Development of a method for GC/MS analysis of PAHs and alkylated PAHs for use in characterization and source identification of PAH contaminated sites

Transcription

1 Project in Chemistry: 15 HP Development of a method for GC/MS analysis of PAHs and alkylated PAHs for use in characterization and source identification of PAH contaminated sites Hanne Vestlund Supervisor: Maria Larsson

2 TABLE OF CONTENTS ABSTRACT... 2 SAMMANFATTNING Introduction PAHs PAH contaminated soils sources and characterization GC/MS Objective Materials and Method Chemicals Method development Development of method for the PAH-mix Development of method for the Alkyl-Mix Development of method for the Alkyl-PAH mix Quality of Data Analysis of soil samples Soil samples GC/MS Analysis Source identification and characterisation Results and Discussion Method development PAH method Alkyl method Alkyl-PAH method Quality Assurance and Quality control Concentration of PAHs in soil samples Source identification and characterisation of soil samples Conclusion Consideration Acknowledgements References Appendix

3 ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are toxic and carcinogenic environmental contaminants originating from different sources; petrogenic, pyrogenic or biogenic. Depending on the source of contamination there will be different ratios of PAHs and the effects on the environment will differ. Petrogenic sources will be higher in concentration of alkyl substituted PAHs (APAHs) while pyrogenic sources will be higher in parent PAHs. In the present study a GC/MS method was developed to separate and calibrate PAHs, dibenzothiophenes and alkyl substituted PAHs in a mix containing 49 standards. The method was able to differentiate between PAHs and APAHs with the same mass number; up to six different compounds with the same mass number was separated. The developed method was used to analyse six different soil samples from various contamination sites. PAHs, APAHs and dibenzothiophenes were identified and quantified in all samples. In order to establish the source of contamination, the distribution pattern, the ratio between different PAHs, and the ratio between APAHs and parent PAHs were used. There was a higher ratio of APAHs/PAHs and a lower ratio between the parent PAHs in the soil samples from sites contaminated with oils compared to the other samples, indicating petrogenic source. 2

4 SAMMANFATTNING Polycykliska aromatiska kolväten (PAH) är giftiga och cancerframkallande miljögifter som härstammar från olika källor; petrogena, pyrogena eller biogena. Olika föroreningskällor kommer att ha olika förhållanden av PAH och effekterna på miljön kommer att skilja. Petrogena källor innehåller högre koncentrationer av alkylsubstituerade PAH (APAH) medan pyrogena källor kommer att ha högre koncentration av PAH. I denna studie har en GC/MS-metod utvecklats för att separera och kalibrera PAH, dibensotiofener och alkylsubstituerade PAH i en blandning innehållande 49 standarder. Metoden kunde skilja mellan PAH och alkylsubstituerade PAH med samma masstal; upp till sex olika föreningar med samma masstal särskildes. Den utvecklade metoden användes för att analysera sex olika jordprover från olika föroreningsplatser. PAH, APAH och dibensotiofener identifierades och kvantifieras i samtliga prover. För att fastställa föroreningskällan användes fördelningsmönstret för APAH och PAH, förhållandet mellan olika PAH och ration mellan APAH och PAH. Det fanns en högre kvot APAH/PAH i jordprover från områden som var förorenade med olja, vilket indikerar på petrogena föroreningskällor. 3

5 1. Introduction 1.1 PAHs Polycyclic aromatic hydrocarbons (PAHs) consist of two or more aromatic rings. More rings usually equals higher lipophilicity and lower vapour pressure. PAHs with lower molecular weight (2-3 ringed PAHs) exist in gaseous form for the most part while the PAHs with higher molecular weight ( 4 ringed PAHs) are adsorbed to particles in air. PAHs are formed, for example, by incomplete combustion of biomass or fossil fuels. There are hundreds of different PAHs in the environment (Britten and Naikwadi, 2009). They have been produced commercially to make dyes, pesticides, and plastics. (Ravindra et al., 2008). PAHs can also be found in the environment because of petroleum spills or discharges. The most salient source is the incomplete combustion of wood, petroleum and coal causing PAHs in the atmosphere, adsorbed to particles like soot (Lima et al., 2005). PAHs can be found all over the globe, from the arctic ice and ocean sediment to high altitude lake sediments (Lima et al., 2005). They are found in soil, sediment, water and air and are especially high in concentration in urbanised or industrialised regions. This is because of the anthropogenic PAHs that arrive from vehicle engines that run on gasoline or diesel and industrial operations and power plants using fossil fuels (Lima et al., 2005) Pyrolysis and pyrosynthesis are the two main mechanisms that explain the formation of PAHs. That is, the decomposition of a compound by heat or the fusion of simpler compounds into more complex ones by heat (Ravindra et al., 2008). Low saturated hydrocarbons form PAHs under oxygen-deficient conditions by pyrosynthesis. The hydrocarbons are fragmented into free radicals that react to form a stabile ring structure; further reactions between these rings and small hydrocarbons create the high molecular weight PAHs (Lima et al., 2005). Lower temperature burning, like wood burning, will usually create the low molecular weight PAHs while higher temperature burning, like the combustion of fuel, will create high molecular weight PAHS. These PAHs are called pyrogenic (Tobiszewski and Namieśnik, 2012). PAHs found in crude oil and petroleum products, which can be found at places such as oil spill sites, are petrogenic PAHs, they are formed slowly under moderate temperatures (Saha et al., 2009). The naturally occurring PAHs from forests fires and such sources are called biogenic PAHs. PAHs are known to be toxic and carcinogenic (Straif et al., 2005). They are metabolized in the body through oxidation by P450 enzymes and may ultimately produce carcinogenic metabolites. These metabolites have been shown to induce lung and skin tumours in animals (Ravindra et al., 2008). People can be exposed through polluted air from urban or industrial environments, tobacco smoke, and diet (Straif et al., 2005). The carcinogenicity of the PAHs usually increases with increased number of aromatic rings and higher molecular weight, while low molecular weight PAHs are more acute toxic (Ravindra et al., 2008). Out of all the PAHs there are 16 that are considered priority pollutants by the U.S. Environmental Protection Agency (EPA) (Figure 1), because of the likelier risk to be exposed to them, the high amount of information about them, and that they are believed to be more harmful. They also exist in high concentration in the 4

