol were added as surrogates prior to extraction, and 2-fluorophenol, terphenyl-d14 and dibenzo-(a,h)anthracene-d14 were used as internal standards for analysis.
Samples were analyzed using a gas chromatograph (7890B, Agilent, Santa Clara, CA, USA) equipped with a 5977A mass spectrometer (GC-MS, Agilent, Santa Clara, CA, USA) and DB-5MS capillary column (30 m × 0.25 mm (i.d.) with 0.25 μm film thickness, Agilent, Santa Clara, CA, USA). A small sample extract (l µL) was injected in splitless mode with injector temperature was 300 °C. The oven temperature was programmed as follows: 45 °C for 2 min, then increase to 265 °C at a rate of 20 °C/min and ramped at 6 °C/min to 285 °C and increase at a rate of to 320 °C at 10 °C/min. The carrier gas was high-purity helium (99.999%) at a flow of 1 mL·min−1. The MS was operated in electron impact mode at 70 eV with an ion source temperature of 230 °C.
2.4. Chemical Analysis of Soil Samples
Soil sample extraction and analysis were performed according to USEPA Method 3570 [14
] and Method 8270D [15
], with some modifications. In order to extract PAHs, approximately 20 g of soil sample dried for 3–4 h at (105 ± 5) °C with 10 g anhydrous sodium sulfate and 100 mL dichloromethane/acetone (1/1, v
) was mixed with 50 µL 100 mg/L surrogates. The mixture was shocked for 2 h at a speed of (30 ± 2) r/min (28 hole reversing machine, Guohuangaoke, Beijing, China). The extracts were condensedusing a K-D concentrator (B type, shanghaihuake, Shanghai, China). The solvent was exchanged with dichloromethane, and further reduced to 1 mL before GC/MS analysis. The internal standards of 5 µL 200 mg/L were added prior to analysis using a GC-MS, for determination of the 16 PAHs.
The blanks, spiked blanks and duplicate samples were processed during the extraction and analysis procedures. The recovery efficiencies (Table 1
) were checked by analysing blank samples (PAH concentration < 0.01 mg/kg) with 16 PAH standard samples, the use of which conforms to the EPA methods mentioned above. The coefficient of variation of PAH concentration in duplicates was less than 15%. In this study, the detection limit of PAHs in the soil was 0.01 mg/kg.
2.5. Data Analysis
Data were subjected to correlation analysis, a Kaiser-Meyer-Olkin (KMO)-Bartlett test of Sphericity, principal component analysis (PCA), and multiple linear regression (MLR) analysis using Microsoft Excel 2007 (Microsoft, Redmond, WA, USA) and SPSS 19.0 (SPSS Inc., Chicago, IL, USA). The chart in the paper was produced using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA).
2.6. Analysis and Evaluation Method
The diagnostic ratios and receptor oriented models used to reveal the sources of PAHs in this paper are well known. In this paper, the diagnostic ratio method and PCA-MLR model, one of the receptor models, were used to evaluate the sources of PAHs in the Momoge Wetland soil samples [16
For the diagnostic ratio method, low molecular weight (2–3 rings, LMW)/High molecular weight (≥4 rings, HMW), Ant/(Ant + Phe) or Ant/Phe, Fla/(Fla + Pyr) or Fla/Pyr, BaA/(BaA + Chr) or BaA/Chr, Ind/(Ind + Bghi) or Ind/Bghi and BaA/Bghiwere used [17
]. The source is believed to be mainly from the combustion of coal, biomass or petroleum when the LMW/HMW ratio is <1. The source is considered to be mainly petroleum spills when the LMW/HMW ratio is >1. When the ratio of Ant/(Ant + Phe) is <0.1, a petroleum source is indicated. When the ratio is >0.1, a combustion source is considered primary. When the ratio of Fla/(Fla + Pyr) is <0.4, a petroleum source is indicated. When the ratio is >0.4 and <0.5, the source of the PAHs is considered mainly petroleum combustion. When the ratio is >0.5, PAHs mainly from coal and biomass combustion are indicated. When the ratio of BaA/(BaA + Chr) is <0.2, a mainly petroleum source is indicated. If the ratio is between 0.2 and 0.35, the source indicated is petroleum combustion. When the ratio is >0.35, the coal and biomass combustion make a greater contribution to the PAHs in the environment. When the ratio of Ind/(Ind + Bghi) is <0.2, the meaning is the same as that for BaA/(BaA + Chr), mainly petroleum sources are indicated. When the ratio is >0.5, the production of PAHs is caused by the combustion of coal and biomass. When the ratio is between the two, the main source is likely petroleum combustion. Finally, when the BaA/Bghi ratio is >0.9, coal and biomass combustion is an important source. If it is <0.9, the PAHs are mainly from gasoline and diesel combustion (i.e., a traffic emission source).
