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Faculty of Bioscience Engineering Academic year 2014 2015 Biomonitoring of polycyclic aromatic hydrocarbons and derivatives in Belgium Zhimiao Zuo Promotor: Prof. dr. ir. Herman Van Langenhove dr. ir. Christophe Walgraeve Master’s dissertation submitted in partial fulfillment of the requirements for the degree of Master of Environmental Sanitation
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Faculty of Bioscience Engineering

Academic year 2014 – 2015

Biomonitoring of polycyclic aromatic hydrocarbons

and derivatives in Belgium

Zhimiao Zuo

Promotor: Prof. dr. ir. Herman Van Langenhove

dr. ir. Christophe Walgraeve

Master’s dissertation submitted in partial fulfillment of the

requirements for the degree of

Master of Environmental Sanitation

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I

Acknowledgement

The dissertation is almost finished, which means the two-year study in the University of

Ghent and the living in Belgium are going to the end as well. The experience of studying

abroad is one of the most important parts of my life. The people I met here will not be

forgotten ever. At the end of my student period, lots of people should be showed of my

gratitude.

Professor Herman Van Langenhove is the promoter of my thesis. He is modest and gentle.

Besides the thesis, several lectures were given by him. The erudite of knowledge, the precise

on research, the diligent on work, the humorous on speech and the optimistic on living

impressed me a lot. I learnt not only knowledge from him, but also knew better on how to

behave correctly. An old saying in Chinese ‘先生之风,山高水长’ describes Professor Herman

Van Langenhove perfectly. The meaning of the sentence is: ‘The prestige of the master will

be spread and remembered forever’.

My tutors Christophe Walgraeve and Dohai Duc guided me pretty well while the thesis

proceeded. Christophe worked with me from the very beginning of the thesis. He was so

scrupulous and conscientious. Even though the experiments always went late to dark, he was

there in the lab until the last moment to give help and guidance. While dissertation

compiling, Christophe was so nice that he gave up part of his summer vacation to read and

modify the text.

My gratitude goes to Professor Kristof Demeestere as well, who always listened to my report

and gave some constructive suggestions kindly. His professional opinion enlightened me to

avoid the detour while thesis designing. He was so responsible to all the students in the lab,

and tried to help the best all the time.

The staff from The Department of Sustainable Organic Chemistry and Technology, such as

Wouter De Soete, Joren Bruneel and so on, although I hardly talked to all of them, the smile

and warm greeting they gave encouraged me every time I arrived at the lab. Especially, my

special thanks to Patrick De Wispelaere, who supported me on GC-MS operation.

Professor Peter Goethals, the coordinators of Master of Science in Environmental Sanitation

(IMENVI) Sylvie Bauwens and Veerle Lambert. Thank you so much for approving my

application and giving such a wonderful and rare opportunity to study in the best university

around the world, and to work with so many amiable and brilliant brains.

I want to show my great thanks to my lovely classmates, we came together from different

continents. Every one of you gave me lots of help and encouragement while I was

disoriented. Daniel Paul Odhiambo Ombaka, Audisny Apristiaramitha Teddy and Workineh

Mengesha Fereja who were working together in the same lab for thesis, gave me lots of

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II

suggestion and introduction about lab work as well.

My parents and grandfather are the inner pillar supported and motivated me to finish this

program straight. My friends in Beijing are the ones released my pressure in the two-year.

My girlfriend is the one consoled and accompanied me while I was frustrated. Without them,

I would never success.

At last, the great thanks to the committee. Thank you for the time and patience to read my

dissertation.

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III

List of Abbreviations

ACN Acetonitrile SEC Size Exclusion Chromatography

AC Adsorption Chromatography SPE Solid Phase Extraction

ASPEC Automated Solid-Phase Extraction Clean-up USEPA U.S. Environmental Protection Agency

DCM Dichloromethane VOCs Volatile Organic Compounds

DNA Deoxyribonucleic Acid WHO World Health Organization

d.w. Dry Weight

F-PAHs Mono-fluorinated PAHs

FW Fresh Weight

GC Gas Chromatography

EI Electron Ionization

GMF Glass Microfiber Filter

GPC Gel Permeation Chromatography

HEX Hexane

HPLC High Performance Liquid Chromatography

IARC International Agency for Research on Cancer

IS Internal standard

LRAT Long-range Atmospheric Transport

MS Mass Spectrometry

NF Not Found

NPAHs Nitro-polycyclic Aromatic Hydrocarbons

oxy-PAHs Oxygenated Polycyclic Aromatic Hydrocarbons

PACs Polycyclic Aromatic Compounds

PAHs Polycyclic Aromatic Hydrocarbon

PCBs Polychlorinated Biphenyls

PLE(ASE)

Pressurized Liquid Extraction (Acceleration

Solvent Extraction)

PM Particulate Matter

POPs Persistent Organic Pollutants

PTFE Polytetrafluoroethylene

RPLC Reversed Phase Liquid Chromatography

RPA Relative Peak Area

RSRF Relative Sample Response Factor

S_B Spiked Blank

S_SAM_A Sample Spiked After

S_SAM_B Sample Spiked Before

SAM Sample

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IV

Abstract

The aim of this study was to develop a bio-monitoring method to analyze polycyclic aromatic

hydrocarbons (PAHs) and oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs). Taxus

baccata (European Yew) was chosen as the bio-monitor species in this thesis. This is the first

time Taxus baccata applied on PAHs and oxy-PAHs researches. The 16 PAHs mentioned in

this thesis are the 16 U.S. Environmental Protection Agency (USEPA) priority PAHs. The

oxy-PAHs and PAHs in samples were analyzed by Gas Chromatography Mass Spectrometry

(GC-MS). The approach of sample preparation, extraction solvent choice, and solid phase

extraction (SPE) clean-up were optimized and developed. PAHs and oxy-PAHs were

quantified by internal standards calibration with deuterated PAHs. The recovery and matrix

effects of PAHs and oxy-PAHs were obtained. The concentration of target compounds in

sample was obtained and expressed as in mass of leaf dry weight.

Air pollution, PAHs, oxy-PAHs, vegetation, Taxus, bio-monitoring.

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V

CONTENT

Acknowledgement ............................................................................................................................. I

List of Abbreviations ......................................................................................................................... III

Abstract ........................................................................................................................................... IV

CHAPTER 1 Introduction................................................................................................................- 1 -

CHAPTER 2 Literature Review .......................................................................................................- 9 -

2.1 Sampling ..........................................................................................................................- 9 -

2.2 Sample Preparation & Extraction ................................................................................. - 13 -

2.3 Clean-up ....................................................................................................................... - 19 -

2.4 PAHs and oxy-PAHs Analysis ......................................................................................... - 24 -

2.5 Concentration Levels .................................................................................................... - 28 -

CHAPTER 3 Chemical Materials .................................................................................................. - 32 -

3.1 Chemicals & Reagents .................................................................................................. - 32 -

3.2 Standards Solution ....................................................................................................... - 33 -

CHAPTER 4 Results & Discussion ................................................................................................ - 34 -

4.1 Selection of Plant Species ............................................................................................ - 35 -

4.2 Dry Matter Content ...................................................................................................... - 36 -

4.3 Solvent Selection .......................................................................................................... - 36 -

4.4 Size of the Sample Determination ................................................................................ - 37 -

4.5 Extraction ..................................................................................................................... - 39 -

4.6 Clean-up & Concentration ............................................................................................ - 40 -

4.6.1 Clean-up & Concentration Procedure ............................................................... - 40 -

4.6.2 Determination of Eluent Volume ...................................................................... - 41 -

4.7 GC-MS Analysis ............................................................................................................. - 47 -

4.8 Recovery and Matrix Effects ......................................................................................... - 47 -

4.8.1 Experiment Design ............................................................................................ - 48 -

4.8.2 Data Processing Method ................................................................................... - 49 -

4.8.3 Results of Analysis ............................................................................................. - 50 -

4.9 Discussion ..................................................................................................................... - 53 -

CHAPTER 5 Conclusions & Prospect ........................................................................................... - 55 -

5.1 Conclusions .................................................................................................................. - 55 -

5.2 Prospect ....................................................................................................................... - 55 -

REFERENCES ............................................................................................................................... - 56 -

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CHAPTER 1 Introduction

In industrialized and densely populated areas, air pollution has been regarded as one of the

most important environmental issues, especially in recent decades (Rodriguez et al., 2010).

Reviewing the top 10 of the pollution incidents in past century, half of them were related to

air pollution, which are listed in Table 1-1 (Zuo, 2010). With the evolution of human

technology, increasing pollutants were produced due to the increasing natural resource

consumption (Agrillo et al., 2013; Yu, 2014). Moreover, human health and air quality are

associated intimately (Esposito et al., 2014; Matus et al., 2012). Epidemiological studies,

which carried out to investigate the relationship between health risks and air pollution,

indicated that ambient air pollution have a high possibility to be responsible for the

increasing rates of lung cancer and respiratory system disease (Du Four et al., 2004; Goldberg

et al., 2001). Therefore, the researches on air quality, air pollution and air purification were

highlighted and focused by more and more scientists.

Air pollutants can be classified in different ways. Firstly, by formalism, air pollutants can be

divided into two groups, primary pollutants and secondary pollutants. Primary pollutants are

the ones emitted from the pollution sources directly, for instance carbon monoxide (CO),

sulfur dioxide (SO2) and nitric oxide (NO) etc. Secondary pollutants are the ones produced by

the chemical or photochemical reactions of primary pollutants, for instance, ozone (O3),

H2SO4, aerosol, etc. Secondly, by physical property, air pollutants can also be divided into two

groups, gaseous pollutants and particulates. A large proportion of air pollution is caused by

gaseous pollutants, for instance, SOx, NOX, chlorofluorocarbon (CFCs), etc. Thirdly, by

chemical property, air pollutants can be divided into two groups, organic pollutants and

inorganic pollutants. (Hao, 2010; Kallenborn et al., 2012; Xia, 2003).

Previous reports have studied the major air contaminants, for instance, O3, NOx, SOx, COx,

volatile organic compounds (VOCs), Particulate Matter (PM), heavy metals, etc. (Cheng et al.,

2008a; Cheng et al., 2008b; Sujaritpong et al., 2014). Various organic compounds are known

to be present as airborne particles. Of primary concern are the polycyclic aromatic

hydrocarbons (PAHs) and their derivatives, for instance nitro-polycyclic aromatic

hydrocarbons (NPAHs) and oxygen containing polycyclic aromatic hydrocarbons (oxy-PAHs).

They are known as polycyclic aromatic compounds (PACs) collectively (Du Four et al., 2004).

PAHs and oxy-PAHs have drawn a lot of attention recently. With the speedy development of

modern industry and transportation, more fuel is required and consumed, thus more air

pollutants are produced nowadays (Srogi 2007; ötvös et al., 2004). Among the air pollutants,

PAHs and their derivatives oxy-PAHs are reported to be one of the biggest environmental

risks (Nocun and Schantz, 2013; Wang et al., 2006). The properties of high carcinogenic

possibility and toxicity to the environment are concerning people (Niu et al., 2003;

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Pongpiachan, 2013). Therefore, PAHs and oxy-PAHs are chosen as the main object of this

study.

Table 1-1 the Air Pollution Incidents among the Top 10 Pollution Incidents in the 20th

Century

PAHs is the name of a large group constituted of more than 100 organic compounds having

Year Subject Region Inducement Details

1930 The 1930

Meuse Valley

Fog

Belgium,

Industrial

Temperature

Inversion

The first public nuisance recorded in the

20th century. It was induced by the

temperature inversion. The valley was full

of heavy industries. 60 people were killed

by the trapped gaseous emission in one

week.

1943 Los Angeles

Photochemical

Smog Episode

U.S., City Heavy Traffic The hydrocarbon, emitted by petrol

burning, catalyzed by ultraviolet

irradiation, resulted in the production of

‘blue smog’, which was known as

photochemical smog later. Two similar

incidents were reported in 1955 and

1970, more than 75% population of the

city was recorded disease.

1948 The 1948

Donora Smog

Donora,

Pennsylvania,

U.S., Industrial

Temperature

Inversion

The city of Donora was well-known by lots

of large-scale iron works, smelters and

sulfuric acid plants. The foggy morning in

1948, controlled by the anticyclone and

temperature inversion, the plants’

emission was trapped in lower

atmosphere, which brought disaster to

the city. More than 20,000 people were

affected.

1952 The London

Smogs

Britain, City Coal Burning 12 severe smog incidents were recorded

in London since 1952. All of them were

induced by the emission of sulfur dioxide

and particulate matter while coal burning.

Reported, more than 12,000 people dead

in the smog of 1952.

1984 Bhopal

Disaster

Bhopal, India,

City

Explosion The city of Bhopal was attacked by 45

tons highly toxic methyl isocyanate smog

which was produced by the explosion of

the farm chemical plant. 20,000 casualty,

50,000 blindness were reported after the

disaster. More than 200,000 people were

affected.