6 environment and the harmful effects are considered representative for PAHs in general (Ravindra et al., 2008). In recent years it has been researched if the carcinogenic effect can be further affected by the substitution of different chemical groups, for example alkyl groups (Braga et al., 1999). Alkylated PAHs (APAHs) are PAHs that have been substituted with a methyl group, ethyl group or other alkyl-group. APAHs and other substituted groups of PAHs are often found in nature together with native PAHs. The alkylated PAHs are formed either from chemical reactions with the parent PAHs or like the PAHs directly from incomplete combustion (Braga et al., 1999). Adding alkyl groups will influence the electronegativity of the PAHs and the various positions on the ring structures can have different biological effects. The alkylated PAHs are classified as C1, C2, C3 and C4 according to how many alkyl carbons present. The effects of these substitutions are not fully understood and the focus is mainly put onto the 16 priority ones. A study by (Sun et al., 2014) showed that methylated forms of phenanthrene were 2 to 5 times more potent in AhRmediated toxicity than its parent form. 5-methylchrysene has been shown to be a more potent carcinogenic than chrysene (Yang et al., 2014). This shows the importance of being able to identify and analyse alkylated PAHs in environmental samples in order to undergo further studies of the toxicity and carcinogenic effects. In this study several alkylated PAHs as well as the priority ones were analysed (Figures 1, 2, and 3). Figure 1. The 16 priority PAHs 5

7 1.2 PAH contaminated soils sources and characterization Concentration and composition of PAHs in soil are dependent on factors like soil type, altitude, humidity, and nutrition. These factors can control the biodegradation by bacteria and fungi over time. It also affects the weathering. Some studies say that certain PAHs and even different isomers of the same homologue degrade faster than others, making it difficult to predict the change in concentration over time and determine a site remediation strategy (Antle et al., 2013; Tobiszewski and Namieśnik, 2012). In particular, levels of naphthalene and other low weight molecular PAHs will because of their water solubility decrease more than high molecular weight PAHs in weathered soil samples (Zhendi and Carl, 2008). The main source of PAH contamination in soils is atmospheric deposition (Tobiszewski and Namieśnik, 2012). The fact that high molecular weight PAHs can be adsorbed to particles makes them possible to transfer far distances in the air and once they hit the ground they can remain there without being decomposed. Another source is contamination by running water carrying PAHs bound to particles in the water and sediment. Some studies suggest that petrogenic PAHS might be more available for biological uptake because of their tendency to bind more strongly to sediment particles (Saha et al., 2009). Soils contaminated by PAHs are generally higher in concentration around industrial areas than the background levels. Around wood-preservation sites a common contamination source is creosote, a compound containing over 85 % PAHs by weight (Murphy and Brown, 2005). According to (Mueller et al., 1989) there are some PAHs and APAHs that are more common than others in creosote, among these are Naphthalene, 2- Methylnaphthalene, 1-Methylnaphthalene, Acenaphthene, Anthracene, 2-Methylanthracene, Phenanthrene, Fluorene, Fluoranthene, Chrysene, Pyrene and, Benzo(a)pyrene. There are relative ratios between different PAHs that can be used as indicators of the pollution emission source, to determine if the source is petrogenic or pyrogenic (Ravindra et al., 2008). For example the ratio between Fluoranthene to Fluoranthene + Pyrene (FLA/(FLA+PYR)) with a threshold of 0.4 (Banger et al., 2010), or the use of Anthtracene to Anthracene and Phenanthrene (ANT/(ANT+PHE)) with a threshold of 0.1 (Pies et al., 2008). One source of contaminated soils is discharged or spilled oil. In oil and petroleum alkylated PAHs account for a large quantity of the total amount of PAHs (Sun et al., 2014). The ratio between alkylated PAHs and their parent PAHs can be used to determine the source of the pollution. Alkylated PAHs are more thermodynamically unstable and will therefore deplete during combustion and be more dominant in petrogenic sources compared to pyrogenic sources (Saha et al., 2009). Higher ratios between the alkylated and the parent PAHs indicate higher concentrations of alkylated PAHs, and petrogenic sources. The ratio between methylated Phenanthrenes and Phenanthrenes (MPHE/PHE) has been commonly used. In a study by (Saha et al., 2009) on sediments in India and Southeast Asia a threshold of MPHE/PHE = 0.4 was used to determine exclusive pyrogenic origin. In soils contaminated by pyrogenic sources the concentration of the PAHs and APAHs is C0>>C1>C2>C3>C4, creating a slope-like distribution pattern. The petrogenic contaminated soil on the other hand creates a bell-like distribution pattern (Stout and Wang, 2008). Weathering tends to change the distribution pattern in the petrogenic contaminated soils to a more reversed slope one, especially for low molecular weight PAHs like naphthalene (Wang et al., 6

8 2008). Soil contaminated with oil has according to (Wang and Stout, 2006) after weathering and biodegradation high concentrations of Fluoranthene, Pyrene and C4-Phenanthrene. The Pyrogenic Index, PI ( 3-6 ring EPA PAHs/ 5 alkylated PAHs) is a method which has shown great consistency in differentiating between pyrogenic and petrogenic sources (Wang et al., 1999). The five alkylated PAHs included in the PI method are C1-C4 Naphthalene, Dibenzothiophene, Phenanthrene, Fluorene, and Chrysene. PI is superior compared to other methods of destinguishing between pyrogenic and petrogenic sources because it takes into account more PAHs and APAHs and will thefore not be as effected by individual uncertenties (Wang et al., 2008). This will also reduce the effects of weathering and biodegradiation on the ratio. Pyrogenic and petrogenic PAHs will affect the aquatic ecosystems differently because of their different tendency to bind to sediment particles (Saha et al., 2009). Knowing the source of contamination will therefore help in the understanding of the impact on the water, air and soil. This shows the importance of knowing the emission source of PAH contamination in the environment. It is crucial for the right risk assessment and risk management. Figure 2. Additional PAHs, methylated PAHS and dibenzothiophenes analysed in this study. 7

