Biomonitoring of polycyclic aromatic hydrocarbons and derivatives in Belgium

67 Pages·2015·2.24 MB·English

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
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
I
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.

II
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
Pressurized Liquid Extraction (Acceleration
PLE(ASE)
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

III
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.

IV
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 -

V
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 (SO ) and nitric oxide (NO) etc. Secondary pollutants are the ones produced by
2
the chemical or photochemical reactions of primary pollutants, for instance, ozone (O ),
3
H SO , aerosol, etc. Secondly, by physical property, air pollutants can also be divided into two
2 4
groups, gaseous pollutants and particulates. A large proportion of air pollution is caused by
gaseous pollutants, for instance, SO , NO , chlorofluorocarbon (CFCs), etc. Thirdly, by
x X
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, O , NO , SO, CO ,
3 x x x
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;
- 1 -
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
Year Subject Region Inducement Details
1930 The 1930 Belgium, Temperature The first public nuisance recorded in the
Meuse Valley Industrial Inversion 20th century. It was induced by the
Fog temperature inversion. The valley was full
of heavy industries. 60 people were killed
by the trapped gaseous emission in one
week.
1943 Los Angeles U.S., City Heavy Traffic The hydrocarbon, emitted by petrol
Photochemical burning, catalyzed by ultraviolet
Smog Episode 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, Temperature The city of Donora was well-known by lots
Donora Smog Pennsylvania, Inversion of large-scale iron works, smelters and
U.S., Industrial 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 Britain, City Coal Burning 12 severe smog incidents were recorded
Smogs 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 Bhopal, India, Explosion The city of Bhopal was attacked by 45
Disaster City 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.
PAHs is the name of a large group constituted of more than 100 organic compounds having
- 2 -
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.
- 3 -
Table 1-2 the Properties and Structure of the 16 EPA PAHs
PAHs Formula Boiling Point/℃ Melting Point/℃ Structure IARC Classification
Naphthalene C H 217.9 80.5 n.e.*
10 8

Acenaphthylene C H 280 93 n.e.
12 8

Acenaphthene C12H10 279 95 CH2 CH2 n.e.

Fluorene C13H10 295 116 CH2 3**

Phenanthrene C H 340 100.5 3
14 10

Anthracene C H 342 216.4 3
14 10

Fluoranthene C H 375 108.8 3
16 10

Pyrene C H 150.4 393 3
16 10

Benzo[a]anthracene C H 400 160.7 2A***
18 12

Chrysene C H 448 253.8 NA
18 12

Benzo[b]fluoranthene C H 481 168.3 2B****
20 12

Benzo[k]fluoranthene C H 480 215.7 2B
20 12

Benzo[a]pyrene C H 496 178.1 2A
20 12

Dibenzo[a,h]anthracene C H 524 266.6 2B
24 14

Benzo[g,h,i]perylene C H NA 277 3
22 12

Indeno[1,2,3-c,d]pyrene C H 536 163.6 2A
22 12

* n.e.: not evaluated
** 3: not classifiable
*** 2A: probably carcinogenic
**** 2B: possibly carcinogenic
- 4 -