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two or more fused aromatic rings (Qiu et al., 2013). The properties were described as

relatively low solubility in water, but highly lipophilic (Srogi, 2007). Vapor phase and the

condensed (aerosol) phases are the two main present states of PAHs in the atmosphere

(Inomata et al., 2013). To specify, low molecular weight PAHs have the tendency to be more

concentrated in the vapor phase. The ones often associated with particulates are higher

molecular weight PAHs (Inomata et al., 2013; Ravindra et al., 2006).

PAHs can be arisen either naturally or as the result of anthropogenic activities (Węgrzyn et al.,

2006; Zhan et al., 2013). Naturally, PAHs can be the products of thermal decomposition; can

be formed during incomplete combustion of organic materials (Niu et al., 2003; Ratola et al.,

2006) and geochemical formation of fossil fuels (Foan and Simon, 2012). On the other hand,

domestic heating, power plants, industrial processes, waste incineration, and most

importantly, the emissions of motor vehicles can be the major anthropogenic sources of

PAHs (Mu et al., 2013; Qiu et al., 2013; Ravindra et al., 2006).

The 16 individual PAHs, which are defined as priority by US Environmental Protection Agency

(USEPA) (Ma et al., 2010), involved in the present studies are naphthalene, acenaphthylene,

acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene (PYR),

benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,

dibenz[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-cd]pyrene (Ziegenhals et

al.,2008; Srogi, 2007). The basic physical properties and structure of the 16 selected PAHs

are described in Table 1-2 (Working Group on Polycyclic Aromatic Hydrocarbons, 2001).

Previous studies have proved that PAHs, as persistent organic pollutants (POPs) could affect

human health by bio-accumulation through food chain (Mu et al., 2013). Also, adverse

characters of PAHs, such as toxicity, slow rates of degradation and the potential for either

bioaccumulation in living organisms or long-range transport were observed recently (Foan

and Simon, 2012). Furthermore, PAHs were proved to be responsible for carcinogenic,

mutagenic (Hubert et al., 2003; Navarro-Ortega et al., 2012; Węgrzyn et al., 2006),

immunotoxic (Rodriguez et al., 2012; Sanz-Landaluze et al., 2010), endocrine disruption,

reproductive and developmental toxicity (Pongpiachan, 2013). Besides, some of the PAHs

may also act as co-carcinogens or tumor promoters (Rodriguez et al., 2010), which are

detrimental to human health and non-human organisms (Zhan et al., 2013).

Among them, the best known carcinogenic PAHs compound is benzo[a]pyrene which has

been used as a leading substance, due to its reasonably well-known emission inventory and

toxicological significance (Pongpiachan, 2013; Ziegenhals et al., 2008; Van Jaarsveld et al.,

1997). Base on the classification of the International Agency for Research on Cancer (IARC),

some of the PAHs compounds are classified as probable or possible to human carcinogens

(Du Four et al., 2005). The details of the PAHs compounds classification about carcinogens of

IARC can be found also in Table 1-2.

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Table 1-2 the Properties and Structure of the 16 EPA PAHs

PAHs Formula Boiling Point/℃ Melting Point/℃ Structure IARC Classification

Naphthalene C10H8 217.9 80.5

n.e.*

Acenaphthylene C12H8 280 93

n.e.

Acenaphthene C12H10 279 95 CH2 CH2

n.e.

Fluorene C13H10 295 116 CH2

3**

Phenanthrene C14H10 340 100.5

3

Anthracene C14H10 342 216.4

3

Fluoranthene C16H10 375 108.8

3

Pyrene C16H10 150.4 393

3

Benzo[a]anthracene C18H12 400 160.7

2A***

Chrysene C18H12 448 253.8

NA

Benzo[b]fluoranthene C20H12 481 168.3

2B****

Benzo[k]fluoranthene C20H12 480 215.7

2B

Benzo[a]pyrene C20H12 496 178.1

2A

Dibenzo[a,h]anthracene C24H14 524 266.6

2B

Benzo[g,h,i]perylene C22H12 NA 277

3

Indeno[1,2,3-c,d]pyrene C22H12 536 163.6

2A

* n.e.: not evaluated

** 3: not classifiable

*** 2A: probably carcinogenic

**** 2B: possibly carcinogenic

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Oxy-PAH is a group of compounds having the structure of aromatic rings attached with

oxygen atom(s). Besides oxygen, other chemical groups are possible to attach (O’Connell et

al., 2013). Normally, oxy-PAHs were commonly observed in highly polluted areas, especially

the industrial areas with PAHs emission (Lundstedt et al., 2007).

Oxy-PAHs can be both primary pollutants and secondary pollutants. There are several

sources of oxy-PAHs formation, for instance, petrogenic process, pyrogenic process,

biological transformation, photo-oxidation and chemical oxidation (Kojima et al., 2010;

Lundstedt et al., 2007; O’Connell et al., 2013). It is notable that, the emission sources of

oxy-PAHs were reported as same as PAHs’. Incomplete combustion was regarded as one of

the major sources of oxy-PAHs. It was also indicated that the gas-phase reactions between

PAHs and ozone or between PAHs and different radicals (such as hydroxyl radicals, nitrate

radicals), were contributed to the oxygenated derivatives formation (Nocun and Schantz,

2013). Besides, oxy-PAHs were found persistent in the environment (O’Connell et al., 2013).

Therefore, oxy-PAHs are also worthy to be studied carefully.

Recent researches showed that the derivatives of PAHs, such as oxy-PAHs, are much more

toxic to both human and environment than the un-substituted PAHs (Lundstedt et al., 2014;

Nocun and Schantz, 2013). Diseases induced by allergy can be caused by exposure to

oxy-PAHs (Chung et al., 2007). Mutagenic was reported as one of the most adverse effects

can be caused by several oxy-PAHs, for instance, polycyclic aromatic ketones, quinones and

anhydrides (Kojima et al., 2010; Wei et al., 2012).

Oxy-PAHs have been noticed by more and more scientists, but the regulations and standards

of oxy-PAHs were not established completely (Wei et al., 2012), different oxy-PAHs were

analyzed in different researches. 9-Fluorenone, 9,10-anthraquinone, 1,8-naphthalic

anhydride and benzanthrone were analyzed by Kojima et al., (2010). 24 compounds of

oxy-PAHs were analyzed by O’Connell et al., (2013), which are shown in Table 1-3. Only 3

compounds, benzanthrone, 9,10-anthraquinone, and 9-fluorenone were identified and

quantified by Souza et al., (2014). Some of the important oxy-PAHs are listed in Table 1-3.

It comes to be a top priority task to monitor the concentration of PAHs and its derivatives in

all programs for evaluating environmental hazards and the human health risk. Traditionally,

PAHs and oxy-PAHs had been monitored in soils, water, air and sediments etc. (Cochran et al.,

2012; Lang et al. 2008; Navarro-Ortega et al., 2012). The result of multi-year systematic air

sampling has revealed the seasonality in PAHs concentrations, the levels were observed

highest during the colder winter months (Halsall et al., 2001).

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Table 1-3 Some of the Important oxy-PAHs Species

Oxy-PAHs Structure Oxy-PAHs Structure

1,4-Benzoquinone O

O

Chromone

O

O

1,2-Naphthoquinone O

O

1,4-Naphthoquinone O

O

Perinaphthenone O

9-Fluorenone O

1,2-Acenaphthenequinone O O

Xanthone

O

O

9,10-Phenanthrenequinone O

O

1,4-Anthraquinone O

O

9,10-Anrhraquinone O

O

Phenanthrene-1,4-dione O

O

2-Ethyl-9,10-Anthraquinone O

O

4H-Cyciopenta[d,e,f]phenanthrenone

O

1,9-Benz-10-Anthrone

O

Benzo[a]-11-fluorenone

O

Pyrene-4,5-dione

O

O

Aceanthrenequinone O O

5,12-Naphthacenequinone O

O

Benzo[c]phenanthrene-1,4-quinone O O

6H-Benzo[c,d]pyrenone O

Benz[a]anthracene-7,12-dione O

O Benzo[a]pyrene-7,8-dione

O

O

Benzo[a]pyrene-1,6-dione O

O

A range of different passive sampling devices have been used to evaluate the air quality

(Seethapathy et al., 2008). The relevant information was provided by the existence of

worldwide natural bio-monitoring matrices (Ratola et al., 2012). Two main limitations of

PAHs and oxy-PAHs urban monitoring have been presented to the researches. Firstly, the

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wide variation of PAHs and oxy-PAHs concentration bothered a lot. Secondly, the monitoring

in urban is an intensive task in labor, equipment and time (Noth et al., 2013). Therefore,

plant bio-monitors are commonly used, and regarded as the complementary to automatic

monitoring devices (Rodriguez et al., 2012; Wang et al., 2008). Time-integrated information

of a large number of persistent pollutants on atmospheric depositions is provided by plant

bio-monitors, furthermore, the pollutants deposition maps at different scales are permitted

while bio-monitoring (De Nicola et al., 2013). Therefore, considering the large scale of

landscape plants distribution in modern cities, bio-monitors are chosen to discuss in this

study.

Some articles reported a direct relationship between PAHs and oxy-PAHs concentrations in

soil and plants. They believed a strong absorption of PAHs in soil was conducted by root cells

(Fismes et al, 2002; Lundstedt et al., 2014; Srogi, 2007; Zhan et al., 2013). But the primary

pathway PAHs transport into the leaves is air which has been verified by both field and

chamber studies recently. According to the articles, “most PAHs (80%) were accumulated in

the wax fraction, and most of the remainder (17%) penetrated the inner tissues of the leaves”

(Murakami et al., 2012). This conclusion makes vegetation bio-monitoring a feasible way to

examine the spatial distribution and composition of PAHs and oxy-PAHs (Noth et al., 2013).

Various groups of plants (e.g. moss, aquatic plants, grasses, vegetable sand trees) have been

recommended as bio-monitors for PAHs by researchers (De Nicola et al., 2005; De Nicola et

al., 2013; Noth et al., 2013; Rinaldi et al., 2012; Rodriguez et al., 2012; Sanz-Landaluze et al.,

2010). They are able to accumulate in vegetation after deposition. Some articles indicated

that leaves which possess a high surface area can be contaminated by PAHs more seriously

(Ziegenhals et al., 2008).

Pine needles can be applied in the PAHs monitoring because of the strong tendency to

accumulate airborne contaminants with the cover of lipidic-waxy, also its perennial character

enable the monitor in the high-pollutant-concentration-winter (Ratola et al., 2012; Ratola et

al., 2006; Navarro-Ortega et al., 2012). For these reasons, pine needles may provide

complementary monitoring information. In some regions, pine needles have already been

used as passive samplers to monitor the concentration, the sources and the spatial

distribution of PAHs (Navarro-Ortega et al., 2012). Specifically, spruce needles are highlighted

in the study of Niu et al., (2003), because its leaf surface is rich with wax components, which

can accumulate many kinds of lipophilic organic compounds. On the other hand, the

bio-monitor species for oxy-PAHs were hardly reported.

Gas chromatography mass spectrometry (GC-MS) is the technique used most widely to

analyze PAHs and oxy-PAHs (Bamford et al., 2003; Forsberg et al., 2014; Zhang et al., 2011).

Before GC-MS analysis, purification was recommended to minimize the influence of the

biological material. Solid phase extraction was recommended as the purification method

(Augusto et al., 2009; Cochran et al., 2012; Ratola et al., 2012).

The advantages on economic aspect of using bio-monitor could be seen easily as well. Due to

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the costly price of technical equipment used in conventional atmospheric pollutants

measurement for PAHs, the use of bio-monitors is important and helpful in developing

countries. (Wannaz et al., 2012).

This thesis aims to develop the existing bio-monitoring method for PAHs quantificational

determination; to introduce the new species Taxus baccata for PAHs and oxy-PAHs

bio-monitoring; to innovate the method for oxy-PAHs bio-monitor.

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CHAPTER 2 Literature Review

2.1 Sampling

Needle-shape leaf species and broad leaf species were applied widely as biological material

for analyzing PAHs and the derivatives. For instance, Pinus pinea, the representative of

needle species and Quercus ilex, the representative of broad species have been used

commonly. Besides, moss is another species which attracted scientists a lot (Augusto et al.,

2013; Martins et al., 2014). However, the species of moss used in different researches were

not consistent.

As for the sampling strategy, some studies pointed that the leaves should be picked from all

parts of the tree and mixed homogeneously (Rodriguez et al., 2012). While others indicated

that only the leaves from outer part or from the canopy should be collected, to ensure the

accuracy of the result (De Nicola et al., 2005; Sun et al., 2010). Consistently, nearly all of the

experiments made the choice to collect leaves growing at the height of 2-4 meter from the

ground, because the comparison between the leaves from bottom branches and the leaves

from higher branches showed that the bottom branches leaves provided less information

than the higher ones (Ratola et al., 2006).

The age of leaf was also mentioned by many researches. Based on different species and

location information, leaf aged ranged from 5 month to 3 years was mostly used. In some

particular cases, the age of leaf was defined as the period of exposure, for example, 1 week

after the fire disaster (Rey-Salgueiro et al., 2008) or 12 weeks after transplanting from

cultivating field to monitoring point (Rinaldi et al., 2012).