9 Figure 3. Additional PAHs, methylated PAHS and dibenzothiophenes analysed in this study. 1.3 GC/MS Gas chromatography coupled to mass spectrometry (GC/MS) is a well-established technique for analysing PAHs. It is cheap, time efficient and provides good sensitivity, selectivity and resolution. The difficulty with analysing PAHs in GC/MS lies in that many PAHs have the same mass (Britten and Naikwadi, 2009). The isomeric PAHs have the same ion fragmentation and chemical structure. This will make them impossible to separate in the mass spectrometer, and importance of a good chromatographic separation is imperative. In order to get the best possible separation, selection of column and oven program optimisation is necessary (Britten and Naikwadi, 2009). Recent years several selective columns have been developed for GC analysis, in order to optimise the separation. In this study a PAH selective column with built in PAH blockers on a siloxane stationary backbone was used. 1.4 Objective The aim of this study is to develop a method to separate PAHs and alkylated PAHs with GC/MS and to use this method on PAH contaminated soil samples. The ratio between the alkylated and the parent PAHs will be used to try and establish the source of the contamination. 8

10 2. Materials and Method All glassware was rinsed in ethanol, n-hexane and dichloromethane before use. All preparation of standards and samples was performed in fume hoods. The standards were kept at -4 C. 2.1 Chemicals The standards (Table 1) were purchased with the highest purity available from Chiron Laboratory, Sigma-Aldrich and LGC standards (Ultra Scientific and Labor Dr.Ehrenstrofer- Schäfers). Two different mixes of quantification standards (QS) were tested, one containing only PAHs and one containing a mix of alkylated PAHs and Dibenzothiophenes (Table 2). Additionally, one mix with all of the compounds was tested, with a total of 49 standards. Toluene was used as a solvent. Deuterium-labelled internal standard (IS), and a recovery standard (RS) of 500 ng were used. A seven point calibration curve at 10 ng, 50 ng, 250 ng, 500 ng, 1000 ng, 2500 ng and 5000 ng with a concentration ranging from ng/µl was established for each mix. Because of lack of standard material a three point curve at 10 ng, 50 ng and 250 was established for Dibenz(a,j)anthracene and a four point curve at 10 ng, 50 ng, 250 ng and 500 ng for Dibenz(a,c)anthracene in the Alkyl-PAH mix. Table 1. List of standards, supplier and purity Supplier Trade Name Name CAS Purity Chiron K-IO 9-Methylanthracene-d atom% D Chiron K-IO 1-Methylnaphthalene-d atom% D Chiron K-IO Dibenzothiophene-d atom% D Chiron T 2,3-Dimethylanthracene % Chiron S T PAH/Dibenzothiophenes Mixture 20 analytes Various >96.5% to >99.5% Ultra Scientific US-9196 Naphtho[2,3-a]pyrene % Ultra Scientific U-RAH-005 Benzo[a]fluorene % Ultra Scientific US-106N PAH mixture 16 analytes Various n/a Fluka, Sigma-Aldrich Naphthacene % Sigma-Aldrich M Methylanthracene % Sigma-Aldrich Cyclopenta[d,e,f]phenathrene % Sigma-Aldrich Methylbenzo[a]pyrene % Sigma-Aldrich S Methylbenz[a]anthracene n/a Sigma-Aldrich ,12-Dimethylbenz[a]anthracene % Sigma-Aldrich BCR077R 1-Methylchrysene % Sigma-Aldrich BCR078R 2-Methylchrysene % Sigma-Aldrich BCR079R 3-Methylchrysene % Labor Dr. Ehrenstorfer Schärfers DRE-C Dibenzo[a,c]anthracene % Labor Dr. Ehrenstorfer Schärfers DRE-L IO Dibenzo[a,j]anthracene % Labor Dr. Ehrenstorfer Schärfers US-106N PAH mix 9 deuterated (16 IS) Various 97.1% to 98.8% 9

11 Table 2. List of quantification standards, internal standards and recovery standard Quantification Standards Internal Standards PAH Mix: Trace: Alkyl mix: Trace: Trace: Naphtalene Methylnaphthalene 142 Naphthalene-D8 136 Acenaphtylene Methylnaphthalene Methylnaphthalene-D Acenaphtene 154 1,6-Dimethylnaphthalene 156 Acenaphthylene-D8 160 Fluorene 166 2,3,5-Trimethylnaphthalene 170 Acenaphthene-D Phenanthrene 178 Dibenzothiophene 184 Fluorene-D Anthracene Methyldibenzothiophene 198 Dibenzothiophene-D H-Cyclopenta(d,e,f)phenanthrene Methylphenanthrene 192 Phenanthrene-D Fluoranthene Methylanthracene 192 Anthracene-D Pyrene 202 2,8-Dimethyldibenzothiophene 212 Fluoranthene-D Benzo(a)fluorene 216 2,4-Dimethylphenanthrene Methylanthracene-D Benzo(a)anthracene 228 2,4,7-Trimethyldibenzothiophene 226 Pyrene-D Chrysene 228 2,3-Dimethylanthracene 206 Benzo(a)anthracene-D Naphtacene 228 1,2,8-Trimethylphenanthrene 220 Chrysene-D Benzo(b)fluoranthene 252 1,2,6-Trimethylphenanthrene 220 Benzo(b)fluoranthene-D Benzo(k)fluoranthene Methylfluoranthene 216 Benzo(k)fluoranthene-D Benzo(a)pyrene 252 Benzo(c)phenanthrene 228 Benzo(a)pyrene-D Dibenz(aj)anthracene 278 Triphenylene 228 Indeno(1,2,3-c,d)pyrene-D Dibenz(ac)anthracene Methylbenz(a)anthracene 242 Dibenz(a,h)anthracene-D Indeno(1,2,3-cd)pyrene Methylchrysene 242 Benzo(g,h,i)perylene-D Dibenz(ah)anthracene Methylchrysene 242 Benzo(ghi)perylene Methylchrysene 242 Recovery Standard Naphtho(2,3-a)pyrene Ethylchrysene 256 Perylene-D ,12-Dimethylbenz(a)anthracene 256 Benzo(j)fluoranthene 252 Benzo(e)pyrene 252 Perylene Methylbenzo(a)pyrene Method development For analysis an Agilent 7890A gas chromatograph coupled to a 5975 low-resolution mass spectrometer using electron ionization (EI) at 70 ev was used. The separation capillary column was a Select PAH column, 30m x 0.25 mm, df = 0.15 µm provided by Agilent. Detection was made in single ion monitoring mode (SIM). MassLynx was used to calibrate the standards. In order to optimise the separation different temperature programs were tried Development of method for the PAH-mix The PAH mix (Table 2) was tested with 8 different oven temperature programs in order to acquire the best separation and fastest analyse time. The first temperature programs (Test 1 and 2) were based on methods for separating PAHs and alkylated PAHs, found in published literature (CARB method 429; EPA method 8272; Oostdijk, 2010). The remaining are tests altered after the results of Test 1 and 2. 10

