Evaluation of Chemical Analysis Method and Determination of Polycyclic Aromatic Hydrocarbons Content from Seafood and Dairy Products
1Department of Food Science and Biotechnology and Food and Bio Safety Research Center, Dongguk University Seoul, Seoul, Korea
2Nutrition Policy & Promotion Team, Korea Health Industry Development Institute, Chungcheongbuk-do, Korea
Han-Seung Shin, Department of Food Science and Biotechnology and Food and Bio Safety Research Center, Dongguk University Seoul, Seoul 04620, Korea E-mail: ude.kuggnod@natraps
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Received 2015 Aug 9; Revised 2015 Sep 20; Accepted 2015 Sep 23.
Copyright © 2015, The Korean Society Of Toxicology
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This study was carried out to investigate contents of 8 polycyclic aromatic hydrocarbons (PAHs) from frequently consumed seafood and dairy products and to evaluate their chemical analysis methods. Samples were collected from markets of 9 cities in Korea chosen as the population reference and evaluated. The methodology involved saponification, extraction with n-hexane, clean-up on Sep-Pak silica cartridges and gas chromatograph-mass spectrometry analysis. Validation proceeded on 2 matrices. Recoveries for 8 PAHs ranged from 86.87 to 103.57%. The limit of detection (LOD) 8 PAHs was 0.04~0.20 µg/kg, and limit of quantification (LOQ) of 8 PAHs was 0.12~0.60 µg/kg. The mean concentration of benzo[a]pyrene (BaP) was 0.34 µg/kg from seafood and 0.34 µg/kg from dairy products. The total PAHs concentration was 1.06 µg/kg in seafood and 1.52 µg/kg in dairy products.
Keywords: Polycyclic aromatic hydrocarbon, Seafood, Dairy products, Carcinogen
Polycyclic aromatic hydrocarbons (PAHs) constitute a class of carcinogenic and mutagenic organic compounds based on two or more aromatic rings and belonging to the Food and Environment Contaminants (1,2).
The International Agency of Research on Cancer classification of benzo[a]pyrene (BaP) was changed from group 2A (probably carcinogenic to humans) to group 1 (carcinogenic to humans), chrysene (CHR) was changed from group3 (not classifiable for humans) to group 2B (possibly carcinogenic to humans), and benzo[a]anthracene (BaA) was re-grouped from 2A to 2B (3,4). According to the EU Scientific Committee on Food (SCF), BaP can be used as a marker for the occurrence and effect of carcinogenic PAHs in food. Maximum levels of BaP in a range of foodstuffs are now specified in a Commission Regulation (Regulation EC No 1881/2006) (5). However, the European Food Safety Authority in 2008 (6,7) concluded that BaP is not a suitable indicator for the occurrence of PAHs in food, and that 4 PAH subgroup (the sum of BaA, chrysene (CHR), benzo[b]fluoranthene (BbF) and BaP) and 8 PAHs subgroup (the sum of BaA, CHR, BaF, benzo[k]fluoranthene (BkF), BaP, dibenzo[a,h]anthracene (DahA), benzo[g,h,i]perylene (BghiP), and indeno[1,2,3-c,d]pyrene (IcdP)) should be used (8).
They are formed at high temperatures in natural processes (fires, volcanic eruptions, etc.) and in anthropogenic processes (burning of fossil fuels, vehicles emissions, plants of petroleum processing, etc.) due to the incomplete combustion of organic matter. PAHs are largely known as ubiquitous environmental contaminants due to their ability to be sorbed onto atmospheric particulate matter and become transported all over the planet (9). Soils, surface waters, and sediments may be contaminated by PAHs due to atmospheric fallout, urban runoff, deposition from sewage, and by oil or gasoline spills. Hence, there is a potential for ingredients like food crops to become environmentally contaminated as a result (10).
In fact, the main source of exposure to PAHs for nonsmokers and non-occupationally-exposed adults is food. Diet contributes to more than 90% of total PAHs exposures of the general population in various countries (11).
Foods of animal origin are recognized as one of the main PAHs givers being fatty foods such as eggs or dairy products like whole milk, yoghourt, butter, or cheese. Especially, the presence in cow’s milk is probably due to the feeding of dairy cattle in grass and soil polluted with air-borne PAHs.
Also, these PAHs are easily accumulated in fish and shellfish, especially by bivalve mollusc like clams, oysters, mussels and scallops, species that are exposed to various kind of PAHs following oil spills at ocean and be contaminated as a result (10,12). And seafood can be contaminated by the marine food web. Concentration of BaP and other PAHs can be various in fish based on the source and preparation of fish.
Due to stable structure and lipophilic character of PAHs, they are apt to concentrate and intensify in the food chain notably related to fat. However, a few regulations of the maximum allowable levels for PAHs in dairy products and seafood have been established (12). European Union has stressed and recommended that PAHs to be measured in as wide as possible in food products in order to obtain result on the occurrence and specific concentrations in a various matrices (13). Therefore, dairy products and marine products being upper predators require monitoring and regulations about them should be set based on this study.