There was a procedure of sampling recommended by most of the authors. Briefly, 1) the

leaves were picked by hand, tried to minimize the contact with leaf surface, gloves were

highly recommended; 2) the leaves were wrapped by aluminum foil; 3) the aluminum foil

packages were sealed in plastic bags and stored in cool box while transport; 4) if the tests

didn’t proceed at the day of sampling, the plastic bags with leaves would be stored in freezer

under -20℃ in the lab until analyzing. More information can be found in Table 2-1.

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Table 2-1 Literature Review of Sampling

Location Species and Type Plant Part Amount Age Sample method Reference

Spain, Barcelona Needle: Pinus pinea L. Randomly from single

tree

NA*

NA NA Ratola et al.,

2006

Portugal (rural/seaside),

Porto; Spain, Barcelona

Bottom branches NA NA NA

Fresno, California,

consider population and

traffic, 91 locations

totally

Needle: Jeffrey pine trees (Pinus

jeffreyi)

The 2 branches from

different sides of one

tree.

99 samples All in same old Wrapped by solvent-washed

aluminum foil, samples were

labeled and tape sealed,

transported with dry ice

Noth et al.,

2013

Spain, the Ebro River

basin, urban, industrial,

and agricultural area

from source to mouth

Needle: Pinus halepensis, Pinus

pinea and Pinus nigra

NA 30 samples NA NA Navarro-Ortega

et al., 2012

Greater Cologne

Conurbation, Industry

and residence combined

Needle: Pine trees NA 3 trees for each

sites

Different ages

collected

separately

Sampled twice in winter and

summer

Lehndorff &

Schwark, 2009

Industrial site Needle: Masson pine (Pinus

massoniana L.)

Outer part of the

middle canopy

5 similar mature

trees/site

Distinct

different ages

Wrapped and sealed by

polyethylene bags, stored in a

homemade cryogenic storage

container

Sun et al., 2010

Canada, Ottawa,

municipal sanitary landfill

(high way and agriculture

land nearby)

Needle: Norway spruce (Picea abies) NA NA NA Wrapped by solvent-cleaned

aluminum foil, stored under -80℃

St-Amand et al.,

2008

Portugal, Spain, and

Greece, 4 urban and 5

non-urban areas in each

country

Needle: Pinus pinea L. Outer branches about

2m height

NA 1-year old

needles

Sampled from the bottom of each

needle, wrapped by aluminum foil

and plastic bag, frozen to transport

& store

Ratola et al.,

2012

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Table 2-1 continue

Location Species and Type Plant Part Amount Age Sample method Reference

Argentina, coastal area Needle: Pinus radiata;

Broad leaf: Populus hybridus;

Long slim leaf: Eucalyptus rostrata

Randomly from single

tree

150-200 g

plant/site

1-year old

needles

Collected a distance between 2.5-3

m in each direction from each plant.

Rodriguez et

al., 2012

Germany, Stuttgart,

Urban (traffic,

background), suburban

(background, rural,

traffic)

Needle: Tillandsia capillaris;

Long slim leaf: Lolium multiflorum

(both are cultivated, not natural)

NA NA 12 weeks Wrapped by labeled paper

bags and kept in a cooling box

Rodriguez et

al., 2010

Brazil, Cubatao, 12km to

costal, surround by

mountain

Needle: Lolium multiflorum;

Broad leaf: P. guajava (tropical

biomonitors)

(both are cultivated, not natural)

Randomly from single

tree

NA NA Wrapped by aluminum foil-freezer Rinaldi et al.,

2012

Germany, Leipzig–Halle

region

Needle: Pinus sylvestris L.;

Broad leaf: Maple leaves (Acer

campestre)

NA 5 trees/site, 300g

needle,10g leaves

1) 5 month

leaves; 2)

years needles

Stored under -25℃ Hubert et al.,

2003

Mexico, Coatzacoalcos

Veracruz, industrial area

Broad leaf: white mangrove

(Laguncularia racemosa), red

mangrove (Rhizophora mangle),

medlar (Eriobotrya japonica)

Randomly from single

tree

NA NA Wrapped by Aluminum foil, plastic

bag and cold boxes successively,

one single tree from one site

Sanz-Landaluze

et al., 2010

Italy, Naples Broad leaf: Quercus ilex L.

(Mediterranean evergreen oak)

Outer part of canopies,

2-4m height

40 leaves/tree, 8

trees/site

1-year old Minimize contact with the leaf

surface while sampling, stored

under -20℃ in polyethylene bags

De Nicola et al.,

2005

China, Beijing, urban

roadsides and inside a

university campus

Broad leaf: Gingkgo (Ginkgo biloba),

peach (Prunus persica), Japanese

pagodatree (Sophora japonica),

purple leaf plum (Prunus cerasifera),

Peking lilac (Syringa pekinensis) and

green spire (Euonymus japonicus).

1.5 m height 50 leaves from 4-6

trees, each site

each species

Annual fresh

leaves

NA Wang et al.,

2008

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Table 2-1 continue

Location Species and Type Plant Part Amount Age Sample method Reference

Italy, Naples and Salerno

in Campania region 19

sites in urban, 2 sites in

the remote

Broad leaf: Quercus ilex L. Outer part of the

canopies, about 2m

height

NA One-, two-

and

three-year-old

leaves

Leaves picked by hand, minimize

contact with the leaf surface,

transported and stored under -20℃

in polyethylene bags, avoid light

De Nicola et

al., 2011

Estuary, towns and

industry area

Broad leaf: Cabbage, maize, grape,

‘‘Padron-type” pepper and tomato

NA NA 1 week after

fire

Stored in a room under 0-4℃ Rey-Salgueiro

et al., 2008

Italy, Campania &

Tuscany, urban,

periurban (5km away) &

extraurban (70-100km)

area

Broad leaf: Quercus ilex;

Moss: leptodon smithii (epiphytic

moss)

1) leaves: randomly

from 15-20 trees,

canopy 3-4 m height; 2)

moss: from same tree

as leaves, 1-2m height

NA 1) leaves:

1-year old; 2)

moss: 2-3

years

Sample stored under -20℃, without

washing the leaves

De Nicola et

al., 2013

Portugal highly

industrialized coast

Moss: Parmotrema hypoleucinum

(Steiner) Hale

Collected from

branches and trunks of

Quercus suber L. &

Pinus pinea L

34 samples NA Leaves were put in brown glass

bottle after picking, stored under

4℃

Augusto et al.,

2009

Spain, Navarra, Bertiz

Nature Reserve

Moss: Isothecium myosuroides Brid.

and Hypnum cupressiforme Hedw

Brown part are not

included

9 samples of Brid,

3 of Hedw

Less than 3

years,

according to

the duration

of research

NA Foan et al.,

2012

*NA: Not mentioned in the articles.

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2.2 Sample Preparation & Extraction

First of all, the dry weight of the sample was determined. The temperature of oven was

suggested to be set at 80 - 85℃ (Hubert et al., 2003; Ratola et al., 2012), while others did

not mention clearly. Secondly, to make the sample prepared, reduce the size of leaf was

applied by most of the literature. Two mainstreams of particle size were adopted: cut into 1

mm sections and grinded into powder under 0.05 mm. In most cases, pulverization was

performed after liquid nitrogen freezing.

Secondly, to separate the target compound from the sample leaves, extraction procedure

was taken. The conventional methods are ultra-sonication and Soxhlet extraction, which

have been widely applied on biological materials extraction. The results of the traditional

ones were considered reliable (De Nicola et al., 2011; Rodriguez et al., 2012; Sun et al., 2010).

However, shortages were reported as well, for instance, cost of time and solvent (Augusto et

al., 2009; Hubert et al., 2003), loss of volatile compounds and filtration is required

mandatorily (Wang et al., 2008), etc. The extraction temperature and power of

ultra-sonication were not clearly stated in literature. The time of extraction normally should

be set at 10 minutes and repeated 3 times (Ratola et al., 2012; Ziegenhals et al., 2008). Little

information about Soxhlet extraction was introduced. The extract time of 24 hours was

suggested by some researches (Rinaldi et al., 2012).

The advanced extraction methods pressurized liquid extraction (PLE) was suggested by

recent researches (Foan et al., 2012; Lehndorff & Schwark, 2009). The accelerated solvent

extractor (ASE 200 and ASE 300) manufactured by Dionex were applied in these researches.

The configuration of the device was found similar after comparing different literature. The

temperature was around 100℃, the pressure was around 100bar, the flush volume was 60%,

the time was 10min, 2-3 static cycles were set and the purge time was 120s.

Once, saponification was also applied for extraction. The details of the extraction were not

introduced. 30mL of cyclohexane was applied as extraction solvent (Ziegenhals et al., 2008).

Lots of solvents were applied as extraction solvents. The mixture hexane:dichloromethane

(1:1, v/v), dichloromethane and hexane were the most commonly used extraction solvents.

Besides, varies choices of solvents were reported, for instance, acetone and the mixture

dichloromethane:acetone (1:1,v/v), etc. The amount of solvent was not clearly explained.

Anhydrous sodium sulfate was applied as desiccant and was suggested to be added while

concentrating (De Nicola et al., 2013; Sun et al., 2010). Filters with 0.22μm or 0.45μm pore

diameter were used for the extracted solvent filtration. The filter composited by Na2SO4 and

celite (70:30, w/w) applied by Ziegenhals et al (2008) only.

To concentrate the extract, nitrogen flow performed by TurboVap and rotary evaporator

were suggested mostly. The temperature, vapor pressure and rotate speed were not

described clearly. The application of combining the TurboVap and the rotary evaporator was

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recommended several times. The solvent was evaporated down to 4-5mL by rotary

evaporator and then blow down by nitrogen flow until the extract dryness (De Nicola et al.,

2011; De Nicola et al., 2013; Rinaldi et al., 2012). The endpoint of concentration was

introduced lower than 1mL by lots of authors.

The comparison between various extract methods was made previously. The articles with

comparison were bold framed in the following table. The difference between PLE and

Soxhlet was studied by Hubert et al., (2003) and Foan and Simon (2012). The recovery (about

70%) and total PAHs concentration (200ng/g) were found similarly when PLE and Soxhlet

were performed, however, the advantages of less consumption of solvent and time were

found, it was 30mL for PLE against 100mL for Soxhlet and 24 samples in 8 hours for PLE

against 2 samples in 3.5 hours for Soxhlet, therefore, PLE extraction was recommended

(Foan and Simon, 2012). The difference between PLE, ultra-sonication and Saponification

was studied by Ziegenhals et al., (2008). PLE was recommended after comparing the

efficiency of extraction. The difference between PLE, Soxhlet and ultra-sonication was

studied by Ratola et al., (2006). The recoveries and relative standard deviation (RSD) of the 3

different extraction technologies were 65-102% with RSD <7.5% (Soxhlet), 72-100% with RSD

<8% (ultra-sonication), 70-137% <11% (PLE), therefore, ultra-sonication was recommended

considering accessibility, lower cost and less time (Ratola et al., 2006).

Two different technologies PLE and non-device extraction was applied on different parts of

the sample leaf by Wang et al., (2008). PLE was applied on inner part of the leaf, while

non-device extraction was applied on the cuticle. Therefore, the differences between the

two technologies were not stated clearly in the article. More information can be found in

Table 2-2.

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Table 2-2 Literature Review of Sample Preparation & Extract

Sample Preparation Extract method & description Solvent information Extra information Evaporation after

extraction

Filter after

extraction

Reference

No Ultra-sonication 2mL of homogenized

n-hexane:acetone (1:1, v/v)

NA Nitrogen used to blow

down solvent

0.22μm Rodriguez et al.,

2012

No Ultra-sonication n-hexane NA Nitrogen used to blow

down solvent

NA Rey-Salgueiro et

al., 2008

No Ultra-sonication dichloromethane:acetone (1:1,

v/v)

5g leaves, 3g moss,

anhydrous sodium

sulfate (=mass of

sample)

Rotary evaporator

dried to 4 mL, then

blew down to dryness

by nitrogen

NA De Nicola et al.,

2013

FW/DW determined Ultra-sonication: repeated 3 times 100mL of

dichloromethane:acetone (1:1,

v:v)

5 g sample Rotary evaporator

dried to 5 mL, then

blew down to dryness

yes De Nicola et al.,

2011

Dried in room

temperature for 1 week,

grilled and sieved by

63цm, stored under 4℃

until analysis

Ultra-sonication: 30s, 20% amplitude,

25℃

Acetone, dichloromethane,

n-hexane, methanol, iso-octane

and n-hexane:acetone (1:1, v/v)

NA Nitrogen used to blow

down solvent

0.22μm Sanz-Landaluze et

al., 2010

Defrosted in room

temperature, FW/DW

determined under 80℃

Ultra-sonication: 10min, repeated 3

times

30mL of hexane:dichloromethane

(1:1, v/v)

5g sample Rotary evaporator to

dryness

NA Ratola et al., 2012

Samples divided to 2

groups, one for PAHs,

one for FW/DW

Soxhlet extraction: 24h 250mL of dichloromethane and

50mL of hexane

0.2g sodium

sulfate

1) rotary evaporator to

10ml; 2) add Na2SO4

then centrifugal to

separate; 3) evaporate

to dryness

0.45μm Rinaldi et al.,

2012

No Soxhlet extraction: 24h 200mL of acetonitrile 2g sample Rotary evaporator NA Augusto et al.,