12 Test 1: 90 C (1 min), 8 C/min to 300 C (10 min), 5 C/min to 315 C (0 min) Test 2: 70 C (0.7 min), 85 C/min to 180 C (1 min), 3 C/min to 230 C (7 min), 23 C/min to 280 C (10 min), 14 C/min to 325 C (3 min) Test 3: 70 C (2 min), 70 C/min to 180 C (1 min), 7 C/min to 230 C (7 min), 50 C/min to 280 C (10 min), 30 C/min to 325 C (5 min) Test 4: 70 C (2 min), 40 C/min to 180 C (1 min), 5 C/min to 230 C (5 min), 20 C/min to 280 C (10 min), 7 C/min to 325 C (10 min) Test 5: 70 C (2 min), 50 C/min to 180 C (1 min), 7 C/min to 230 C (7 min), 20 C/min to 280 C (10 min), 5 C/min to 325 C (5 min) Test 6: 70 C (1 min), 8 C/min to 300 C (10 min), 5 C/min to 325 C (3 min) Test 7: 70 C (2 min), 40 C/min to 180 C (0 min), 7 C/min to 230 C (7 min), 20 C/min to 280 C (10 min), 5 C/min to 325 C (7 min) Test 8: 70 C (2 min), 40 C/min to 180 C (0 min), 7 C/min to 230 C (5 min), 20 C/min to 280 C (7 min), 3 C/min to 325 C (6 min) The PAH-mix elution was finally run under an optimised temperature program, with the standards divided into windows according to Table 3. Table 3. Conditions of the GC/MS method for PAH: PAH method Conditions Technique: GC/MS Column: Select PAH, 30 m x 0.25 mm df = 0.15 µm (Part number CP7462) Sample Concentration: ng/µl Injection volume: 1 µl Temperature: 70 C (2 min), 40 C/min to 180 C (0 min), 7 C/min to 230 C (7 min), 20 C/min to 280 C (10 min), 5 C/min to 325 C (7 min) Carrier gas: Helium, constant flow 2 ml/min Injector: 250 C, Splitless mode, 1 50 ml/min Detector: Triple Quad, EI in SIM mode, ion source 230 C, transfer line 300 C SIM Parameters: Group 1: (128.00, 30) (136.00, 30) (152.00, 30) (Mass, Dwell time) (154.00, 30) (160.00, 30) (164.00, 30) (166.00, 30) (176.00, 30) (178.00, 30) (188.00, 30) (190.00, 30) Group 2: (202.00, 20) (212.00, 30) (216.00, 30) (228.00, 30) (240.00, 30) (252.00, 30) (264.00, 30) Group 3: (276.00, 30) (278.00, 30) (288.00, 30) (292.00, 30) (302.00, 30) 11

13 2.2.2 Development of method for the Alkyl-Mix The Alkyl-mix (Table 2) was tested with both the method developed for the PAHs and with a method (Test 6) based on a standard method for PAHs and oxygenated PAHs (Lundstedt et al., 2014) and the standard one proved to give the best separation. Altering the start temperature and the holding time at some temperatures in order to get the fastest analyse resulted in a final optimised temperature program with standards divided into windows according to Table 4. Table 4. Conditions of the method for Alkyl-mix: Alkyl method Conditions Technique: GC/MS Column: Select PAH, 30 m x 0.25 mm df = 0.15 µm (Part number CP7462) Sample Concentration: ng/µl Injection volume: 1 µl Temperature: 90 C (1 min), 8 C/min to 300 C (4 min), 25 C/min to 325 C (1 min) Carrier gas: Helium, constant flow 2 ml/min Injector: 250 C, Splitless mode, 1 50 ml/min Detector: Triple Quad, EI in SIM mode, ion source 230 C, transfer line 300 C SIM Parameters: Group 1: (142.00, 30) (152.00, 30) (156.00, 30) (170.00, (Mass, Dwell time) 30) Group 2: (184.00, 30) (192.00, 30) (198.00, 30) (204.00, 30) (206.00, 30) (212.00, 30) (216.00, 30) (220.00, 30) (226.00, 30) Group 3: (228.00, 50) (242.00, 50) (252.00, 50) (256.00, 50) (264.00, 50) (266.00, 50) Development of method for the Alkyl-PAH mix Both methods developed for PAHs and APAHs were tested for the Alkyl-PAH mix. A combination of the two methods proved to be the most successful. The final temperature program for the Alkyl-PAH mix is shown in Table 5. Table 5. Conditions of the method for Alkyl-PAH mix: Alkyl-PAH method Conditions Technique: GC/MS Column: Select PAH, 30 m x 0.25 mm df = 0.15 µm (Part number CP7462) Sample Concentration: ng/µl Injection volume: 1 µl 12