MATERIALS AND METHODS
Chemicals and materials. The 8 PAHs used in this study were BaA, CHR, BbF, BkF, BaP, DahA, BghiP, and IcdP supplied as individual stock solutions by Supelco (Bellefonte, PA, USA). And CHR-d12, BaP-d12 (obtained from Supelco) were used as an internal standard. All solvents (dichloromethane, ethyl alcohol, methanol, and nhexane) were of HPLC grade and were purchased from Burdick & Jackson (Muskegon, MI, USA). Water was purified by a Milli-Q water purification system (Billerica, MA, USA). Sodium sulfate (Na2SO4, minimum 99% purity) used for dehydration and potassium hydroxide (KOH, minimum 85% purity) used for saponification were obtained from Junsei (Chuo-ku, Tokyo, Japan). Sep-Pak Silica cartridges, supplied by Waters (Milford, MA, USA), were used as solid phase extraction for purification. PTFE membrane filters (25 mm, 0.45 µm) were purchased from Agela Technologies (Wilmington, DE, USA).
Sample preparation. Food samples were collected from September 2014 to March 2015, in 18 large supermarkets located in 9 cities, Korea. Samples were homogenized in a blender and stored in a freezer at −20℃ in tightly sealed bottles prior to extraction and analysis. Foods samples were classified under two groups. The first group included 15 samples of seafood. The second group included 15 samples of dairy products.
Extraction and clean-up. A 10 g of the homogenized sample was weighed into a round bottom flask (300 mL), spiked with 1 mL of deuterated internal standard (100 µg/kg CHR-d12, BaP-d12. A 100 mL of 1 M potassium hydroxide (Junsei) in ethanol (Burdick & Jackson) was added for alkaline degradation under reflux at 80℃ for 3 hrs to isolate PAHs bound to the sample and to eliminate the matrix that would interrupt the PAH analysis. After cooling the flask with cold water, pre-weighed amount of n-hexane (50 mL) (Burdick & Jackson) and 1 : 1 ethanol/n-hexane (50 mL) (Burdick & Jackson) were added. Then the solution was filtered through filter paper (Filter paper 110 mm; Advantec, Toyo Roshi Kaisha, Ltd., Japan) and transferred to a separating funnel. This solution was liquid-liquid extracted two times with 50 mL n-hexane (Burdick & Jackson), after washes three times with 50 mL of distilled water (Milli-Q water purification system). The clear upper hexane layer was dried using anhydrous sodium sulfate (Junsei) and collected into a round bottom flask (250 mL). The extracts were reduced to a small volume using a rotary evaporator (Rotary vacuum evaporator N-N series SB-100; EYELA, Tokyo, Japan) at 37℃. The extract contains not only PAHs, but also numerous other hydrophobic and slightly non polar compounds. These components must be removed in further steps of analysis in order to facilitate the separation and quantification of single PAHs. Samples were first eluted using an activated solid phase extraction cartridge (Sep-Pak Silica cartridges, Waters) with a 5 mL of n-hexane (Burdick & Jackson) and mixture of 15 mL nhexane-dichloromethane (3 : 1) (Burdick & Jackson). This resulting solution was taken up to dryness using a gentle stream of nitrogen gas at 37℃, re-dissolved in 1 mL of dichloromethane (Burdick & Jackson). The solution was passed through a 0.45 µm PTFE membrane filter (Agela Technologies) and transferred to 2 mL amber screw-cap vials (Agilent Technologies,USA). An aliquot of 1 µL of this solution was injected into the GC/MS system Agilent Technologies 7820A/5975 MSD GC-MS apparatus (Santa Clara, CA).
GC-MS analysis of PAHs. The sample extracts were analyzed using an Agilent Technologies 7820A/5975 MSD GC- MS apparatus (Santa Clara, CA) with the conditions listed in Table 1. The used column is a HP-5MS column (30 m × 0.25 mm, ID particle size 0.25 µm). Ultra pure (99.999%) helium is used as a carrier gas (1.0 mL/min). The solutions of the extracted are injected in the splitless mode. The mass spectrometer was operated in the electron ionization (EI) mode using selected ion monitoring (SIM). Typically, two to four ions are monitored per compound, target ions were 228, 252, 276, 278, 240 (IS), and 264 (IS) for the 8 PAHs. The list of analyzed compounds and internal standards employed, the quantification ion and the confirmation ion are shown in Table 2.
Analysis conditions of gas chromatography-mass spectroscopy for the eight polycyclic aromatic hydrocarbons
List of 8 PAHs, the deuterated standards employed (underlined), the quantification ion and confirmation ion for SIM (single ion monitoring) GC-MS mode
Identification and quantification of PAHs. The identification of individual PAH was performed by comparison of the substance retention time and their retention time obtained with true standards in the same conditions. The way to prove the identification of the PAHs is molecular mass or characteristic mass fragments using a library database. To obtain standard calibration curves, PAH standard solutions relative to the two internal standard compounds were determined at five PAHs concentrations (1, 2, 5, 10, and 20 µg/kg). For validation of recovery, all standards containing 100 µg/kg of CHR-d12 and BAP-d12 as an internal standard.