2009

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Table 2-2 continue

Sample Preparation Extract method & description Solvent information Extra information Evaporation after

extraction

Filter after

extraction

Reference

Samples were separated

into two groups, one

washed with deionized

water, another left

original, while testing,

cut into 1-cm sections,

freeze-dried

Soxhlet extraction: 48h dichloromethane 5g sample, Na2SO4 NA NA Sun et al., 2010

Dried spruce needles PLE*: ASE

** 200, 140℃, 1000psi

(≈69bar)***

, 10min, 2 static cycles,

purged time 120 s

hexane:dichloromethane (1:1, v/v) NA Concentrated using a

TurboVap

NA St-Amand et al.,

2008

FW/DW determined

under 50℃

PLE: 120℃, 75bar hexane:dichloromethane (99:1,

v/v)

Centrifugation to

remove waxes

NA NA Lehndorff &

Schwark, 2009

1) FW/DW determined

under 85℃; 2)

ultra-sonication with

100mL of DCM for

10min, the wax layer was

filtered; 3) inner

substance shredded

PLE: ASE 200, two steps at 40 and

120℃, 15MPa (≈150bar)****

, 10min, 3

static cycles

n-hexane NA NA NA Hubert et al.,

2003

No Soxhlet extraction: 20h n-hexane 100g sample NA NA

Freeze-dried

recommended, grinded

to <0.05mm

PLE: ASE 200, 80℃, 150bar, warm up

for 5min, 2 static cycles, purged with

purified nitrogen for 120s

n-hexane 1.5g sample, 0.75g

Na2SO4

Nitrogen used to blow

down to 1mL

NA Foan et al.,

2012

No Soxhlet extraction: Soxtec System

HT2, immersed in boiling solvent 2h,

rinsed for 1h

100mL of n-hexane 1.5g sample, 50ng

surrogate standard,

0.75g Na2SO4

Nitrogen used to blow

down to 1mL

NA

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Table 2-2 continue

Sample Preparation Extract method & description Solvent information Extra information Evaporation after

extraction

Filter after

extraction

Reference

Homogenized, levigate

with acrylic acid

PLE: ASE 200, 100℃, 100 bar, 10 min,

4-cycle, flush volume 60%, 2 static

cycles, purge time 120s

n-hexane, n-hexane:acetone (1:1,

v/v)

1.5–2 g tea NA Glass

microfiber

filters

Ziegenhals et

al., 2008

Samples were heated

with 30 mL methanolic

(KOH) under reflux 1 h,

filter by glass wool

Saponification: after extraction,

washed 3 times with 30mL portions of

distilled water

30mL of cyclohexane 1.5–2 g tea Rotary evaporator Na2SO4/Celite

(70:30, w/w)

No Ultra-sonication: repeated 3 times. dichloromethane:acetone (1:1,

v/v)

1.5-2g sample Rotary evaporator Na2SO4/Celite

(70:30, w/w)

Cut into 1cm bits,

analysis immediately

after sampled

Soxhlet extraction: 24 h 100mL of

hexane:dichloromethane (1:1, v/v)

10g sample Rotary evaporator to

0.5mL

NA Ratola et al.,

2006

Cut into 1cm bits,

analysis immediately

after sampled

Ultra-sonication: 360W Selecta

ultrasonic bath, 10min, repeated 3

times.

30mL of hexane:dichloromethane

(1:1, v/v)

10g sample Rotary evaporator to

dryness

NA

Grind and fill to the top

with hydro matrix

PLE: ASE 200, 150℃, 1500psi

(≈103bar), warm up for 7min, run for

10min, 2 static cycles, purge time 90s

hexane:dichloromethane (1:1, v/v) NA TurboVap LV

evaporator with

Nitrogen to 0.5mL

NA

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Table 2-2 continue

Sample Preparation Extract method & description Solvent information Extra information Evaporation after

extraction

Filter after

extraction

Reference

Fresh leaves were

air-dried (25℃) for 1 h

NA 150mL of dichloromethane 1-6g sample 50mL extraction dried

by nitrogen, weighted

as cuticle wax

0.45цm Wang et al.,

2008

Step1: Fresh leaves were

air-dried (25℃) for 1h;

Step 2: (extracted leaves)

freeze-dried and

pulverized for 40-mesh

sieve

PLE: ASE 300 dichloromethane:acetone (1:1,

v/v)

0.5-2g sample NA NA

* PLE: Pressurized Liquid Extraction

** ASE: Accelerated Solvent Extractor

*** 1bar ≈ 14.5psi

**** 1bar ≈ 0.1MPa

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2.3 Clean-up

The co-extraction and co-elution of plant lipids and pollutants can lead to great interference

on GC–MS analysis (Hubert et al., 2003). Therefore, chromatographic columns packed with

Silica/Alumina/Florisil cartridges (Solid Phase Extract-SPE cartridges) were applied to remove

the influence of polar compound and gel permeation chromatography (GPC) was applied to

eliminate the influence caused by lipids (Sun et al., 2010).

According to the literature, the procedure of SPE clean-up can be concluded as following: 1)

the devices and tools were washed by distilled water; 2) the devices and tools were

conditioned by solvent; 3) the solvent after extraction was added into the SPE columns

followed by the eluents; 4) every drop of the liquid was collected into one tube; 5) collected

solvent was concentrated to almost dryness by rotary evaporator or TurboVap; 6) the

residue after concentration was reconstituted by 1mL of solvent; 7) the prepared sample

was transferred to vials and stored in freezer waiting for further analysis (Ratola et al., 2006;

Ratola et al., 2012; Ziegenhals et al., 2008). Dichloromethane, cyclohexane and the mixture

hexane:dichloromethane (1:1, v/v) were the eluents which widely used for SPE (Ratola et al.,

2006; Ratola et al., 2012; Ziegenhals et al., 2008). The eluent volume was not explained

clearly in the articles.

GPC was recommended by some articles. Briefly, the residue from extraction was eluted and

filtered, and then the liquid was transferred for GPC clean up. The product of GPC was

concentrated to 0.5mL or to dryness by rotary evaporator. Further purification maybe

performed depends on different situation. Cyclohexane:ethylacetate (1:1 v/v) was used as

the eluent for GPC (Rodriguez et al., 2010).

Other clean up method were introduced, for instance, size exclusion chromatography (SEC),

medium pressure liquid chromatography etc., some researchers also suggested it is not

necessary for cleaning up (Rodriguez et al., 2012; Sanz-Landaluze et al., 2010).

The comparison between various extraction methods was made previously. The literature

with comparison was bold framed in the following table. The difference of Florisil, alumina

and silica between cartridges format and glass chromatography was studied by Ratola et al.,

(2006). The similar results were obtained by glass chromatography and by alumina and silica

in cartridges format. The recovery was 100% for most of the 16 PAHs with RSD <13%, except

for the ones less volatile which the recovery was around 50%. Inconsistence and higher high

molecular weight PAHs recovery were obtained by Florisil SPE cartridge. ’Due to the more

time-consuming set-up and operation of this approach, it was decided to consider alumina

cartridges as the best and most selective clean-up method.’ (Ratola et al., 2006).

The difference between SEC and conventional method was studied by Hubert et al., (2003).

The concentration of each PAHs compound was improved while comparing SEC clean up to

the conventional clean up. The RSDs for SEC (10%) were found lower than the conventional

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(20-30%). Therefore, SEC was recommended for clean-up (Hubert et al., 2003). Both GPC

and SPE were applied by Ziegenhals et al., (2008), but the difference was not clearly stated.

More information can be found in Table 2-3.

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Table 2-3 Literature Review of Clean-up

Clean-up method Extra information Program Reference

No clean up NA NA Rodriguez et al.,

2012

No clean up NA NA Sanz-Landaluze et

al., 2010

Centrifuge NA 3500rpm, -2℃, 15min, twice Rinaldi et al., 2012

GPC* NA 1) residue from extraction eluted with 10mL of cyclohexane:ethylacetate (1:1 v/v); 2) 5mL of above used

in GPC and concentrate to 0.5mL in 40℃ water bath with rotary evaporator; 3) further purify with

adsorption chromatography (AC) with silica gel of petrolether:dichloromethane (4:1, v/v); 5) mix with

propan-2-ol for final purification on Sephadex LH-20 column; 6) the rest was re-constituted by 1mL of

hexane.

Rodriguez et al.,

2010

SPE**

(Polypropylene) 5g, 25mL of Alumina

cartridges

1) 50mL of hexane:dichloromethane (1:1, v/v) to condition; 2) extract add to column, elute with 50mL of

hexane:dichloromethane (1:1, v/v) and 50mL of dichloromethane; 3) pre-concentrate to 0.5mL with

rotary evaporator; 4) re-constituted by 1mL of hexane.

Ratola et al., 2012

SPE cartridges of Florisil 30mL of acetonitrile

as eluting solvent

NA Augusto et al., 2009

SPE cartridges of silica NA NA Rey-Salgueiro et al.,

2008

Silica column chromatography column information:

10mm i.d. × 350mm

length, 10g of silica

gel and 20mm length

of Na2SO4

1) concentrated using rotary evaporator; 2) transferred with cyclohexane and eluted with 25mL of

n-hexane, followed by 50mL of pentane:dichloromethane (3:2, v/v) at the rate of 2mL/min; 3) evaporated

to dryness by rotary evaporator; 4) rinsed 3 times in Kudema-Danish concentrator with n-hexane; 5)

blown down to 1mL by nitrogen

Wang et al., 2008

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(Table 2-3 continue)

Clean-up method Extra information Program Reference

Small-deactivated silica column NA 1) eluted with hexane; 2) concentrated to approximately 200μl using a TurboVap St-Amand et al.,

2008

Medium pressure liquid

chromatography

NA NA Lehndorff &

Schwark, 2009

GPC NA 1) 4.5mL of cyclohexane:ethyl acetate (1:1, v/v); 2) polytetrafluoroethylene (PTFE) filter (pore size 1mm)

with flow rate of 5mL/min, waste time 0–36min, collect time 36–65min; 3) rotary evaporator used to

remove solvent, then blew down to dryness by nitrogen

Ziegenhals et al.,

2008

SPE: ASPEC***

Xli Before clean-up, 1)

1g silica dried for 12h

under 550℃; 2)

deactivate with 15%

distilled water into

8mL column

1) condition column with 3mL of cyclohexane; 2) 10mL of cyclohexane added to the sample as solvent

SPE cartridges of Florisil 5g 1) conditioned with 50mL of hexane:dichloromethane (1:1, v/v); 2) sample added and eluted with 50mL

of hexane:dichloromethane (1:1, v/v), followed by 50mL of dichloromethane; 2) Rotary evaporator dried

to 0.5mL, then blew down to dryness by nitrogen; 3) reconstitute in 1mL of hexane.

Ratola et al., 2006

SPE cartridges of silica 5g

SPE cartridges of alumina 5g

Glass column with Florisil, silica

and alumina

5g Florisil / Silica /

Alumina and 1cm

high of Na2SO4

Activated at 400℃ for 12h and deactivated with 1.2% ultrapure water

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(Table 2-3 continue)

Clean-up method Extra information Program Reference

SEC****

The extracted solvent

was concentrated to

dryness first

1) 600μL THF was used to dissolve the solvent; 2) SEC condition: flow rate 5mL/min, run for 15min; 3) the

solvent collected between 0-9.2min was discarded; 4) the solvent between 9.2-12min was collected and

concentrated to dryness by rotary evaporator.

Hubert et al., 2003

Conventional The extracted solvent

was concentrated to

2mL first

1) extracted solvent was mixed with 15g deactivated Florisil; 2) eluted with 160mL

n-hexane:dicholoromethane (1:1, v/v); 3) the first 60mL eluate were mixed with 3.5g deactivated Florisil

and eluted with 60mL same eluent; 4) concentrate to dryness.

* GPC: Gel Permeation Chromatography

** SPE: Solid Phase Extraction

*** ASPEC: Automated Solid-Phase Extraction Clean-up

**** SEC: Size Exclusion Chromatography

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2.4 PAHs and oxy-PAHs Analysis

Two technologies were applied to analyze PAHs and oxy-PAHs, High Performance Liquid

Chromatography (HPLC) and GC-MS. GC-MS was applied more widely than HPLC.

Apparently, GC-MS is combined by two parts. Gas chromatography (GC) is the first part

which is applied to analyze the chemical species existed in the sample. Firstly, the sample

was heated into gaseous phase. The gaseous sample was carried by inert gases, such as

helium, and was transported into the chromatographic column. The absorption ability of

absorbent to the components existed in the sample was different, resulting in that the speed

of various components was different in GC. The components that have the weakest

absorption ability to the adsorbent were discarded the earliest. (Wiersma, 2004; Zeng, 2010).

Mass spectrometry is one of the quantitative approaches based on the measurement of

mass-to-charge ratio. The components are ionized into positive ions with different

mass-to-charge ratio first. (Wiersma, 2004; Zeng, 2010).