14 Temperature: 70 C, 8 C/min to 205 C (2 min), 8 C/min to 250 C, 3 C/min to 270 C (2 min), 9 C/min to 279 C, 1 C/min to 280 C (3 min), 5 C/min to 325 C (5 min) Carrier gas: Helium, constant flow 2 ml/min Injector: 250 C, Splitless mode, 1 50 ml/min Detector: Triple Quad, EI in SIM mode, ion source 230 C, transfer line 300 C SIM Parameters: (Mass, Dwell time) Group 1: (128.00, 30) (136.00, 30) (142.00, 30) (152.00, 30) (154.00, 30) (156.00, 30) (160.00, 30) (164.00, 30) (166.00, 30) (170.00, 30) (176.00, 30) Group 2: (178.00, 30) (184.00, 30) (188.00, 30) (190.00, 30) (192.00, 30) (198.00, 30) (202.00, 20) (204.00, 30) (206.00, 30) (212.00, 30) (216.00, 30) (220.00, 30) (226.00, 30) Group 3: (228.00, 30) (240.00, 30) (242.00, 30) (252.00, 30) (256.00, 30) (264.00, 30) (266.00, 30) (276.00, 30) (278.00, 30) (288.00, 30) (292.00, 30) (302.00, 30) Quality of Data The internal standard method using deuterium labelled standards was used for quality control and assurance. In lack of labelled standards, relative response factor (RRF) values for the compounds were calculated using the compound nearest in retention time (EPA method 8272; EPA method 8000B). Target compounds were quantified by the use of a seven point calibration curve. Because of lack of standard compounds a three point calibration curve were used for Dibenzo(a,j)anthracene and a four point calibration curve for Dibenzo(a,c)anthracene. Points should have linearity in order to be used for quantitative purposes, correlation coefficient r must be greater than or equal to Analysed compounds should be in the concentration range of the calibration curve. Relative standard deviation (RSD) of the relative response factors (RRF) should be less than or equal to 15 % for PAHs and less than or equal to 30% for the alkylated PAHs. In order to test the repeatability of the GC/MS and the standards a 500 ng Alkyl- PAH standard was analysed 5 consecutive times. Limit of detection (LOD) was defined as three times signal to noise ratio and limit of quantification (LOQ) as 10 times signal to noise ratio. The IPUAC 10 % valley definition was used in order to determine separation. Isomers were determined separated if the valley did not exceed 50% of the taller of the two peaks (60 % for Benzo(k)fluoranthene and Benzo(j)fluoranthene) (CARB method 429). 2.3 Analysis of soil samples The samples were spiked with 500 ng of internal deuterium labelled standards (Table 2) before analysed with GC/MS. MassLynx was used to integrate the peaks. The internal standards as well as the calibration curve from the PAH-Alkyl method were used to quantify PAHs and APAHS in the samples. 13

15 2.3.1 Soil samples Six samples from different contamination sites were analysed (Table 6). Table 6. List of the different soil samples tested, their weight and origin. Samples Dry matter (%) Dry weight (g) Origin SAKAB PAH-contaminated soil from different PAHcontaminated areas. SWE Soil from a wood-preservation site L1: Soil from surrounding of a leaking oil boiler central L2: Soil from surrounding of a leaking oil boiler central NM Soil from old gas stations and residuals from treatment of oil contaminated soils AB Soil from old gas stations and residuals from treatment of oil contaminated soils GC/MS Analysis The soil samples were analysed by use of the developed method for the Alkyl-PAH mix (Table 5) Source identification and characterisation Distribution patterns, FLA/(FLA+PYR) ratio, ANT/(ANT+PHE) ratio and the ratio between 8 alkylated PAHs and their parent PAHs as well as methylated dibenzothiophenes were examined; Methylnaphthalene and Naphthalene, Methylphenanthrene and Phenanthrene, Alkylanthracene and Anthracene, Methylfluoranthene and Fluoranthene, Methylchrysene and Chrysene, Methylbenzo(a)anthracene and Benzo(a)anthracene, Methyldibenz(a,h)anthracene and Benz(a,h)anthracene and, Methyldibenzothiophene and Dibenzothiophene (Figures 2 and 3). 14

16 3. Results and Discussion Results from the method development and the analysis of the six soil samples are summarised in tables and figures below. The full results are given in Appendix. 3.1 Method development GC/MS methods of the two separate mixes (Table 2) were established first, due to the risk of not being able to separate all PAHs, APAHs and Dibenzothiophenes. All three methods were used to calibrate the three different mixes of quantification standards. The calibration curve finally used for the soils samples was the Alkyl-PAH method. The calibration accomplished with this method can be seen in Appendix. The column used had a capillary film thickness of 0.15 µm compared to the standard 0.25 µm. Decreased thickness would usually result in shorter retention time but with this column the retention times are prolonged compared to a standard method using a standard column. This could be due to the stationary phase binding stronger to the compounds. After discovering some peaks that were cut at the top, the dwell time was set to 30 ms instead of 100 ms, resulting in better peaks and better separation (Figure 4). The dwell time was then changed in all three methods giving better peaks and separation in all of them. Alkyl 500ng HV_ Scan EI+ TIC 3.28e HV_ Scan EI TIC 2.96e5 % % Time Figure 4. Comparison between dwell time 30 (top) and dwell time 100 (bottom), for mass number 216 and 220 in the PAH-Alkyl mix 500 ng PAH method Problem with co-eluting PAHs was solved by gradually increasing the temperature to when they were eluting and by holding the temperature there long enough for them to elute. Another problem that occurred during the first temperature programs was that Naphtho(2,3-a)pyrene did not elute. After trying several tests it was made clear that increasing the temperature slowly up to 325 o C (5 o C/min) and holding for some time was more successful than increasing faster and holding longer at 325 o C. The identification of individual PAHs was done by comparison with 15

17 chromatograms in published literature (Yang et al., 2014). With the method developed for PAHs (Table 3) all 22 standards in the PAH-mix were separated and quantified. All PAHs had a relative standard deviation (RSD) of relative response factor (RRF) under accepted limit (15%) with the exception of Dibenzo(a,j)anthracene Alkyl method The identification of the individual APAHs was done by comparison with chromatograms in published literature (Yang et al., 2014). For 1-Methylchrysene, 2-Methylchrysene, 3- Methylchrysene, and 7-Methylbenz(a)anthracene (molecular weight 242) and 7,12- Dimethylbenz(a)anthracene, and 6-Ethylchrysene (molecular weight 256) a GC/MS elution with the individual APAHs was done in order to establish the retention time of the individual alkyl substituted PAHs. With the method developed (Table 4) all 27 standards in the Alkyl-mix were separated and quantified. All standards had RSD of RRF under accepted limit (30%) Alkyl-PAH method In order to get the best separation for the mix containing both APAHs and PAHs, a total of 49 standards, a combination of the method for the individual mixes was used. The method for the Alkyl mix showed better result for the lighter molecular weight PAHs eluting in the beginning and the PAH method showed better result for the heavier weight PAHs that elutes in the end at higher temperatures (Figures 5 and 6). After changing the dwell time, the separation and calibration of all 49 quantification standards with the Alkyl-PAH method was possible (Figure 7). Several of the PAHs and APAHs in the mix had the same mass or masses close to each other. The biggest challenge lay in separating Benzo(b)fluoranthene, Benzo(j)fluoranthene, Benzo(k)fluoranthene, Benzo(e)pyrene, Benzo(a)pyrene and Perylene which all had the mass number 252. After optimising the temperature and changing the dwell time, a separation of these 6 compounds were possible (Figure 8). All calibration curves for the individual PAHs and APAHs had a RSD of RRF under the accepted limit (15 % for PAHs and 30 % for APAHs), with the exception of Naphtacene and Dibenz(a,j)anthracene, see Appendix. When it came to the alkyl substituted PAHs the standard deviation increased with higher mass numbers and higher GC temperature, tentatively arriving from the instability to heat of the alkylated PAHs. 16