Method validation and analytical quality assurance. Method was validated for accuracy, precision, linearity, limit of detection (LOD) and limit of quantification (LOQ). Validation proceeded on 2 matrices including seafood and dairy product. All standard mixtures were injected at a volume of 1 µL in triplicate to construct calibration curves. Accuracy (%) and precision (%) were evaluated by repeating the spiked samples run. The spiked samples were analyzed in 3 times during the same day (intra days) and in three different days (inter days).
RESULTS AND DISCUSSION
Linearity of the calibration curve. The chromatograms of the 8 PAHs in the standards and samples are shown in Fig. 1. The PAHs showed a wide spectrum and all 8 PAHs behaveed the same in the standard and the sample. The recoveries were obtained using an internal standard method, assuming that PAHs and d-PAHs appear to behave in a similar during extraction. For calibration curves, the response factors of PAHs relative to the two internal standard were assessed at five different PAH concentration levels (1, 2, 5, 10, and 20 µg/kg). The squared correlation coefficient of determination (R2) measures the chromatographic area as the concentration of the calibration curve. The correlation coefficient was observed for PAHs at all concentrations with R2 > 0.99.
GC/MS chromatograms of 8 PAH standards (100 μg/kg) (A), 100 μg/kg standard spiked sample (B) and BaA, CHR peak in sample (C) 1, benzo[a]anthracene; 2, chrysene-deuterium12; 3, chrysene; 4, benzo[b]fluoranthene; 5, benzo[k]fluoranthene;...
LOD and LOQ. LOD is defined as the lowest concentration leading to a signal-to-noise ratio of 3 whereas the LOQ is defined as the concentration leading to a signal-to-noise ratio of 10. The LOD of seafood matrix was 0.12~0.20 µg/kg, and the LOQ was 0.36~0.60 µg/kg. The LOD of dairy product matrix was 0.04~0.20 µg/kg, and the LOQ was 0.12~0.60 µg/kg (Table 3).
Recovery. The recovery values were measured using the peak area of CHR-d12 and BaP-d12. Average recovery varied between 90.99~103.57% from seafood matrix and 90.43~102.67% from dairy product matrix. And average relative standard deviation was 8.08~15.31% and 6.75~13.26%, respectively (Table 3).
Accuracy (%) and precision (%) analysis. Accuracy (%) and precision (%) were performed for at 5 concentrations. Intra-day accuracy and precision were evaluated by analyzing one sample on 3 different days. Inter-day accuracy and precision were performed by running tree analyses on the same day under the same conditions. Results are presented in Table 4.
Comparison of accuracy and precision (CV) of the polycyclic aromatic hydrocarbons (PAHs)
PAH content in seafood and dairy products. The concentration of the 8 PAHs in seafood and dairy products were determined. GC-MS chromatograms of the 8 PAHs for the standards and spiked samples are given in Fig. 1. The concentrations of the 8 PAHs in 15 samples of seafood are presented in Table 5, then 21 samples of dairy products are presented in Table 6. All of the experiments were carried out in triplicate. As seen in Table 5, BghiP were not detected in all seafood samples. The mean concentration of BaP in seafood samples was 0.34 µg/kg, 4 PAHs were presented at 0.67 µg/kg, and the total concentration of 8 PAHs was 1.06 µg/kg. The mean concentration of BaP in dairy product samples was 0.34 µg/kg, 4 PAHs were presented at 1.02 µg/kg, and the total concentration of 8 PAHs was 1.52 µg/kg, in Table 6. Among seafood samples, the highest BaP concentration in sea eel and the highest total PAHs concentration in Gray mullet. Among dairy product samples, concentration of BaP was the highest in coffee cream and concentration of total PAHs was the highest in whipping cream.
Concentration of polycyclic aromatic hydrocarbons (PAHs) in seafood (μg/kg)
Concentration of PAHs in dairy products (μg/kg)
DouAbul et al. revealed levels of PAHs in edible muscle of fishes collected from the Red Sea. Mean concentrations for individual PAHs in fish were; BaA 0.4, CHR 1.9, BbF 0.5, BkF 0.5, BaP 0.5, and IcdP 0.1 µg/kg dry weight respectively (14).
Dhananjayan and Muralidharan investigates the concentrations of 15 PAHs in 5 species of fish samples collected the Harbour, Mumbai. The levels of 6 carcinogenic PAHs (BaA, BbF, BkF, BaP, IcdP, DahA) ranged from 9.49 to 31.23 µg/kg and the maximum concentration of 15PAHs in marine fish species was found in Goldspotted grenadier anchovy (70.44 µg/kg wet wt.) (15).
Lawrence and Weber has studied determination of PAHs in dairy product samples collected from local outlets in Canada. The concentration of 5 PAHs (BaA, BbF, BaP, DahA, IcdP) ranged from ND to 1.9 µg/kg, in skim milk samples and 7.8 µg/kg in infant formula sample (16). Cho and Shin carried out evaluation of the contents of the 7 PAHs in infant formulas and mixed milk powder. The concentration of 7 PAHs ranged from 0.064~0.968 µg/kg in the infant formula group and 0.244~0.775 µg/kg in mixed milk powder samples (17).