HP-5 MS capillary column with 30m × 0.25mm i.d. × 0.25μm film thickness GC-MS column

was applied in most of the experiments. TR-50MS 10m × 0.1mm i.d. × 0.1μm film thickness

GC-MS column was applied by Ziegenhals et al., (2008). Fused-silica capillary column with

15m × 0.25mm i.d. × 0.25μm film thickness GC-MS column was applied by St-Amand et al.,

(2008). Vydac 201TP C18, 250mm × 2.1mm (length i.d.), 5μm particle size column was

applied by Rodriguez et al., (2012). SupelcosilTM LC-PAH C18 column with 25m × 0.32mm i.d.

× 0.52μm film thickness HPLC column was applied by Foan et al., (2012). More information

can be found in Table 2-4.

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Table 2-4 Literature Review of Detection & Separation

Equipment Re-constitution Column Flow Program Reference

GC-MS*

NA HP-5MS capillary column (30m ×

0.25mm i.d. with a 0.25μm film

thickness)

NA 1) 33℃, 1min; 2) 20℃/min to 280 ℃; 3) keep for

15min

De Nicola et

al., 2005

GC-MS NA TR-50MS column (10m × 0.1mm ×

0.1μm)

NA 1) 50℃, 1min ; 2) 25℃/min to 280℃; 3) 1℃/min to

330℃; 4) hold 30min

Ziegenhals et

al., 2008

GC-MS NA HP ultra 2 capillary column (25m ×

0.32mm i.d. × 0.52μm film thickness)

NA 1) 60℃, 1min; 2) 10℃/min to 260℃; 3) keep 1min Hubert et al.,

2003

GC-MS NA HP-5 MS capillary column (30m ×

0.25mm i.d. × 0.25μm film thickness)

1mL/min,

helium

Start at 60℃, raise to 280℃ in 6 minutes Wang et al.,

2008

GC-MS The solvent after clean-up was

concentrated and re-dissolved in

4mL of cyclohexane

HP-5MS capillary column (30m ×

0.25mm i.d. × 0.25μm film thickness)

1.11mL/min,

helium

1) started at 70℃; 2) increased by 20℃/min, to

280℃; 3) held for 24min.

De Nicola et

al., 2011

GC-MS 1) The solvent after clean-up was

dried to 1mL by rotary evaporated;

2) further concentrated to 0.2mL; 3)

1μl hexamethyl-Benzene was added

NA 1.2 mL/min,

helium, linear

velocity

25.4cm/sec at

300°C

1) start at 60℃ for 3 min; 2) raised to 200℃ by

20°C/min; 3) to 260℃ by 4℃/min; 4) to 270℃ by

2℃/min; 5) 10 min hold time

Sun et al.,

2010

GC-MS NA DB-5 column (30m × 0.25mm i.d. ×

0.25μm film thickness) coated with 5%

diphenyl-olydimethylsiloxane

Helium 1) 60℃, 1min; 2) 6℃/min to 175℃, hold 4min; 3)

3℃/min to 235℃; 4) 8℃/min to 300℃, hold 5min

Ratola et al.,

2006

GC-MS The solvent after clean-up was

concentrated and reconstituted to

4mL by 0.4mL of internal standard

and cyclohexane

HP-5MS capillary column (30m ×

0.25mm i.d. × 0.25μm film thickness)

1.11mL/min,

helium

1) started with 70℃; 2) up by 20℃/min to 280℃; 3)

keep 24min

De Nicola et

al., 2013

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(Table 2-4 continue)

Equipment Preparation Column Flow Program Reference

GC-MS The solvent after clean-up was

concentrated and re-dissolved in

1mL of hexane

VF-5MS (30m × 0.25mm i.d. × 0.25μm

film thickness)

1mL/min helium 1) 60℃ for 1min; 2) up to 175 by 6℃/min hold 4min;

3) up to 235℃ by 3℃/min; 4) up to 300℃ by 8℃

/min; 5) keep to total time of 60min

Ratola et al.,

2012

GC-MS The solvent after clean-up was

concentrated and re-dissolved in

1mL of hexane

HP-5 MS (30 m × 0.25 mm i.d.,

0.25μm) fused-silica capillary column

1mL/min helium 1) 90℃ for 4 min; 2) raise to 100 by 10℃/min; 3)

raise to 290℃ by 3℃/min; 4) keep 22 min

Rodriguez et

al., 2010

GC–EIMS**

NA Fused-silica capillary column (15m ×

0.25mm) coated with 0.25μm

chemically bonded HP-5MS phase (5%

phenyl methyl siloxane)

NA 1) Initial 150℃ for 2 min; 2) 10℃/min to 240 ℃; 3)

5℃/min to 300℃; 4) held for 5 min.

St-Amand et

al., 2008

HPLC***

The solvent after clean-up was

concentrated and re-dissolved in

2mL of acetonitrile

25cm × 4.6mm (length—i.d.) 5μm

particle Supelcosil LC-PAH coupled

with a guard column

NA NA Rinaldi et al.,

2012

HPLC The solvent after clean-up was

concentrated and re-dissolved in

200μl acetonitrile:water (6:4, v/v)

Vydac 201TP C18, 250mm × 2.1mm;

5μm

0.3 ml/min 1) ACN:water (60:40, v/v); 2) 18min, 100% CAN; 3)

21min back to step one; 4) total time 28min

Rodriguez et

al., 2012

HPLC NA Supelcosil LC-PAH C18 column (250

mm × 4.6mm i.d. 5μm particle size)

and a precolumn (20mm × 4.6mm i.d.

5μm particle size)

NA NA Foan et al.,

2012

HPLC The solvent after clean-up was

concentrated and filled up to a final

volume of 0.5mL with ACN

Supelcosil LC-PAH (25cm × 4.6mm

(length × i.d.), 5μm particle size), kept

constant at 33℃.

Inject volume:

50μl by flow

rate of 1mL/min

Mobile phases were acetonitrile (ACN) and water. 1)

the gradient was: 80:20 ACN/H2O; 2) changed to 95:5

ACN/H2O in 40 min; 3) changed to 80:20 ACN/H2O in 1

min; 4) hold for 10 min giving an analysis time of

51min.

Rey-Salgueiro

et al., 2008

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* GC-MS: Gas Chromatography Mass Spectrometry

** GC-EIMS: Gas Chromatography - Electron Ionization Mass Spectrometry

*** HPLC: High Performance Liquid Chromatography

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2.5 Concentration Levels

The highest total PAHs concentration in industry area was reported in Guangdong Province, China.

PAHs concentration was recorded 1927.2ng/g (d.w.) around a ceramic manufactures (Sun et al.,

2010). On the other hand, the lowest was reported in Argentina, which is 49ng/g (d.w.) monitored

around an aluminum industry (Rodriguez et al., 2012).

The total PAHs concentration in cities was the most common topic which the researches focused on.

The difference between the urban area and suburban area was also usually compared. The highest

PAHs concentration of 8500 ng/g (d.w.) was found by De Nicola et al., (2011) in an urban area of

Campania, Italy. The result was not convinced enough to be the representative to describe the

situation of ‘Urban area’. The average city PAHs concentration was reported range from 154.2 to

4420ng/g (d.w.) depending on different traffic condition, city scale and climates. The highest PAHs

concentration was found in January (De Nicola et al., 2005). The difference age of leaf was verified

having no effect on the accumulation of PAHs (De Nicola et al., 2011). The concentration of oxy-PAHs

in leave samples or other biological samples was rarely found in previous researches. More

information can be found in Table 2-5.

According to the previous studies, phenanthrene was found has the highest concentration in natural

environment, while, indeno[1,2,3-c,d]pyrene and benzo[b]fluoranthene was found the lowest

(Ratola et al., 2006; Navarro-Ortega et al., 2012). Fluoranthene, phenanthrene and pyrene were

found have higher concentration than other PAHs compounds, relating to different traffic condition

in cities (Rodriguez et al., 2012). Naphthalene, dibenzo[a,h]anthracene, and indeno[1,2,3-c,d]pyrene

were found the lowest in cities (Rodriguez et al., 2012). Naphthalene, phenanthrene, pyrene,

chrysene and fluoranthene were found have higher concentration than others relating to different

industrial area (Sun et al., 2010). More information can be found in Table 2-6.

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Table 2-5 Literature Review of Current Results in Sum of PAHs

Total PAHs Concentration in Leaf (ng/g, d.w.)

(Details of the Location Information)

Type of Location Reference

97/111/111.2 (Fresh leaves) 3 different polluted sites in Germany Hubert et al., 2003

290 Along the whole river in Spain Navarro-Ortega et al., 2012

41 Urban area Noth et al., 2013

1927 (ceramic manufactures)

1565 (steel industry)

1144 (petrochemical)

1045 (blank, no industry)

Industry (Guangdong, China) Sun et al., 2010

1100-2076 (May, 2001)

1349-1930 (September, 2001)

1798-4420 (January, 2002)

1038-1962 (May, 2002)

300-500 (control site)

Urban area of Naples De Nicola et al., 2005

1216 (1-year-old leaves)

1126 (2-year-old leaves)

1102 (3-year-old leaves)

500-2000 (urban site mean)

8500 (highest urban value)

Urban area (Campania) De Nicola et al., 2011

382 (moss); 714 (leaves) Urban area (Campania) De Nicola et al., 2013

154 (moss); 365 (leaves) Urban area (Tuscany)

326 (moss); 218 (leaves) Periurban (Campania)

72 (moss); 172 (leaves) Periurban (Tuscany)

125 (moss); 426 (leaves) Extraurban (Campania)

111 (moss); 141 (leaves) Extraurban (Tuscany)

400 Industry (Mexico) Sanz-Landaluze et al., 2010

2080 (heavy traffic)

1031 (moderate traffic)

423 (low traffic)

Urban area (Stuttgard) Rodriguez et al., 2010

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(Table 2-5 continue)

Total PAHs Concentration in Leaf (ng/g, d.w.)

(Details of the Location Information)

Type of Location Reference

49-1830 Aluminum industry (Argentina) Rodriguez et al., 2012

200 Bertiz Nature Reserve (Spain) Foan and Simon, 2012

22 (Costa Nova)

59 (Cerveira)

75 (Barcelona)

339 (Porto)

Natural polluted Ratola et al., 2006

807 (Porto)

454 (Setubal)

309 (Evora)

348 (Faro)

Urban area (Portugal) Ratola et al., 2012

350 (Miranda de Ebro)

620 (El Prat)

791 (Barcelona)

346 (Vic)

Urban area (Spain)

563 (Chania)

713 (Rethymno)

566 (Malia)

344 (Ierapetra)

Urban area (Greece)

121 (Antua)

135 (Quintas)

155(Souselas)

78(Sines)

105(Alcoutim)

Sub-urban area (Portugal)

111 (Villodas)

84 (Monteagudo)

128 (Movera)

83 (Alcolea de Cinca)

135 (Torres de Segre)

Sub-urban area (Spain)

180 (Elafonisi)

98 (Paleochora)

124 (Festos)

87 (Analipsi)

39 (Moni Toplou)

Sub-urban area (Greece)

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Table 2-6 Literature Review of Current Findings in Individual PAHs

Reference Ratola et al., 2006 Rodriguez et al., 2012 Sun et al., 2010 Navarro-Ortega

et al., 2012

Site information Costa Nova

(Natural)

Cerveira

(Natural)

Barcelona

(Natural)

Porto

(Natural)

High

Traffic

Moderate

Traffic

Low Traffic Ceramic Steel Petrochemical No Industry Natural

PAHs (±SD) ng/g d.w.

Naphthalene 4.8 2.3 2.9 14.8 1.9±0.9 2.5±2.2 0.5±0.3 141.0±34.4 116.1±21.6 198.9±77.5 320.1±30.3 12.3±8.6

Acenaphthylene NA 0.9 2.8 7.0 11.9±2.2 6.0±2.2 3.9±0.8 21.9±0.2 9.2±1.8 31.8±3.3 33.1±3.6 4.9±3.2

Acenaphthene NA 6.2 1.8 5.5 67.9±25.4 29.9±15.0 18.6±10.1 46.8±7.9 50.8±0.5 49.8±11.4 55.4±6.5 6.8±8.6

Fluorene 0.9 2.9 5.2 28.9 50.2±17.4 20.9±6.2 8.5±4.3 143±82 69.4±8.3 45.9±24.7 36.6±8.4 52.4±47.7

Phenanthrene 7.3 11.6 20.4 102.9 595±293 263±122 98.6±29.5 467±246 252±29.9 157.5±59.2 203.9±32.83 124±111

Anthracene 0.6 1.4 1.9 7.6 21.8±5.8 10.2±4.4 6.3±1.6 70.2±35.9 76.9±8.9 35.1±11.1 49.1±11.4 4.1±3.5

Fluoranthene 1.3 3.8 8.4 42.9 614±149 335±258 128±43 207±100 198.5±62.3 134.7±70.3 79.3±18.4 20.7±19.8

Pyrene 0.9 4.0 9.1 37.1 311±47 154±107 58.2±17.9 366±172 182.2±15.5 266±115 94.7±27.4 30.8±27.5

Benzo[a]anthracene 0.5 2.3 2.4 10.1 29.8±11.2 12.5±5.1 6.6±0.8 93.3±49.0 150.1±30.4 116.1±76.5 81.1±30.3 7.5±7

Chrysene 2.7 6.2 8.9 45.9 199±81 91.2±34.1 52.4±11.4 287±152 182.6±54.3 61.5±33.2 57.2±21.5 16.9±9

Benzo[b]fluoranthene 0.3 2.5 3.0 7.2 50.8±15.6 22.5±6.4 16.4±1.6 111.3±3.7 93.7±17.5 83.8±28.8 25.0±6.1 3.8±3.1

Benzo[k]fluoranthene 0.5 2.3 3.0 7.9 16.6±2.1 9.3±2.8 9.0±3.3 69.5±13.1 79.0±7.6 73.7±9.6 33.9±7.0 3.7±1.8

Benzo[a]pyrene NA 3.5 1.8 6.8 22.4±8.6 10.8±4.4 7.4±2.4 72.6±22.6 53.5±12.1 44.0±10.1 21.2±11.8 1.4±0.9

Dibenzo[a,h]anthracene 0.7 2.8 1.3 5.7 65.2±8.1 NA NA 47.6±43.6 40.9±10.8 NA 10.9±3.5 0.4±0.5

Benzo[g,h,i]perylene 0.7 3.1 1.2 4.9 12.7±6.3 36.3±21.0 8.4±3.7 126.9±20.1 NA NA NA 0.6±0.4

Indeno[1,2,3-c,d]pyrene 0.5 3.4 0.8 4.3 10.3±1.1 6.5±4.6 NA 61.4±4.4 37.7±9.7 NA 39.3±114.8 0.4±0.3

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CHAPTER 3 Chemical Materials

3.1 Chemicals & Reagents

The chemicals and reagents used in this thesis are listed in Table 3-1. Except

dichloromethane is purchased from ACROS, others reagents are purchased from SIGMA –

ALDRICH.