PCA is a multivariate statistical tool used to transform the original data set into a smaller one [19
], and it has been proven that a PCA-MLR model can be a good method for analysing the source of PAHs [20
]. The main steps in the use of PCA-MLR are as follows. First, the KMO-Bartlett test should be conducted before PCA, and if the p
value > 0.6, it is suitable for PCA. The PCA was found appropriate for use with the data in this study. Next, the principal components with eigenvalues >1 were extracted by PCA, and the main factors, the properties of which can be used to identify sources to a certain extent, of each principal component were determined according to different factor loading. Then, the factors extracted by PCA and the content of PAHs were analysed by MLR to determine the contribution of each principal component. Finally, the source, and the contribution of each source, was determined.
2.6.2. Evaluation of Wetland Soil Pollution
Comparative analysis using environmental standards is used to evaluate the PAH pollution level. This was needed because there is no PAH standard value or background value for China. Because the PAH standard in The Netherlands is sound, the national standards of PAHs in soils by the Dutch government was used [22
]. Through comparative analysis, the pollution status in this area of China was obtained.
In addition, the PAH pollution in soil was divided into four grades by the contamination interval method (proposed by Maliszewska-Kordybach, 1996) [23
]. When the content of Σ16 PAHs is <0.2 mg/kg, no pollution is present. When Σ16 PAHs is >0.2 mg/kg and <0.6 mg/kg, the area is slightly polluted. When the Σ16 PAHs is >0.6 mg/kg and <1.0 mg/kg, the area is polluted; and when the value is >1.0 mg/kg, the area is heavily polluted.
2.6.3. Ecological Risk Assessment
The biological toxicity evaluation shows that the concentration of pollutants present will produce toxic effects on organisms, and in order to measure the magnitude of the toxicity, the effects range-low (ERL) and the effects range-median (ERM) were proposed [24
]. When the pollutant concentration is lower than ERL, no toxic effects on biological toxicity are produced and the incidence of toxicity is 10%. When the pollutant concentration is higher than ERL, but lower than that of ERM, it will produce toxic effects on living organisms and the occurrence probability of toxicity is 10%–50%. When the concentration is >ERM, the toxicity (poisoning rate) is >50%, showing a strong toxic effect.
The organic carbon normalization method is a risk assessment criterion for the summing of organic carbon normalized concentration of 13 PAHs (Nap, Acl, Ace, Flu, Phe, Ant, Fla, Pyr, BaA, Chr, BbF, BkF and BaP). It was suggested by Swartz in 1999 [25
]. It uses three indicators: the critical effect content (CEC), medium effect content (MEC) and extreme effect content (EEC) to assess the risk level. The concentrations of (0.29, 1.8 and 10) mg/kg were defined as the TEC, MEC and EEC value, respectively. The Σ13 PAH concentration measured at each sampling site was compared with the TEC, MEC and EEC values. If the Σ13 PAHs was <TEC, there was no ecological risk. If the Σ13 PAHs >TEC but <MEC, it is defined as accidental ecological risk. However, when the Σ13 PAHs is between MEC and EEC, there is high ecological risk, and when the Σ13 PAHs >EEC value, there is serious ecological risk.