It is known that PAHs can be produced during drying processes in direct heating (18). Whereas Aguinaga et al. reported that no PAHs was detected in the half-fat milk and skimmed milk samples, perhaps since PAHs are reduced during the skimming process (19).
According to the Commission Regulation (EC) No. 1881/2006, the maximum tolerance limit for BaP of infant formula, follow-on formula, and baby foods is 1 µg/kg (5). In this study, no sample exceeded the maximum permitted level of 1 µg/kg.
The main aim of the present work is to evaluate an analytical method for the determination of 8 PAHs and the content of carcinogenic PAHs including BaA, CHR, BbF, BkF, BaP, DahA, BghiP and IcdP in milk products and seafood using the GC/MS. In this way, the results of PAHs concentration can be used to select those contributing to minimize their presence, and to establish the limits of PAHs in this kind of food products.
This research was supported by a grant (13162MFDS049) from Ministry of Food and Drug Safety in 2013-2015.
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This paper aims to provide a review of the analytical extraction techniques for polycyclic aromatic hydrocarbons (PAHs) in soils. The extraction technologies described here include Soxhlet extraction, ultrasonic and mechanical agitation, accelerated solvent extraction, supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction. The influencing factors in the extraction of PAHs from soil such as temperature, type of solvent, soil moisture, and other soil characteristics are also discussed. The paper concludes with a review of the models used to describe the kinetics of PAH desorption from soils during solvent extraction.
Polycyclic aromatic hydrocarbons or polynuclear aromatic hydrocarbons (PAHs) are compounds produced through incomplete combustion and pyrolysis of organic matter. Both natural and anthropogenic sources such as forest fires, volcanic eruptions, vehicular emissions, residential wood burning, petroleum catalytic cracking, and industrial combustion of fossil fuels contribute to the release of PAHs to the environment . The presence of PAH compounds in soils is an issue of concern due to their carcinogenic, mutagenic, and teratogenic properties. In 2008, 28 PAHs have been identified as priority pollutants by the National Waste Minimization Programme, a project which is funded by US Environment Protection Agency .
PAHs which consist of fused benzene rings are hydrophobic in nature with very low water solubility and high octanol-water partition coefficient (). Hence, they tend to adsorb tightly to organic matter in soil rendering them less susceptible to biological and chemical degradation. Prolonged aging time in contaminated soil promotes the sequestration of PAH molecules into micropores and increases the recalcitrance of PAHs towards treatment . Thus the extraction process of PAHs from soil for analysis is made more complicated due to these factors. In this paper, various analytical extraction techniques for PAHs in soils will be reviewed, ranging from more widely applied methods such as Soxhlet extraction, sonication, mechanical agitation, and accelerated solvent extraction to alternative ones such as supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction. The influencing factors in the extraction of PAHs from soil such as temperature, type of solvent, soil moisture and other soil characteristics are also discussed. Finally, a review of the models used to describe the kinetics of PAH desorption from soils during solvent extraction will be provided.
2. Extraction Techniques
2.1. Soxhlet Extraction
The Soxhlet extraction has been vastly used as a benchmark technique in the extraction of PAHs from soils and sediments. Basically, in the Soxhlet extraction technique, the solid sample is placed into an extraction thimble which is then extracted using an appropriate solvent via the reflux cycle. Once the solvent is boiled, the vapour passes through a bypass arm into the condenser, where it condenses and drips back onto the solvent in the thimble. As the solvent reaches the top of the siphon arm, the solvent and extract are siphoned back onto the lower flask whereby the solvent reboils, and the cycle is repeated until all the sample is completely extracted into the lower flask.
The main disadvantage of this extraction process is the use of large volumes of solvent, which can be more than 150 mL for the extraction of PAHs from a mere 10 g of soil sample. In addition to that, this method is very labour intensive and time consuming, as the solvent has to be refluxed up to 24 hours to achieve considerable extraction efficiencies [4, 5]. The Soxhlet extraction too has been shown to have relatively poor selectivity for PAHs compared to bulk soil organic matter, with approximately a quarter to one third of bulk soil organic matter removed during extraction . Studies have indicated that the chromatograms of extracts produced via Soxhlet using GC-MS and GC-FID yielded more artefact peaks with branched alkane “humps,” demonstrating that compounds such as n-alkanes and humic substances other than PAHs are coextracted using the Soxhlet technique [6, 7]. Other minor drawbacks of using the Soxhlet apparatus include the likelihood of sample carryover, the need to fractionise extracts to avoid heavy contamination of GC injection port, and the unfeasibility of redissolving dried Soxhlet extracts [8, 9].