18 Figure 5. Chromatogram of Alkyl-PAH mix 500 ng after GC/MS with Alkyl method, mass numbers 252 to Benzo(b)fluoranthene-D12, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(j)fluoranthene, 2. Benzo(e)pyrene, Benzo(a)pyrene-D12, 3.Benzo(a)pyrene, 4. Perylene-D12, 5. Perylene, Methylbenzo(a)pyrene, 7. Dibenz(ac)anthracene 8. Indeno(1,2,3-cd)pyrene, Dibenz(ah)anthracene 9. Benzo(g,h,i)perylene-D12, 10. Benzo(g,h,i)Perylene Figure 6. Chromatogram of Alkyl-PAH mix 500 ng after GC/MS with PAH method, mass numbers 252 to Benzo(b)fluoranthene-D12 2. Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(j)fluoranthene, 3. Benzo(e)pyrene, 4. Benzo(a)pyrene-D12, 5. Benzo(a)pyrene, 6. Perylene-D12, 7. Perylene, Methylbenzo(a)pyrene, 9. Dibenz(ac)anthracene 10. Indeno(1,2,3-cd)pyrene, Dibenz(ah)anthracene 11. Benzo(g,h,i)perylene-D12, 12. Benzo(g,h,i)Perylene 17

19 Figure 7. Chromatogram of Alkyl-PAH mix 500 ng after GC/MS with Alkyl-PAH method, mass numbers 252 to 278, dwell time Benzo(b)fluoranthene-D12 2. Benzo(b)fluoranthene, 3. Benzo(k)fluoranthene, 4. Benzo(j)fluoranthene, 5. Benzo(e)pyrene, 6. Benzo(a)pyrene-D12 7. Benzo(a)pyrene, 8. Perylene-D12, 9. Perylene, Methylbenzo(a)pyrene, 11. Dibenz(ac)anthracene 12. Indeno(1,2,3-cd)pyrene 13. Dibenz(ah)anthracene, 14. Benzo(g,h,i)perylene-D12, 15. Benzo(g,h,i)perylene Figure 8. Comparison between dwell time 100, alkyl method (top) and dwell time 30, alkyl-pah method (bottom) for mass number 252 in the PAH-Alkyl mix 500 ng containing a total of 6 PAHs with mass number Benzo(b)fluoranthene, 2. Benzo(k)fluoranthene, 3. Benzo(j)fluoranthene, 4. Benzo(e)pyrene, 5. Benzo(a)pyrene, 6. Perylene. 18

20 3.1.4 Quality Assurance and Quality control Relative Standard deviation (RSD) of the relative response factors (RRF) was less than 15 % for the PAHs with the exception of Naphtacene and Dibenz(a,j)anthracene (37 % and 34 %, respectively), and less than 30% for all APAHs (Table 7). According to (EPA method 8000B) in those instances where the RSD for one or more analytes exceeds the accepted limit the initial calibration is still acceptable if the mean of the RSD values for all analytes (including PAHs, APAHs and dibenzothiophenes) in the calibration is less than or equal to 20 %. The mean of the RSD values for all analytes is 10.7%, so the calibration is within accepted quality. The 5 consecutive analyses of 500 ng Alkyl-PAH standard showed good repeatability. All standard calibration curves showed linearity (Figure 9) as correlation coefficient, r was greater than 0.99 in all. Table 7. RSD (%) of RRF for all standard compounds Name RRF % RSD Name RRF % RSD Naphtalene 7.5 Benzo(b)fluoranthene Methylnaphthalene 7.3 7,12-Dimethylbenz(a)anthracene Methylnaphthalene 6.2 Benzo(k)fluoranthene 9.8 1,6-Dimethylnaphthalene 8.9 Benzo(j)fluoranthene 8.9 Acenaphtylene 8.4 Benzo(e)pyrene 15.6 Acenaphthene 9.2 Benzo(a)pyrene ,3,5-Trimethylnaphthalene 9.0 Perylene 12.4 Fluorene Methylbenzo(a)pyrene 26.1 Dibenzothiophene 5.7 Dibenz(a,j)anthracene 34.0 Phenanthrene 7.3 Dibenz(a,c)anthracene 6.9 Anthracene 9.3 Indeno(1,2,3-cd)pyrene Methyldibenzothiophene 9.9 Dibenz(ah)anthracene Methylphenanthrene 9.7 Benzo(g,h,i)perylene Methylanthracene 9.3 Naphtho(2,3-a)pyrene 9.2 4H-Cyclopenta(d,e,f)phenanthrene 11.9 Naphthalene-D ,8-Dimethyldibenzothiophene Methylnaphthalene-D ,4-Dimethylphenanthrene Methylanthracene-D ,4,7-Trimethyldibenzothiophene 21.1 Acenaphthylene-D ,3-Dimethylanthracene 25.7 Acenaphthene-D Fluoranthene 7.5 Dibenzothiophene-D8 5.7 Pyrene 9.2 Anthracene-D ,2,8-Trimethylphenanthrene 14.3 Benzo(a)anthracene-D ,2,6-Trimethylphenanthrene 14.5 Benzo(b)fluoranthene-D Methylfluoranthene 13.5 Benzo(k)fluoranthene-D Benzo(a)fluorene 9.9 Benzo(g,h,i)perylene-D Benzo(c)phenanthrene 10.0 Benzo(a)pyrene-D Benzo(a)anthracene 9.7 Chrysene-D Triphenylene 5.1 Dibenz(a,h)anthracene-D Chrysene 7.7 Fluoranthene-D Naphtacene 37.2 Fluorene-D