Table 3-1 Chemicals & Reagents

Subject Manufacture Description

cyclohexane SIGMA - ALDRICH ACS reagent ≥99%

hexane SIGMA - ALDRICH CHROMASOLV® Plus, for HPLC, ≥95%

dichloromethane ACROS 99.9% for residual analysis

SPE Tube SIGMA - ALDRICH LC-Florisil®, 1g, 6mL

Sodium Sulfate SIGMA - ALDRICH ACS reagent ≥90%, anhydrous, powder

Two internal standards were applied in this thesis, which are mono-fluorinated PAHs and

deuterated PAHs. The relationship between the 16 kinds of PAHs and the internal standards

(IS) were shown in the following table. The internal standards were chosen follow the rules

of minimum difference of molecule weight between the analytes and the internal standards.

The details can be found in Table 3-2 and Table 3-3.

Table 3-2 Internal Standards Information (1)

Internal standards Concentration

ng/mL

Analytes

Deuterated PAHs Naphthalene-d8 196 Naphthalene

Biphenyl-d10 199 Acenaphthene

Acenaphthylene

Phenanthrene-d10 198 Anthracene

Phenanthrene

Fluorene

Pyrene-d10 199 Pyrene

Fluoranthene

Benz[a]anthracene-d12 197 Benz[a]anthracene

Chrysene

Benzo[a]pyrene-d12 197 Benzo[b]fluoranthene

Benzo[k]fluoranthene

Benzo[a]pyrene

Benzo[g,h,i]perylene-d12 197 Dibenz[a,h]anthracene

Indeno[1,2,3-cd]pyrene

Benzo[g,h,i]perylene

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Table 3-3 Internal Standards Information (2)

Internal standards Concentration

ng/mL

Analytes

Mono-fluorinated

PAHs

1-Fluoronaphthalene 200 NA

4-Fluorobiphenyl 201 NA

3-Fluorophenanthrene 201 NA

1-Fluoropyrene 199 NA

3-Fluorochrysene 199 NA

9-Fluorobenzo[k]fluoranthene 100 NA

According to Lundstedt et al., (2014), Kojima et al., (2010), Cochran et al., (2012) and O’

Connell et al., (2013), pyrene-d10, was chosen as the single IS for oxy-PAHs analysis.

3.2 Standards Solution

The stock solution was diluted 7 times which were named standard 1 to standard 7. In this

thesis, standard 1 and standard 4 were chosen to be the standard solutions. Standard 1 was

prepared by blending 50μL of PAHs stock solution, 50μL of oxy-PAHs stock solution, 900μL of

dichloromethane, 1μL of internal standard mono-fluorinated PAHs and 1μL of internal

standard deuterated PAHs. Standard 4 was prepared by blending 20μL of standard 1, 980μL

of dichloromethane, 1μL of internal standard mono-fluorinated PAHs and 1μL of internal

standard deuterated PAHs. The two standards were prepared by the PAHs and oxy-PAHs

stock purchased from SIGMA – ALDRICH, in the lab of the Department of Environmental

Organic Chemistry and Technology. The details of the stocks are shown in Table 3-4.

Table 3-4 Details of PAHs and oxy-PAHs Stock Solution

PAHs Conc. in Toluene µg/ml oxy-PAHs Conc. in DCM µg/ml

Naphthalene 100 Napthalene-1-carboxaldehyde 100

Acenaphthylene 100 Napthalene-1,4-dione 105.2

Acenaphthene 100 Fluorene-9-one 130

Fluorene 100 Fluorene-2-carboxaldehyde 107.6

Phenanthrene 100 Anthracene-9,10-dione 114.8

Anthracene 100 1,8-napthalic anhydride 99.6

Fluoranthene 100 Phenanthrene-9-carboxaldehyde 108

Pyrene 100 Phenanthrene-9,10-dione 106.8

Benz[a]anthracene 100 7H-benzo[d,e]anthracene-7-one 102.4

Chrysene 100 Pyrene-1-carboxaldehyde 115.6

Benzo[b]fluoranthene 100 Benz[a]anthracene-7,12-dione 104.4

Benzo[k]fluoranthene 100 Napthacene-5,12-dione 105.6

Benzo[a]pyrene 100 Anthracene-9-one 114.4

Dibenz[a,h]anthracene 100

Benzo[g,h,i]perylene 100

Indeno[1,2,3-cd]pyrene 100

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CHAPTER 4 Results & Discussion

The design of this thesis contains four main parts, which are sample preparation, extraction,

SPE clean-up and concentration. The experiment begins with the preparation of sample. The

size of leaves was tested first. Three leaf shapes, ‘intact leaf’, ‘1mm leaf particle’ and ‘N2

freeze-dry grinded leaf’ were selected. Samples consisting of 5g leaves of each size, 1g of

Na2SO4, 9mL extraction solvent and internal standard were prepared and extracted by

ultra-sonication.

Secondly, the extraction solvent was determined. Three alternatives were chosen according

to the literature, which were the mixture hexane:dichloromethane (1:1, v/v), hexane and

dichloromethane. The prepared samples were extracted in 10mL BD PlastipakTM syringe by

CPX2800H-E BRANSONIC ultra-sonication bath, and were filtered by 0.45μm GMF WHATMAN

syringe filter. The rest was concentrated to 0.5mL by nitrogen on Caliper TurboVap®LV

concentration evaporator workstation.

Florisil solid phase extraction cartridge supplied by Supelco (LC-Florisil) was used to perform

clean up. The device was cleaned by acetone first. Then the cartridge and the device were

conditioned by the extraction solvent. The eluent was the mixture

dichloromethane:cyclohexane (1:1, v/v). The amount of eluent was determined during the

experiments. The clean-up samples were concentrated by TurboVap to the last drop. The

samples were reconstituted using 1mL of dichloromethane, then transferred into CleanPack®

vials and were stored in freezer for further GC-MS analysis.

Figure 4-1 is given to draw a clear image of the procedure of PAHs and oxy-PAHs analysis.

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Figure 4-1 Flow Chart of the Design

4.1 Selection of Plant Species

Taxus baccata, which is shown in Figure 4-2, is an evergreen species growing widely in

western, central and southern Europe, northwest Africa and southwest Asia. It is also known

as ‘yew’ or ‘European yew’ which can be found easily in Ghent, Belgium. It can be seen in

most of the urban landscape designs, for instance parks, lawns, belts between both way

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motor roads etc. Taxus baccata is a medium size species which can reach 10-20m tall. The

leaves are green or dark green and flat. The size of the leaves can be 1-4cm long and 2-3mm

broad. Taxus baccata is considered as one of the most longevity species around the world.

Figure 4-2 Taxus baccata

4.2 Dry Matter Content

5g of leaf was taken and was left in oven under 110℃ for 24 hours. Triplicates were

performed. The water content of Taxus baccata was calculated as 55.11% in average, which

was applied in further experiments. The water content and dry weight can be calculated by

Equation (1) and Equation (2).

𝑴𝒂𝒔𝒔 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑳𝒆𝒂𝒗𝒆𝒔 = 𝑩𝒆𝒂𝒌𝒆𝒓 𝒘𝒊𝒕𝒉 𝑭𝒓𝒆𝒔𝒉 𝑳𝒆𝒂𝒇 − 𝑩𝒆𝒂𝒌𝒆𝒓 𝒘𝒊𝒕𝒉 𝑫𝒓𝒚 𝑳𝒆𝒂𝒇 (𝟏)

𝑫𝒓𝒚 𝑴𝒂𝒕𝒕𝒆𝒓 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = (𝑴𝒂𝒔𝒔 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑳𝒆𝒂𝒗𝒆𝒔

𝑴𝒂𝒔𝒔 𝒐𝒇 𝑭𝒓𝒆𝒔𝒉 𝑳𝒆𝒂𝒗𝒆𝒔) × 𝟏𝟎𝟎% (𝟐)

4.3 Solvent Selection

According to the literature, hexane, dichloromethane and the mixture of

dichloromethane:hexane (1:1 v/v) were nominated as extraction solvent. To determine

which solvent is the most suitable for Taxus baccata, the following experiment was designed.

Three piles of 5g intact leaf were put into tubes which filled with 50mL of three different

solvents. The tubes were soaked in ultra-sonication bath to extract. The extracts were

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transferred into 50mL BD PlastipakTM syringes. The syringes were equipped with 0.45μm

syringe filter and filled with 2g of Na2SO4. The filtered solvents were sunk in BUCHI Heating

Bath B-490 water bath kettle and were rotary evaporated by BUCHI Rotavapor R-200 rotary

evaporator. Triplicates were performed for each solvent. The results are shown in Figure 4-3.

Figure 4-3 the Result of Solvent Test.

Visual test was performed. Figure 4-3 shows the turbidity of each extract after evaporation.

From left to right they were the mixture of hexane:dichloromethane (1:1, v/v),

dichloromethane and hexane. The highest turbidity was given by the mixture. The highest

clarity was given by dichloromethane. Therefore, dichloromethane and hexane were feasible

in this case. The following experiments performed base on these two solvents. The efficiency

and recovery of the two were compared.

4.4 Size of the Sample Determination

3 different sample preparations were compared.

1) Intact leaf (intact)

1g fresh leaves and 1g Sodium Sulfate were weighed and transferred into a syringe. The

plunger of syringe was removed in advance. The syringe was blocked by a stopper at the

mouth. 9mL solvent, dichloromethane or hexane, was filled in next. Afterwards, the syringe

was sealed by the syringe plunger. The prepared syringe was shown in Figure 4-4.

2) Cut into 1mm cube leaf particles (cut 1mm)

The fresh leaves were sliced down to 1mm parts by Swann Morto Ltd. surgical blades on

‘everyday’ 15 micrometer, 30cm × 30m Aluminum foil. The following operation was as same

procedure as 1).

The Mixture

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3) Liquid nitrogen dried and grinded (N2)

The whole Taxus baccata branches were left under room temperature for 1 week to dry.

Leaves were picked from the branches and collected in a mortar. Liquid nitrogen was applied

to freeze. The frozen leaves were grinded into particles smaller than 1mm. Then following

operation was as same procedure as 1).

Figure 4-4 the Prepared Syringe

To make the results easy to be interpreted, the ratio of peak area was calculated. The results

of ‘intact’ were regarded as reference, and were compared with the results of ‘cut 1mm’ and

‘N2’. The interpreted results are shown in Table 4-1 and Table 4-2. The best results extracted

by dichloromethane are highlighted in green, while the best ones extracted by hexane are

highlighted in blue.