Nonetheless, the Soxhlet extraction is still the preferred method because of its comparative extraction results despite the nature of matrix sample. Not only does the Soxhlet extraction yields similar results with methods such as the supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), and ultrasonic methods, but the results also show small variations with low relative standard deviations [10–12]. Statistically, Berset et al.  showed that the Soxhlet method resulted in median values which corresponded to the overall mean of other extraction procedures including ASE, SFE, MAE and sonication. The efficiency of the Soxhlet extraction increases with molecular weight, reaching an efficiency range of 84–100% for PAHs with more than 4 rings .
To further improve the Soxhlet extraction technique, Edward Randall patented the automated Soxhlet extraction method in 1974. This is a two-step procedure which combines boiling and rinsing such that the total extraction time is reduced while the evaporated solvent condenses rapidly for reuse, reducing the amount of total solvent required. In this improved technology, the extraction thimble is initially lowered directly into the flask containing the boiling solvent to remove residual extractable material while the extractable materials pass readily from the sample and dissolve into the solvent simultaneously. The level of solvent is then reduced to a level below the extraction thimble such that the configuration mimics the traditional Soxhlet extractor whereby the PAH is extracted by refluxing condensed solvent and collected in the solvent below the extraction thimble. With this improvisation, the PAH extraction efficiencies and precisions were statistically improved, with almost 100% recovery rates . In addition to that, the compact design of the automated system also allows several samples to be extracted simultaneously with its multiple extraction cells assembly while being run unattended [4, 5].
2.2. Ultrasonic Agitation/Sonication
The ultrasonic agitation, also known as sonication, is a technique which engages the acoustic energy of ultrasonic waves with a minimum frequency of 16 kHz in fluid, causing rapid compression and rarefaction of fluid movement which results in the cavitation phenomenon, that is, the reoccurring formation and collapse of microbubbles. This agitation can be performed either by immersing a sonicator transducer also known as an ultrasonic horn into the sample solvent mixture or placing the sample solvent mixture directly into a sonication bath. The desired ultrasound is generated by means of piezoelectric ceramic attached either to the ultrasonic horn or the walls of the sonication bath.
Sun et al.  claimed that sonication was better than the Soxhlet because it provided higher extraction efficiencies, was more economical and easily operated. Likewise, Guerin  noted that similar levels of extraction efficiency to the Soxhlet extraction method could be attained through vigorous sonication. However, the level of extraction efficiency was observed to be highly dependent on the sample matrix and concentration of contaminants in the sample. Contrary to these observations, other studies have indicated that sonication was less efficient than the Soxhlet with relatively low recoveries particularly for lower molecular weight PAHs (44–76%) [13, 16].
The power amplitude and duration of sonication need to be carefully controlled in order to avoid extensive exposure to the irradiation which may degrade the contaminants in the sample and reduce the extraction rates of PAHs. The decrease in efficiency during excessive sonication is due to an increase in broken carbonaceous particles and additional contact surface area which adsorbs the PAHs more readily, causing a reversed adsorption cycle of PAHs . Additionally, further separation techniques such as centrifugation or filtration are required after the extraction process.
2.3. Mechanical Agitation
This simple, low-cost method uses agitation or mixing action to extract the PAHs from samples in a shake-flask placed onto a rotary shaker, or with a magnetic stirrer submersed into the flask directly. Although it is an easy handling method with minimal glassware and smaller volumes of extraction solvent, this method has not been as widely used as the Soxhlet and sonication due to the lower extraction efficiency and unsatisfactory quantitative results [5, 7]. Although some studies reported that this method was comparable to the Soxhlet technique, the results obtained using mechanical shaking showed larger variations and less selectivity due to the difficulty in quantifying the PAH extracts [12, 17]. Comparable results were only attainable with long shaking times to extend the contact time with solvent [18, 19].
2.4. Accelerated Solvent Extraction (ASE)/Pressurised Fluid Extraction (PFE)
Accelerated solvent extraction (ASE) or pressurised fluid extraction (PFE) is a fairly new technology which raises the solvent temperature above its boiling point but maintains it in the liquid phase by elevating the pressure. As a result, the high pressure aids in the solubilisation of air bubbles, thereby exposing more of the sample to the extraction solvent while increasing the capacity of the heated solvent to impart better solubility.
Today, ASE systems are commercially available for extracting organic compounds from a variety of solid samples. The ASE system is built up of several extraction cells on a loading tray proximate to an oven. During extraction, organic solvent is pumped into the extraction cells preloaded with soil samples while increasing the temperature and pressure to the desired values. Once extraction is completed, a nitrogen cylinder is used to purge the samples of residual solvent.
With the usage of the ASE system, the recovery of PAHs from soils and sediments was reported to be two times higher than using the Soxhlet extraction method , while the accuracy was also improved with a relative standard deviation of less than 10% . Other benefits of ASE include reduction of solvent consumption and total time required due to the use of high pressures. The extraction procedure can be fully automated with an online purification column, preventing loss of the volatile PAHs, avoiding tedious preparation and potential contamination as in the case of mechanical shaking [21, 22].