21 7-Methylbenz(a)anthracene 20.4 Indeno(1,2,3-c,d)pyrene-D Methylchrysene 17.8 Phenanthrene-D Methylchrysene 18.9 Pyrene-D Methylchrysene 18.4 RS-Perylene-D Ethylchrysene 19.1 Mean=10.7 Figure 9. Linearity for compound Fluorene with r > Concentration of PAHs in soil samples Figures 10 to 14 and Table A2 show the concentration of the PAHs in the samples, calculated by the internal standard method and the calibration curve of the Alkyl-PAH method. The concentration of lower molecular weight PAHs were lower compared to the higher molecular PAHs in all of the samples. This is most likely because of weathering and biodegradation of the PAHs in the soil (Zhendi and Carl, 2008). Another reason could be that higher molecular ones bind stronger to soil particles while lighter ones can pass through with ground water (Tobiszewski and Namieśnik, 2012). The concentration of higher molecular PAHs being larger than that of lower molecular PAHs in weathered soil have been noticed in studies before (Banger et al., 2010). The structure of Naphtacene with 4 aromatic rings aligned (Figure 3) makes it very unstable, even in the standards. Because of the low peak area and high standard deviation in the quantification standards, the instability of Naphtacene will cause the concentration to be overestimated in all soil samples. According to (Wang and Stout, 2006) Fluoranthene, Pyrene and C4-Phenanthrene should be high in concentrations in weathered soil contaminated with oil. Samples L1:1 and L2:7 showed high concentration of Fluoranthene and Pyrene (no C4- Phenanthrene was measured). The concentration of these two compounds were high in all samples which implies that they are more resistant to weathering in all PAH contaminated soil samples, not just the soils contaminated with oil. 20

22 Figure 10. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample SAKAB. SAKAB (Figure 10) is a sample of PAH contaminated soils from different contaminated sites. As expected the PAHs were high in concentration and the alkyl substituted PAHs lower in concentration. Similar PAH profile was shown in soil sample SWE (Figure 11) from a wood preservation site. This points to creosote being the cause of this contamination, and would explain the high concentrations of Fluoranthene, Pyrene and Chrysene, high molecular weight PAHs that are common in creosote (Mueller et al., 1989). Figure 11. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample SWE. 21

23 Figure 12. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample L1:1. Samples L1:1 (Figure 12) and L2:7 (Figure 13) are both from sites contaminated by a leaking oil boiler central. They should therefore contain higher concentrations of alkylated PAHs then their parent PAH (Wang et al., 1999). This is the case with L2:7, which contained high concentrations of most of the alkylated PAHs. The L1:1 contained higher concentrations of parent PAHs than the L2:7 sample. The oil in this soil sample might have been heated enough so that the alkylated PAHs, because of their thermodynamically instability, have been depleted (Saha et al., 2009). The two samples L1:1 and L2:7 contained noticeable lower concentrations of all compounds compared to the other samples, another sign of petrogenic source. Figure 13. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample L2:7. 22

24 Figure 14. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample NM1. Samples NM1 (Figure 14) and AB5 (Figure 15) are soil samples from old gas stations mixed with residual from treatment of oil contaminated soils. The soil samples contained high concentrations of both PAHs and APAHs. In these samples there is a difference between the higher molecular weight PAHs and APAHs and the lower molecular weight PAHs. The high molecular weight PAHs are much higher in concentration compared to the low molecular weight PAHs. Like mentioned before, the weathering and biodegradation plays a big parts in the degradation of different PAHs and APAHs, changing the ratio between the low molecular weight PAHs and the high molecular PAHs by degrading the low molecular weight PAHs faster. Figure 15. Concentration of the 49 PAHs, APAHs and dibenzothiophenes in the soil sample AB5. 23

25 3.3 Source identification and characterisation of soil samples By comparing the distribution pattern between the samples it is clear that soil collected from oil contaminated sites were more prone to bell-like pattern in concentration (Figure 16). This indicates petrogenic source. Some of the patterns for certain APAHS/PAHs like Naphthalene and Anthracene showed a more reversed-slope pattern, suggesting that the PAHs in the soil have been under the influence of weathering (Wang et al., 2008). The sample SAKAB from known PAH contaminated sites showed a more slope-like pattern, which indicates that the source of the contamination is pyrogenic (Figure 17). Figure 16. PAHs and some of their C 1-C 3 APAHs in a soil sample from surrounding of a leaking oil boiler central. Lines drawn indicate reversed slope - or bell -like distribution patterns. 24

26 Figure 17. PAHs and some of their C 1-C 3 APAHs in a soil sample from different PAH-contaminated areas. Lines drawn indicate slope -like distribution patterns. The ratios between the alkylated PAHs and their parent PAHs are shown in Table 8. If threshold of 0.4 for (Saha et al., 2009 ) Methylphenanthrene/Phenanthrene was used; L1:1, L2:7 and SWE are implied to be of petrogenic souces and SAKAB, NM1 and AB5 of pyrogenic sources. Sample SWE is not of petrogenic sources but originated from creosote contaminated soils which might show the same pattern as a pyrogenic source. The fact that creosote can derive from crude oil (oil-tar creosote) and is known to contain different methylated PAHs could be the reason for the petrogenic ratio (Mueller et al., 1989). The low molecular weight alkyl substituted PAHs were higher in concentration then their parent PAH in the SWE sample. When it comes to the higher molecular weight PAHs in the SWE soil sample the ratio was that of pyrogenic PAH profile. When looking at the other ratios the results show some inconsistency; with Naphthalene there is a higher concentration of methylated Naphthalenes in most samples. Similar ratios are shown for Dibenzothiophene, which once again points to the fact that the low molecular PAHs are more prone to degradation in nature than the high molecular ones (Banger et al., 2010; Zhendi and Carl, 2008). Sample L1:1 shows signs of pyrogenic PAHs as well as petrogenic. The ratios between the APAHs and PAHs in L1:1 show similar patterns to the NM1 and AB5, which are of mixed sources. In sample L2:7 there was a clearer petrogenic pattern in the ratios. Because the alkylated versions of the low molecular weight PAHs are higher in concentration then their parent PAHs this suggests that adding alkyl groups might make them more stable, depending on the position of the substitution. When it comes to the high molecular weight PAHs the APAH/PAH ratio decreases, which could also be due to the increased stability of the parent PAHs. One thing noticeable when it comes to the higher molecular APAHs/PAHs ratios is 25