Table 4-1 Result of Sample Size Selection: PAHs

Preparation

PAHs

DCM HEXANE

Intact Cut 1mm N2 Intact Cut 1mm N2

Naphthalene 1.00 1.26 1.42 1.00 1.16 1.42

Acenaphthylene 1.00 1.53 1.00 1.00 0.86 0.60

Acenaphthene 1.00 1.32 0.53 1.00 0.84 0.61

Fluorene 1.00 1.13 0.66 1.00 1.48 0.88

Phenanthrene 1.00 0.86 0.88 1.00 1.53 1.49

Anthracene 1.00 0.86 NF 1.00 1.36 NF

Fluoranthene 1.00 1.33 1.32 1.00 1.45 1.50

Pyrene 1.00 1.21 1.17 1.00 1.32 1.44

Benzo[a]anthracene 1.00 1.19 1.21 1.00 0.95 0.95

Chrysene 1.00 1.15 1.15 1.00 1.26 1.19

Benzo[b]fluoranthene 1.00 1.30 1.14 1.00 1.14 0.87

Benzo[k]fluoranthene 1.00 1.18 1.39 1.00 0.99 1.45

Benzo[a]pyrene 1.00 1.34 1.54 1.00 0.74 1.20

Dibenzo[a,h]anthracene NF NF NF NF NF NF

Benzo[g,h,i]perylene NF NF NF NF NF NF

Indeno[1,2,3-c,d]pyrene 1.00 1.69 2.04 1.00 0.95 0.96

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- 39 -

Table 4-2 Result of Sample Size Selection: oxy-PAHs

Preparation

oxy-PAHs

DCM HEXANE

Intact Cut 1mm N2 Intact Cut 1mm N2

Napthalene-1,4-dione 1.00 0.89 0.03 1.00 0.96 NF

Fluorene-9-one 1.00 0.89 0.59 1.00 1.13 0.87

Anthracene-9,10-dione 1.00 1.21 0.91 1.00 2.04 2.23

1,8-napthalic anhydride NF NF NF NF NF NF

7H-benzo[d,e]anthracene-7-one 1.00 0.91 0.67 1.00 2.09 1.36

Benz[a]nthracene-7,12-dione 1.00 0.77 0.76 1.00 1.33 NF

Napthacene-5,12-dione NF NF NF NF NF NF

According to the tables, ‘cut 1mm’ and ‘N2’ gave better extraction results than ‘intact’. The

difference between ‘cut 1mm’ and ‘N2’ was not clear. Different behaviors were shown by

different compounds. Therefore, to avoid the unexpected influence, nitrogen freezing was

performed on fresh leaves in this thesis.

4.5 Extraction

Extraction was performed by ultra-sonication bath under 28℃, 4 lever power (maximum) for

20 minutes. The sealed syringes were hold by tube stand and sunk into ultra-sonication bath.

The bath container was filled to 10mL mark line of syringe by deionized water. The syringes

were taken out and shaken for 2-3 seconds every 2-3 minutes. This is aimed to mix the

samples and solvent homogeneously to increase the efficiency of extract.

The stoppers were replaced by 0.8 × 50mm BD MicrolanceTM syringe needles. The needles

were bended manually into a ‘U’ shape conduit. The syringes and needles were connected by

a 0.45μm syringe filter. The design is shown in Figure 4-5. The syringes were held upside

down and squeezed manually until no continuous liquid flow can be observed. The detail can

be seen in Figure 4-6. Air was sucked in aiming to squeeze again. All syringes were squeezed

3 times. The needles were removed, and the syringes were squeezed harshly and directly

from the mouth of syringe filter to tube until the last drop. The extract was concentrated

down to around 0.5mL by TurboVap. Nitrogen was applied as flowing gas under 30℃, 10psi.

Figure 4-5 The Manually Bended ‘U’ Shape Conduit.

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Figure 4-6 the Process of Extract Transfer

4.6 Clean-up & Concentration

4.6.1 Clean-up & Concentration Procedure

Florisil solid phase extraction cartridge was applied to remove the influence made by polar

compounds. The device and configuration were shown in Figure 4-7. The device was washed

by acetone. Then the columns were connected and conditioned by 5mL of dichloromethane

or hexane slowly. The mixture of dichloromethane:cyclohexane (1:1 v/v) was prepared as

eluent. The switches of the columns were switched off at the beginning. The solvents were

transferred into the cartridge by the VWR 230mm disposable glass Pasteur pipettes. The

solvent was slowly collected drop by drop into the VWR 100 × 16.00 × (0.8-1.0) mm tubes.

The switches were switched off immediately the liquid in the cartridge tubes sunk into the

solid phase. Afterwards, the cartridges were filled by eluent and the same operation was

performed.

The collected solvent was concentrated to almost dryness by TurboVap nitrogen gas flow,

under the condition of 30℃ and 10psi. The concentrated clean-up solvent was re-dissolved

in 1mL of dichloromethane. To retrieve all compounds, the re-dissolved tube was sunk into

ultra-sonication bath for 5 seconds under the condition of 25℃, 4 lever power (maximum).

The re-dissolved sample extract was transferred into vials and stored in freezer until analyze.

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Figure 4-7 the Clean-up Device

4.6.2 Determination of Eluent Volume

To decide how much eluent is sufficient for elution, the following experiment was designed.

1mL sample were prepared by 995μL extraction solvent and 5μL standard 1 solutions. Both

hexane and dichloromethane were applied for extraction in this design. To brief the

procedure, 16mL eluent was prepared for elution, 1mL sample was added first, then the

eluent. The eluent was added milliliter by milliliter. The solvent were collected in 16 tubes

separately.

Most of PAHs compounds behaved similarly, most of the compounds were recovered before

the 5th milliliter. The recovery of naphthalene was found differently, but the recovery pattern

was similar to others. Four oxy-PAHs compounds were recovered. They performed similarly

comparing to the PAHs compounds, the recovery peak can be observed about the 6th

milliliter of eluent, except the DCM extraction of 7H-benzo[d,e]anthracene-7-on. The details

of the elution results of each PAHs compound and oxy-PAHs compound are shown in Figure

4-8 to Figure 4-27.

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Figure 4-8 Eluent Volume Test Result: Naphthalene Figure 4-9 Eluent Volume Test Result: Acenaphthylene

Figure 4-10 Eluent Volume Test Result: Acenaphthene Figure 4-11 Eluent Volume Test Result: Fluorene

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Naphthalene

Hexane

DCM

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Acenaphthylene

Hexane

DCM

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Acenaphthene

Hexane

DCM

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Fluorene

Hexane

DCM

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Figure 4-12 Eluent Volume Test Result: Phenanthrene Figure 4-13 Eluent Volume Test Result: Anthracene

Figure 4-14 Eluent Volume Test Result: Fluoranthene Figure 4-15 Eluent Volume Test Result: Pyrene

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Phenanthrene

Hexane

DCM

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Anthracene

Hexane

DCM

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Fluoranthene

Hexane

DCM

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Pyrene

Hexane

DCM

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Figure 4-16 Eluent Volume Test Result: Benz[a]anthracene Figure 4-17 Eluent Volume Test Result: Chrysene

Figure 4-18 Eluent Volume Test Result: Benzo[b]fluoranthene Figure 4-19 Eluent Volume Test Result: Benzo[k]fluoranthene

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Benz[a]anthracene

Hexane

DCM

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Chrysene

Hexane

DCM

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Benzo[b]fluoranthene

Hexane

DCM

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Benzo[k]fluoranthene

Hexane

DCM

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- 45 -

Figure 4-20 Eluent Volume Test Result: Benzo[a]pyrene Figure 4-21 Eluent Volume Test Result: Dibenz[a,h]anthracene

Figure 4-22 Eluent Volume Test Result: Benzo[g,h,i]perylene Figure 4-23 Eluent Volume Test Result: Indeno[1,2,3-cd]pyrene

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Benzo[a]pyrene

Hexane

DCM

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Dibenz[a,h]anthracene

Hexane

DCM

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Benzo[g,h,i]perylene

Hexane

DCM

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

% m

ass

in f

ract

ion

Fraction nr (+/- 1ml)

Indeno[1,2,3-c,d]pyrene

Hexane

DCM

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Figure 4-24 Eluent Volume Test Result: Benz[a]anthracene-7,12-dion Figure 4-25 Eluent Volume Test Result: Anthracene-9,10-dionen

Figure 4-26 Eluent Volume Test Result: Fluorene-9-one Figure 4-27 Eluent Volume Test Result: 7H-benzo[d,e]anthracene-7-on

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Benz[a]anthracene-7,12-dion

Hexane

DCM

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Anthracene-9,10-dione

Hexane

DCM

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fluorene-9-one

Hexane

DCM

0

2

4

6

8

10

12

14

16

18

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

7H-benzo[d,e]anthracene-7-on

Hexane

DCM

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The details of clean-up and concentration procedure applied in further experiments are

described step by step: 1) 16mL eluent were prepared for elution; 2) sample added into the

cartridge; 3) eluent was added into the cartridge following this program: 1mL, 1mL, 1mL,

1mL, 1mL, 2mL, 2mL, 2mL, 2mL, and 3mL; 4) the first and second milliliter of eluent was used

to wash the residual sticking on the wall of tube; 5) the 16mL eluent were collected in two

tubes, the first tube were replaced by the second tube right after the 9th milliliter eluent

added; 6) the first tube was concentrated by nitrogen gas flow under 30℃, 10psi; 7) the

solvent collected in the second tube was transferred into the first tube when more than half

of the solvent in the first tube had been evaporated; 8) the second tube was washed by 1mL

of dichloromethane and the washing liquor was transferred into the first tubes as well.

4.7 GC-MS Analysis

To brief, the concentrated extract was injected in splitless mode via a BEST programmed

temperature vaporizer (PTV) injector (Interscience, Louvain-la-Neuve, Belgium). One μl was

injected. Chromatographic separation was performed on a Restek Rxi-17Sil MS column (30 m;

0.25 mm ID; 0.25 μm; Interscience, Louvain-la-Neuve, Belgium). To protect the analytical

column, a 50 cm guard column was connected in front of the analytical column (deactivated,

0.25 mm ID). The GC (Trace GC Ultra) oven temperature was initially set at 70°C (2.5 min),

and then heated to 320°C at a heating rate of 10°C/min. The final temperature was held for

15 min. The MS-transfer line was heated to 240°C.

After separation, compounds were subjected to Electron Ionization (EI; 70eV). A

perfluorokerosene mix was continuously introduced (1 to 2 μl per 24h of analysis) into the

source via a heated (150°C) capillary leak. The mass fragments of the perfluorokerosene

were used as internal reference ions enabling accurate mass measurements. The mass

spectrometer was run in multiple ion detection (MID) mode with a mass resolution above 10

000 (10% valley definition).

4.8 Recovery and Matrix Effects

The following 5 experiments were designed to obtain the recovery and matrix effects of PAHs

and oxy-PAHs. Five experiments were conducted: 1) ‘Blank’, 2) ‘Standard tests’, 3) ‘Leaves’, 4)

‘Spiked before’ and 5) ‘Spiked after’. Each experiment was carried out with two solvents

dichloromethane and hexane. Each experiment was conducted in three fold. Therefore, 30

tests were carried out in total. Figure 4-28 is given to draw a clear image of the tests. The

details are described in following section.

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Figure 4-28 Flow Chart of the Recovery Determination

4.8.1 Experiment Design

The experiment was carried out without leaves. 9mL solvent and 1g anhydrous sodium

sulfate were added into syringe. The extraction, clean-up and analysis were performed next

as described in previous sections.

The experiment was carried out without leaves. 9mL solvent, 1g anhydrous sodium sulfate,

20μL standard-1 and internal standards (1μL F-PAHs) were added into the syringe. The

extraction, clean-up and analysis were performed next as described in previous sections. 1μL

deuterated-PAHs were added into the reconstituted vials before GC-MS analysis.

The experiment was carried out with leaves. The leaves were prepared as described in

previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate and internal standards

(1μL F-PAHs) were added into the syringe. The extraction, clean-up and analysis were

performed next as described in previous sections. 1μL deuterated-PAHs were added into the

reconstituted vials before GC-MS analysis.

The experiment was carried out with leaves. The leaves were prepared as described in

previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate, 20μL standard-1 and

internal standards (1μL F-PAHs) were added into the syringe. Standard-1 was dropped onto

the leaves first. The syringe was shaken to make the leaves and Standard-1 mix well. F-PAHs

were dropped onto the leaves when the Standard-1 and leaves were well mixed. The solvent

was added next. The extraction, clean-up and analysis were performed next as described in

previous sections. 1μL deuterated-PAHs were added into the reconstituted vials before

GC-MS analysis.

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The experiment was carried out with leaves. The leaves were prepared as described in

previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate and internal standards

(1μL F-PAHs) were added into the syringe. F-PAHs were dropped onto the leaves before the

solvent added. The extraction, clean-up and analysis were performed next as described in

previous sections. 20μl Standard-1 and 1μL deuterated-PAHs were added into the

reconstituted vials before GC-MS analysis.