2.5. Supercritical and Subcritical Fluid Extraction
Supercritical fluids exhibit a continuum of both gaseous and liquid phase properties. Their physical characteristics including liquid-like density, low viscosity, high diffusivity and zero surface tension enable them to penetrate almost anything and dissolve most materials into their components. Carbon dioxide which has a supercritical temperature and pressure of 31°C and 74 bar, respectively, is widely employed in SFE as an environmentaly friendly solvent in its supercritical state .
In a study by Miége et al. , comparisons between Soxhlet and SFE extraction revealed that the recoveries of PAHs for both methods were almost similar. Although the SFE technique was more difficult to optimise, the technique provided extraction results with lower relative standard deviation and better selectivity, due to cleaner extracts. Other studies [6, 23] also indicated that SFE removed only 8% of the bulk organic matrix in comparison with Soxhlet extraction or ASE which extracted a quarter to one third of bulk soil organic matter. Furthermore, integrated SFE systems allow concentrated extracts to be directed straightaway into the cleanup column, reducing the need to remove the eluate manually. In certain SFE systems, the extracts may also be analysed directly by GC without any cleanup. This prevents extra contamination that may occur during manual handling [12, 24]. However, the high complexity of the SFE process may contribute to inconsistent results this system should be carried out in different laboratories .
In the development of SFE, water has also been considered as the extraction fluid. However, the use of supercritical water is limited because of the high temperature (>374°C) and pressure (>218 atm) requirements which creates a highly corrosive environment . Thus, subcritical water extraction (SWE) also known as pressurised hot-water extraction is used instead. As the temperature of water is raised from 100°C to 274°C under pressure, the hydrogen bonding network of water molecules weakens resulting in a lower dielectric constant and simultaneously decreasing of its polarity. Thus, subcritical water becomes more hydrophobic and organic-like than ambient water, promoting miscibility of light hydrocarbons with water . In contrast to SFE which extracts mostly non polar organic compounds, it has been reported that SWE gives better preference to more polar analytes, therefore providing a higher extraction efficiency of PAHs with less or almost no extraction of other alkanes . Wet oxidation or SWE combined with oxidation using oxidising agents such as air, oxygen, or hydrogen peroxide was reported to remobilise bound organic residues, providing a higher extraction capability [27, 28]. In one study, SWE combined with oxidation resulted PAH soil extraction efficiencies within the range of 99.1–99.99% compared to extraction efficiencies within 79–99+% using SWE alone .
2.6. Microwave-Assisted Extraction (MAE)
Another highly instrumental extraction technique is the MAE whereby both solvent and samples are subjected to heat radiation energy attained from electromagnetic wavelengths between 1 m and 1 mm, with frequencies of 300 MHz to 300 GHz. Microwave radiation is preferred compared to conventional heating due to its rapid heating which is reproducible and has less energy losses. Modern designs of the microwave ovens include carousels which can hold at least twelve extraction vessels allowing simultaneous multiple extractions. The main advantages of the MAE method are the reductions in solvent usage and time. In comparison to SFE, the cost of MAE is moderately lower . Additionally, this unique heating mechanism provides selective interaction with polar molecules which greatly enhances the extraction efficiency of PAHs [29, 30].
The major drawback of this method however is that the solvent needs to be physically removed from the sample matrix upon completion of the extraction prior to further analysis. In certain cases whereby samples are pretreated with activated copper bars to assist the extraction process, the removal of this copper is necessary for a cleaner extract . Although a subsequent purification step can be implemented to rectify this problem, there may be possibilities of losing analytes or inducing contaminants with additional cooling time for this extra handling. Furthermore, the sample allowance for analysis is limited to 1.0 g which is insufficient for a homogenous analysis .
2.7. Alternative PAH Extraction Techniques
Solid phase extraction (SPE), a method that is generally used to clean up a sample has been used for rapid and selective extraction of PAHs from soil samples. Soil samples are washed with solvent to leach away undesired components before extraction of PAHs with a different solvent into a collection tube . When this extraction technique is employed, filtering over an empty SPE column or using purified sand prior to extraction is usually recommended to prevent soil samples clogging the SPE column. A variation to the SPE of PAHs from soils is the solid phase microextraction (SPME). Ouyang and Pawliszyn  described the application of the technique on PAH extraction from soils. This solvent free approach utilises a small diameter fused-silica fibre coated with the extracting phase and mounted in a syringe-like device for protection and ease of handling. The depth of injected needle is adjusted for headspace sampling before exposing the fibre which adsorbs the PAHs from the soil. The exposed SPME fibre is then transferred directly to the injection port of an analytical instrument such as a GC for quantitative analysis. The major advantage of the SPME is its fast, simple and convenient extraction which can be done on-site. The configuration of the solid-phase microextractor offers solutions to sampling problems because it allows extraction of small volume of samples which can then be analysed without any pretreatment. When stored properly, the fibre on the needle can also be analysed several days later in the laboratory without significant loss of volatiles. The capability of the SPME device to extract such small volumes of samples requires extreme precision during manufacturing to achieve homogeneity in the construction of the fibre (extraction phase surface) to provide consistency in extraction outcomes and qualities . One study using SPME revealed that only volatile compounds such as lower molecular weight (LMW) PAHs (less than 4 rings) were detected .