27 that even though they are low they are still higher in the two soil samples L1:1 and L2:7, known to originate from a leaking oil boiler central. The Pyrogenic Index (PI) could not be used in the present study since not all C1-C4 compounds for the 5 alkylated PAHs where quantified. The PI would be significantly overestimated considering that the sum of the alkylated PAHs would be underestimated. The ratio between the parent PAHs fluoranthene/fluoranthene + pyrene (FLA/(FLA+PYR)) and anthracene/anthracene + phenanthrene (ANT/(ANT+PHE)) are shown in Table 9. The ratio is lower in the soil samples contaminated by oil, as would be expected. The threshold of 0.1 (Pies et al., 2008) for ANT/(ANT + PHE) or 0.4 for FLA/(FLA+PYR) was however not reached for any of the oil samples. Table 8. Ratio between methylated PAH and parent PAH for different soil samples. RATIO SAKAB SWE L1:1 L2:7 NM1 AB5 2-Methylnaphthalene/Naphtalene Methylnaphthalene/Naphtalene ,6-Dimethylnaphthalene/Naphtalene ,3,5-Trimethylnaphthalene/Naphtalene Methyldibenzothiophene/Dibenzothiophene ,8-Dimethyldibenzothiophene/Dibenzothiophene ,4,7-Trimethyldibenzothiophene/Dibenzothiophene Methylphenanthrene/Phenanthrene ,4-Dimethylphenanthrene/Phenanthrene ,2,8-Trimethylphenanthrene/Phenanthrene ,2,6-Trimethylphenanthrene/Phenanthrene Methylanthracene/Anthracene ,3-Dimethylanthracene/Anthracene Methylfluoranthene/Fluoranthene Methylchrysene/Chrysene Methylchrysene/Chrysene Methylchrysene/Chrysene Ethylchrysene/Chrysene Methylbenz(a)anthracene/Benzo(a)anthracene ,12-Dimethylbenz(a)anthracene/Benzo(a)anthracene Methylbenzo(a)pyrene/Benzo(a)pyrene Table 9. Ratio between FLA/(FLA+PYR) and ANT/(ANT+PHE) SAKAB SWE L1:1 L2:7 NM1 AB5 FLA/(FLA+PYR) ANT/(ANT+PHE)

28 4. Conclusion The GC/MS method developed in this study was capable of separating a mixture of 49 PAHs, alkyl substituted PAHs and Dibenzothiophenes in 55 minutes time. All calibration curves showed linearity, and all compounds with the exception of Naphtacene and Dibenzo(a,j)anthracene had RRF values with standard deviations within accepted limits. Two GC/MS methods, one for only PAHs and one for alkyl substituted PAHs and Dibenzothiophenes were also successfully developed. The PAH method separated all PAHs in 47 minutes, the time mostly because of Naphtho(2,3-a)pyrene eluting several minutes after the compound before it. The method for the Alkyl-substituted PAHs was fast, separating all compounds within 34 minutes. The six samples from different contamination sites were all tested and the PAHs, APAHs dibenzothiophenes in the quantification standards were identified and quantified in all samples. The calculated APAH/PAH ratios was higher and the FLA/FLA+PYR and ANT/ANT+PHE ratios lower in the soil samples originating from oil contamination than in the other samples. This indicates petrogenic source. Because of factors like different soil type, aging of contamination, biodegradation affecting the concentration of alkylated PAHs and PAHs the determination of source is challenging. The use of pyrogenic index and other ratios might have helped further characterise and determine pyrogenic or petrogenic sources of the soil samples. The ongoing understanding of the toxic and carcinogenic effects of alkyl substituted PAHs makes it more important to be able to quantify these in samples from contaminated sites. The result from this study suggests that alkyl substitution might alter the way the PAHs are affected by biodegradation and weathering. In order to decide course of action for the remediation it is therefore important to determine the source of the PAH contamination. 27

29 5. Consideration PAHs are pollutants that exist in over 100 different variants (for example, oxygenated-, alkylated PAHs or nitrogen-, sulphur substituted heterocyclic compounds) which can be found all over the world in air, water, sediment and soil (Britten and Naikwadi, 2009). Many PAHs are toxic and carcinogenic (Straif et al., 2005). Because of human impact PAHs have increased in the environment, especially around urbanised and industrialised areas (Lima et al., 2005). PAHs in soils is a worldwide problem. Contamination free soil is required for clean ground water and agriculture. In order to achieve a non-toxic environment, remediation of contaminated sites is crucial. Because remediation is costly and timely it is necessary to be able to quantify PAHs and different substituted PAHs with certainty. This puts high demands on the methods for identifying and quantifying PAHs in environmental samples. Because PAHs mostly occur in the environment as complex mixes of PAHS and different substituted PAHs, which will be effected by weathering and biodegradation differently, this is a challenging task. In order to quantify PAHs, the use of not only toxic and hazardous solvents but also the handling of toxic and carcinogenic PAHs as standards is necessary. The storage and disposal of these standards must be done in a correct way. The requirements on the analyst doing a correct risk assessment and a proper sample preparation are therefore immense. Different sources of contamination (pyrogenic or petrogenic) will contain different mixes of PAHs and substituted PAHs (Saha et al., 2009; Tobiszewski and Namieśnik, 2012). As these mixes are affected by weathering and biodegradation differently, the characterisation and identification of the contamination source is important in order to decide remediation strategies. Earlier studies, along with this study, have shown that traditional chemical analysis of the 16 priority PAHs, to determine the degree of contamination in soil, usually miss potential pollutants present in the samples (Larsson et al., 2013). Also the focus of study toxicity and carcinogenic effects of PAHs have to most part been put onto the 16 priority PAHs. Only in recent years have different substituted PAHs come into consideration, as studies have shown that the toxicity and carcinogenic effects might increase with substitution (Sun et al., 2014; Yang et al., 2014). The toxic effects by these substitutions are far from understood. The importance of separating alkylated PAHs from parent PAHs is therefore not only important to determine contamination source but also in order to undergo further studies of the toxicity and carcinogenic effects of these substitutions. 28