4.8.2 Data Processing Method

PAHs and oxy-PAHs analysis were carried out by GC-MS. The mass of the compounds were

calculated as following procedure:

𝑹𝑷𝑨∗ =𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅𝒔 𝑷𝒆𝒂𝒌 𝑨𝒓𝒆𝒂

𝑰𝑺∗∗ 𝑷𝒆𝒂𝒌 𝑨𝒓𝒆𝒂 (𝟑)

*RPA: Relative Peak Area

**IS: internal standard

𝑹𝑺𝑹𝑭∗𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 = 𝑹𝑷𝑨 ×

𝑰𝑺 𝑴𝒂𝒔𝒔 (𝒏𝒈)

𝑴𝒂𝒔𝒔 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅(𝒏𝒈) (𝟒)

*RSRF: Relative Sample Response Factor

𝑴𝒂𝒔𝒔 𝒐𝒇 𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅 (𝒏𝒈) = 𝑹𝑷𝑨 ×𝑰𝑺 𝑴𝒂𝒔𝒔

𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒐𝒇 𝑹𝑺𝑹𝑭 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 (𝟓)

𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 =𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒃𝒆𝒇𝒐𝒓𝒆 −

𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔

𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒃𝒆𝒇𝒐𝒓𝒆

𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓 −𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔

𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓

× 𝟏𝟎𝟎 (𝟔)

𝑴𝒂𝒕𝒓𝒊𝒙 𝒆𝒇𝒇𝒆𝒄𝒕𝒔 =𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓 −

𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔

𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓

𝑹𝑷𝑨𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅

× 𝟏𝟎𝟎 (𝟕)

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𝑳𝒆𝒂𝒇 𝑴𝒂𝒔𝒔 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 =𝑴𝒂𝒔𝒔 𝒐𝒇 𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅

𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 × 𝑴𝒂𝒕𝒓𝒊𝒙 𝒅𝒊𝒔𝒕𝒖𝒓𝒃𝒂𝒏𝒄𝒆 (𝒏𝒈) (𝟖)

𝑫𝒓𝒚 𝑾𝒆𝒊𝒈𝒉𝒕 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =𝑳𝒆𝒂𝒇 𝑴𝒂𝒔𝒔 𝑪𝒐𝒏𝒕𝒆𝒏𝒕

𝑳𝒆𝒂𝒗𝒆𝒔 𝑫𝒓𝒚 𝑾𝒆𝒊𝒈𝒉𝒕 (𝒏𝒈/𝒈) (𝟗)

4.8.3 Results of Analysis

The recovery, matrix effects and compounds dry weight on leaves of dichloromethane

extraction are shown in Table 4-3 and Table 4-4.

Table 4-3 DCM extraction results: PAHs

Results

PAHs R

1 (%) SD

2 RSD

3 Matrix

4 SD RSD d.w.

5 (ng/g)

Naphthalene 24.7 0.22 90 0.98 0.39 40 1519

Acenaphthylene 66.0 0.05 8 1.23 0.03 2 3.4

Acenaphthene 73.6 0.07 9 1.06 0.06 5 8.7

Fluorene 66.2 0.08 12 1.20 0.02 1 13.8

Phenanthrene 63.6 0.06 10 1.22 0.01 1 60.6

Anthracene 72.1 0.07 10 1.17 0.06 5 NF

Fluoranthene 69.9 0.06 8 1.37 0.02 2 59.4

Pyrene 64.9 0.07 11 1.28 0.04 3 41.2

Benzo[a]anthracene 69.2 0.07 10 1.20 0.04 3 17.9

Chrysene 70.2 0.07 11 1.22 0.11 9 67.4

Benzo[b]fluoranthene 66.6 0.05 7 1.18 0.02 2 25.8

Benzo[k]fluoranthene 64.1 0.05 8 1.08 0.01 1 14.6

Benzo[a]pyrene 70.1 0.07 10 1.14 0.07 6 4.8

Dibenzo[a,h]anthracene 68.0 0.06 9 1.33 0.00 0 NF

Benzo[g,h,i]perylene 66.1 0.06 9 1.12 0.02 2 5.6

Indeno[1,2,3-c,d]pyrene 67.0 0.05 8 1.25 0.02 2 4.3

Sum 1847

Sum without naphthalene 327

1. R: Recovery 2. SD: Standard deviation

3. RSD: Relative standard deviation 4. Matrix: Matrix effects

5. d.w.: Dry weight concentration on leaf

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Table 4-4 DCM extraction results: oxy-PAHs

Results

oxy-PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)

Napthalene-1,4-dione 45.0 0.03 6 1.62 0.06 4 0.7

Fluorene-9-one 67.3 0.04 6 1.89 0.07 4 10.5

Anthracene-9,10-dione 66.5 0.06 9 2.22 0.11 5 5.6

1,8-napthalic anhydride NF NF NF NF NF NF NF

7H-benzo[d,e]anthracene-7-one 58.6 0.06 10 3.26 0.04 1 5.8

Benz[a]nthracene-7,12-dione 63.2 0.05 7 1.98 0.04 2 4.17

Napthacene-5,12-dione 13.7 0.01 11 2.44 0.02 1 NF

Sum 26.7

As for PAHs, the result of naphthalene was found higher than others, while the recovery was

found lower. The concentration of naphthalene was found 10-100 times higher than others

in blank experiments. Therefore, the sum results were shown in two ways, with naphthalene

and without. The recovery of PAHs compounds ranged between 63-74%. The matrix effects

coefficient ranged between 0.9-1.3, which means the influence given by solvents was not

significant. The sum concentration of PAHs in leaf samples was 327ng/g (d.w.). The

concentration of chrysene, phenanthrene and fluoranthene were found higher than others,

which were 67.4ng/g (d.w.), 60.4ng/g (d.w.) and 59.4ng/g (d.w.) respectively. Anthracene and

dibenzo[a,h]anthracene were not found in the sample leaf.

AS for oxy-PAHs, 1,8-napthalic anhydride was not found in most of the tests, except ‘spiked

after’. It was rarely found in the natural background, and it could be absorbed to the SPE

cartridges while cleaning up. Therefore, 1,8-napthalic anhydride is not discussed. The

recovery of oxy-PAHs ranged between 58-67%, except naphthalene-1,4-dione was 45% and

napthacene-5,12-dione was 14%. The matrix effects coefficient was ranged between 1.6-2.4,

except 7H-benzo[d,e]anthracene-7-one was 3.3. The concentration of oxy-PAHs in leaf

samples was 26.7ng/g (d.w.). Fluorene-9-one was found having the highest concentration

which was 10.5ng/g (d.w.). Napthacene-5,12-dione was not found in the sample leaf.

The recovery, matrix effects and compounds dry weight on leaves of hexane extraction are

shown in Table 4-5 and Table 4-6.

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Table 4-6 hexane extraction results: PAHs

Results

PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)

Naphthalene 33.2 0.30 92 0.99 0.10 11 925

Acenaphthylene 55.7 0.07 12 1.31 0.08 6 4.3

Acenaphthene 59.8 0.08 14 1.14 0.07 6 13.8

Fluorene 60.5 0.08 14 1.13 0.08 8 14.7

Phenanthrene 57.9 0.09 15 1.06 0.10 9 68.6

Anthracene 70.5 0.09 12 1.07 0.05 5 NF

Fluoranthene 59.2 0.09 15 1.50 0.13 8 42.3

Pyrene 57.3 0.08 14 1.39 0.10 7 26.8

Benzo[a]anthracene 60.7 0.06 10 1.05 0.06 6 9.3

Chrysene 56.9 0.08 14 1.08 0.10 9 37.6

Benzo[b]fluoranthene 58.6 0.08 13 1.10 0.06 5 12.6

Benzo[k]fluoranthene 60.8 0.09 14 0.98 0.04 4 9.1

Benzo[a]pyrene 65.2 0.09 14 0.99 0.06 6 3.9

Dibenzo[a,h]anthracene 57.7 0.07 12 1.28 0.06 5 NF

Benzo[g,h,i]perylene 57.9 0.07 12 1.05 0.06 6 3.5

Indeno[1,2,3-c,d]pyrene 57.7 0.07 13 1.20 0.07 5 2.5

Sum 1174

Sum without naphthalene 249

Table 4-5 hexane extraction results: oxy-PAHs

Results

PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)

Napthalene-1,4-dione 35.6 0.04 11 1.51 0.09 6 0.0

Fluorene-9-one 59.2 0.07 11 1.77 0.08 4 13.4

Anthracene-9,10-dione 66.1 0.07 10 2.04 0.08 4 5.3

1,8-napthalic anhydride NF NF NF NF NF NF NF

7H-benzo[d,e]anthracene-7-one 52.8 0.06 11 2.93 0.10 4 2.9

Benz[a]nthracene-7,12-dione 59.6 0.07 12 1.89 0.06 3 0.0

Napthacene-5,12-dione 4.8 0.01 11 2.33 0.13 6 NF

Sum 21.6

The higher d.w. concentration and lower recovery of naphthalene than others, and NF of

1,8-napthalic anhydride were found when extracted by hexane as well. Therefore, neither of

the two was discussed in the sum result. Anthracene, dibenzo[a,h]anthracene, and

napthacene-5,12-dione were not found when hexane was applied as the extraction solvent.

As for PAHs, the recovery ranged between 56-61%, except anthracene was about 71%. The

matrix effects coefficient ranged between 0.9-1.3, except fluoranthene was 1.5 and pyrene

was 1.39. The concentration of PAHs in leaf samples was 249ng/g (d.w.). Different to DCM

extraction, the concentration of phenanthrene was the highest which was 68.6ng/g (d.w.),

and was followed by fluoranthene (42.3ng/g (d.w.)) and chrysene (37.6ng/g (d.w.)).

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As for oxy-PAHs, the recovery of oxy-PAHs ranged between 53-67%, except

Napthalene-1,4-dione was 36% and napthacene-5,12-dione was 5%. The matrix effects

coefficient was ranged between 1.5-2.3, except 7H-benzo[d,e]anthracene-7-one which was

2.93. The concentration of oxy-PAHs in leaf samples was 21.6ng/g (d.w.). Fluorene-9-one was

found having the highest concentration which was 13.4ng/g (d.w.), while

benz[a]nthracene-7,12-dione and n apthalene-1,4-dione was not found.

Comparing the results between two solvents, higher recovery and dry weight concentration

were obtained using dichloromethane. Therefore dichloromethane was recommended to be

the extraction solvent for Taxus baccata.

4.9 Discussion

This thesis is designed to develop a method using Taxus baccata to bio-monitor PAHs and

oxy-PAHs in atmosphere. Existed results were found rarely about oxy-PAHs bio-monitoring

and Taxus baccata PAHs bio-monitoring. To reduce the size of sample leaves, fresh leaves

nitrogen freezing followed by pulverizing was applied because of the better extraction results.

Nitrogen freezing followed by pulverizing provides smaller particles and bigger specific

surface area than other methods. Therefore, more contact area between sample and

extraction solvent was given. Furthermore, freezing and pulverizing broke the lipid layer

covered on leaves and broke the cells to some extent, which made it possible to extract the

PAHs and oxy-PAHs stored in inner parts of the leaves.

Due to the good recovery, 16mL eluent was applied to clean up the extracted solution.

Compared to other PAHs compounds, different pattern of recovery was showed by

naphthalene. Naphthalene is a widely used household substance (Praharaj and Kongasseri,

2012) and chemical intermediate in industries of dye, o-phthalic anhydride, pesticide, etc.

(Gerd et al., 2003; Sava et al., 2014; Zhang and Zhao, 2008). Volatility is one of the important

properties of naphthalene and one of the major reasons that naphthalene can be found

more easily than other PAHs compounds in atmosphere. Another important reason of the

different recovery pattern can be deduced as the adsorption of the Florisil cartridge to

naphthalene, which needs to be studied further. Oxy-PAHs are the derivatives of PAHs, the

recovery and behavior in biological samples were not studied clearly in previous researches.

Further studies are required to explain the different recovery pattern between PAHs and

oxy-PAHs.

Based on visual test, recovery test and the PAHs/oxy-PAHs concentration extracted from

leaves, dichloromethane was chosen as the best extraction solvent for Taxus baccata.

Turbidity was observed reproducibly when the mixture of dichloromethane and hexane was

applied as extraction solvent. Some chemical reactions between the extraction solvent and

part(s) of the leaves may be accelerated by the mixture, which can be deduced as one of the

reasonable explanation. Further studies are required to figure out the exact reasons.

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Comparing to the previous studies, considering the traffic flow and industrial polluted

scenario of Ghent, the total PAHs results of this thesis are higher than the suburban area of

Campania (218ng/g (d.w.)) and Tuscany (172ng/g (d.w.)) (De Nicola et al., 2013), and also

higher than the suburban area of Portugal (mean 118ng/g (d.w.)), Spain (mean 108ng/g

(d.w.)) and Greece (105ng/g (d.w.)) (Ratola et al., 2012).

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CHAPTER 5 Conclusions & Prospect

5.1 Conclusions

Taxus baccata was chosen as the bio-monitoring species for PAHs and oxy-PAHs. The leaves

were taken in the city area of Ghent, Belgium. The methods were optimized in the extraction

step and clean up step. Liquid nitrogen freeze was proved better than other methods for

Taxus baccata. The ‘U’ shape conduit was developed for extraction and filtration. The 16mL

eluent program was innovated for SPE clean-up. Dichloromethane was selected as the most

suitable extraction solvent for Taxus baccata. The recovery of PAHs compounds ranged

63-74%, matrix disturbance ranged 0.9-1.3, and the sum of dry weight concentration was

327ng/g. Oxy-PAHs was first tested by bio-monitoring. The recovery of oxy-PAHs compounds

ranged 58-67%, matrix disturbance was ranged between 1.5-2.4, and the sum of dry weight

concentration was 26.7ng/g.

5.2 Prospect

As for pollutants information, NPAHs are another important and toxic group of PAHs

derivatives. The existence and concentration are recommended to be studied in other

researches. As for extraction efficiency, the temperature, frequency and extraction time of

ultra-sonication bath are expected to be studied further. As for clean-up, automatic

operation is expecting to be applied. At last, the difference between the results of

bio-monitors and conventional chemical methods in the same regions are expected to be

compared.

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