Another alternative PAH extraction technique is thermal desorption which does not use solvents or high-pressure extraction equipments. The thermal desorption technique is commonly coupled with GC by direct injection of solid sample onto the cold injector. The carrier gas is temporarily halted while the injector is rapidly heated to the desired temperature approximately within 200–500°C to volatilise targeted compounds from soil. The carrier gas is then resumed and the isothermally extracted compounds are swept onto the GC column, providing a direct and rapid analysis of the contaminated soil. Thermal desorption and online GC analysis technique has been widely employed in the analysis of PAHs in various matters including fly ash, ambient air particulate matter as well as creosote and petroleum contaminated soil [36–39]. The technique, however, requires prior calibration to allow for nonlinear response to sample size and concentration of contaminants .
Contrary to thermal desorption, pyrolysis (Py) or high temperature distillation (HTD) extraction technique employs high rate temperature ramping or flash pyrolysis at high temperatures. In flash pyrolysis, the sample is heated in a very short time using either inductive heating (also known as Curie point pyrolysis) or Ohmic heating using platinum foil. The significant increase in heat energy in the system causes thermal cracking of larger macromolecules into simpler monomers which are more volatile. Due to its high heating velocity, accurate temperature reproducibility and wide temperature range, the Py has successfully been applied to various nonvolatile compounds and matrices such as synthetic plastics, rubbers and paints. Buco et al.  have demonstrated its novel application in the analysis of PAHs in contaminated soil. Here, induction heating of the soil sample is carried out in a ferromagnetic foil called pyrofoil in an oven equipped with a radio frequency field to reach the Curie point temperature (160–1040°C) whereby the pyrofoil loses its magnetic properties and simultaneously adopts the specific property of a heated alloy. As such, the soil sample which is wrapped inside the pyrofoil is desorbed of the PAHs and the PAH bearing pyrolysates are transferred immediately into an online GC column for further analysis. Pyrolysis methods have been a more popular choice than thermal desorption due to their capabilities in providing greater temperature control. With high temperature Py method, the extraction speed is also significantly reduced, permitting a higher number of samples to be analysed. The main advantages of thermal desorption or pyrolysis with online GC is the exclusion of reconcentration and clean-up steps necessary for some other extraction methods. Therefore, the contamination risks are lower with higher sensitivity and specificity when these methods are employed. Similar to SPME, the use of solvents are also eliminated, which subsequently reduces cost. Nonetheless, the small sample size used (approximately 30 mg) may result in insignificant data analysis errors since it does not provide a good representative of the entire field soil. In addition, the temperature program used has to be carefully optimised to avoid the decomposition of the cellulose filter itself, which may result in formations of undesirable byproducts.
Fluidised-bed extraction (FBE) has also been reported in the specialised literature to extract PAHs from soils . The system is analogous to the automated Soxhlet extraction apparatus whereby the soil sample is loaded into an extraction tube secured with a filter at the bottom while the extraction solvent is filled into the basic vessel beneath the soil sample. The heating block of the device is first heated up to evaporate the extraction solvent through the filter which then condenses when in contact with the cooling bar above the soil sample. The condensed solvent then drips back into the soil sample and further down into the collected solvent. The constant penetrating flow of solvent vapour heats up and agitates the soil mixture, causing it to be fluidised. The collected solvent in the basic vessel is then concentrated for further analysis. In comparison with the conventional Soxhlet extraction, the extraction duration and solvent used is reduced under optimised conditions.
3. Influencing Factors
In the majority of analytical studies using ASE, SFE, and MAE, the PAH extraction efficiencies were observed to generally increase with increasing temperatures, as can be seen in Table 1 [9, 31, 42–46]. Elevated temperatures reduce both fluid density and viscosity, resulting in lower surface tension and improved contact between the solvent and targeted PAH analytes. The diffusion of PAHs through the soil as well as the diffusion of solvent into the interior of the soil matrix is enhanced. Likewise, the desorption of PAHs from the solid matrix and their solubilities in the extraction solvent are improved by increased temperatures. As such, the time to achieve equilibrium is significantly shortened. Unfortunately, 2- and 3rings PAHs are highly volatile and more susceptible to evaporation instead of extraction at higher temperatures. Thus, the reported extraction efficiencies for LMW PAHs were less than the higher molecular weight (HMW) PAHs. A few papers reported that increasing temperatures caused a general decrease in the PAH extraction efficiencies and recovery yields [44, 47]. While there is no certain explanation for this behaviour, it has to be noted that these studies were using SFE.
Table 1: Bibliographic compilation of studies on extraction temperature.
3.2. Solvent Type
Table 2 is a bibliographic compilation of PAH extraction studies from soils using various solvents. Generally, the choice of extraction solvent is dependent on several factors, with one of them being the degree of PAH concentration in the soil. For lowly polluted soil ( dry weight sample), PAHs are mainly found on the surface, therefore a more polar solvent such as acetone is preferred to break up the soil aggregates and to allow intensive contact between particles. For highly polluted soil ( dry weight sample) however, a relatively nonpolar solvent such as toluene or cyclohexane would be a better solvent . Since the principles of solvent extraction are based on the theory of like dissolves like, the polarity of solvent with respect to the polarity of PAH contaminants also plays a role in determining the extent of solubility. For instance, it was shown that dichloromethane as an extraction solvent for PAHs resulted in low recoveries for all compounds, whereas hexane-acetone (1 : 1) was an effective extraction solvent for PAHs [48, 49]. Apart from PAH concentration and polarity of solvents, extraction efficiencies vary from one technique to another. In MAE, for example, solvents are chosen based on their dissipation factor (dielectric constant) which determines the degree of absorption of microwave energy [31, 49].
Table 2: Bibliographic compilation of solvents used in the extraction of PAHs.
3.3. Soil Moisture and Other Soil Characteristics
The effects of soil moisture on PAH extraction efficiencies are dependent on the type of extraction technique employed as shown in Table 3. With MAE studies, PAH extraction efficiencies were generally observed to increase with increasing soil moisture. This is mainly due to the ability of the localised superheating to form gas bubbles from existing water residues in soil and cause expansion of pores, allowing solvent penetration into the matrix. Additionally, the high dielectric constant of water allows more microwave absorption which in turn provides more heating [29, 30]. Similarly, a study using SFE showed that for water content less than 10 wt. %, the water in soil acted as a modifier to the extraction solvent which increased the fluid’s capability to penetrate further into the soil particles . Other SFE and Soxhlet experiments revealed that the presence of soil moisture decreased or did not significantly affect the efficiency of PAH removal from soil. Soil drying is therefore carried out in some cases to eliminate the influence of moisture on the PAH extraction efficiency. Comparisons between various drying methods showed that thermal drying of soil between temperatures of 25°C and 40°C for several days was best for prevention of losses of volatile PAHs while air drying was reasonably sufficient and freeze drying was least preferable due to partial loss of highly volatile PAHs such as naphthalene .
Table 3: Bibliographic compilation of studies on soil moisture.
Apart from soil moisture, the composition of soil affects the extraction of PAHs. The extraction process of PAHs was observed to be significantly more difficult from high clay content soil (>40%) due to the fact that 32% of the total carbon content where most of the HMW PAHs resided in was concentrated in the clay fraction . Strong adsorption of PAHs to clay surfaces also result in reduced desorption during thermal extraction and less detectable hydrocarbons . The size of soil particles also impacts the efficiency of PAH extraction. It has been demonstrated that PAHs accumulate preferentially on smaller particles . As such, PAHs are more easily extracted from fine soil fractions such as fine silts and clays than larger aggregate size fractions. Reduced particle sizes allow ample diffusion and better accessibility of solvent through the matrix, thus increasing the flow rate of solvent and rate of extraction [17, 51].
4. Kinetics Models of PAH Desorption from Soils
4.1. First-order Mass Transfer with Single Equilibrium Desorption
The dissolution and desorption of PAHs can be fitted to a first-order mass transfer coefficient model :
where is the liquid-phase concentration at any point in time, is the lumped mass transfer coefficient, is the equilibrium liquid-phase concentration and is the contact time with the extraction solvent.
4.2. First-order Mass Transfer with Dual Equilibrium Desorption
The desorption process in sediments and soils contaminated with hydrophobic contaminants can be classified as a biphasic process, with a fast and a slow component [53–55]. This two-site kinetic model is described by
where is the liquid-phase concentration at any point in time, is the equilibrium liquid-phase concentration, is the equilibrium liquid-phase concentration of the first stage (rapid), is the mass transfer coefficient of first stage, is the equilibrium liquid-phase concentration of second stage (slow), is the mass transfer coefficient of second stage, and is the contact time with the extraction solvent.
This model treats the process as a combination of two kinetically controlled reactions occurring simultaneously, whereby the first stage is governed by a rapid partitioning between the solid and liquid phases while the latter stage is which generally slower than the first is kinetically controlled by other processes. Equation (2) can also be employed in its fractional form whereby the rapidly desorbing fraction is while the slowly desorbing fraction is ():
where is the fraction of the PAH extracted after time .
Of the PAH extraction technologies discussed here, Soxhlet extraction, ultrasonic and mechanical agitation can be implemented easily since the processes are carried out with minimal instruments or glassware and at ambient pressures. In comparison, ASE and MAE provide a faster extraction with lesser solvent consumption albeit at higher capital costs and possibly operating costs. PAH extraction using supercritical carbon dioxide or subcritical water is an environmentaly friendly technique but entails the use of high pressure equipment. SPE and SPME, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction are novel alternatives which require further in-depth studies prior to wide-scale adoption in laboratories. It has to be recognised that no single extraction technology can be the solution for all extractions of PAHs in soils and sediments. Costs, the required accuracy and precision in results, analysis time, as well as technical competence are factors to be considered in deciding the right extraction technique.