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UPCONCENTRATION OF REALISTIC
ENVIRONMENTAL CONTAMINANT
MIXTURES WITH SILICONE RUBBER
PASSIVE SAMPLERS PROOF OF PRINCIPLE WITH A MIXTURE OF POLYCYCLIC
AROMATIC HYDROCARBONS
Number of words: 29269
Jarno Van de Velde Official code: 01301771
Promotor: Prof. Dr. Karel De Schamphelaere
Master thesis submitted to achieve the master’s degree Master of Science in Industrial Sciences:
Biochemistry
Academic year: 2016 - 2017
UPCONCENTRATION OF REALISTIC
ENVIRONMENTAL CONTAMINANT
MIXTURES WITH SILICONE RUBBER
PASSIVE SAMPLERS PROOF OF PRINCIPLE WITH A MIXTURE OF POLYCYCLIC
AROMATIC HYDROCARBONS
Number of words: 29269
Jarno Van de Velde Official code: 01301771
Promotor: Prof. Dr. Karel De Schamphelaere
Master thesis submitted to achieve the master’s degree Master of Science in Industrial Sciences:
Biochemistry
Academic year: 2016 - 2017
The author and the promoter give the permission to use this thesis for consultation and to copy
parts of it for personal use. Every other use is subject to the copyright laws, more specifically the
source must be extensively specified when using the results from this thesis.
May 28, 2017
Preface After an intensive period of nine months, I’m writing this preface as finishing touch on my thesis.
During these nine months, I had the opportunity to expand my knowledge not only in the scientific
area, but also on a personal level. It was definitely not an easy process, but I completed this thesis
with a positive feeling.
In the inter-semestrial week, I tore the ligaments in my shoulder in a ski accident. This complicated
the practical work, but with some help of the laboratory staff and my tutor, I managed to continue
working in the laboratory without too many complications.
At the start of my thesis, I personally found that my English speaking and writing skills were not of
a sufficiently high level. Nevertheless, I decided to write this thesis in English and I followed the
elective subject ‘Scientific English’ to address this need. This course together with the reading of
English literature, communicating and writing in English definitely helped me to improve my English
language proficiency in a scientific/academic context.
I would like to thank the people who have supported and helped me throughout this period, in the
first place Prof. Dr. Karel De Schamphelaere. I want to thank him for the opportunity he gave me
to perform my thesis at GhenToxLab. Secondly, I want to thank my tutor Samuel Moeris for
introducing me in GhenToxLab, for the daily guidance and for the time and effort he put in explaining
new procedures and techniques. I furthermore want to thank him for proofreading my text and for
the many tips and tricks he gave me. I also wish to thank Nancy De Saeyer for her help with the
chemical analyses and Foppe Smedes for his help in the partitioning calculations. Finally, I would
like to thank all members of GhenToxLab for creating an enjoyable working atmosphere in the
laboratory.
Jarno Van de Velde
Ghent, May 28, 2017
Abstract The combination of passive sampling and dosing has recently been described as a promising
alternative in aquatic toxicity testing. However, in the context of mixture risk assessment it is
required to test concentration series for determination of effect values such as EC50 or NOEC,
which cannot be achieved by simple usage of passive samplers as passive dosing devices. This
thesis responds to this need by investigating the possibility of upconcentrating contaminant
mixtures on silicone rubber passive samplers.
The theoretical upconcentration factor 10 was not fully achieved for any of the five mixture
concentrations. However, results showed a good potential for upconcentrating contaminant
mixtures. There was a trend of increasing upconcentration with decreasing sum PAH mixture
concentration. The upconcentration factors for the five tested concentration treatments were 5.9,
4.3, 3.7, 2.6 and 1.2 from the lowest to the highest concentration treatment, respectively.
Compound losses could be mostly explained by volatilization and capacity limits by showing a clear
correlation between volatility and compound loss.
Further, it was tested if the biological response exerted by the compounds on the samplers was
not influenced by the whole upconcentration procedure. A 72 h growth inhibition experiment in
which the marine diatom Phaeodactylum tricornutum was exposed to the non-upconcentrated
samplers and upconcentrated samplers resulted in a dose-response relationship with EC50 values
of 131.8 µg/L and 109.6 µg/L, respectively. Repetition of the growth inhibition experiment resulted
in slightly lower EC50 values of 93.3 µg/L and 74.1 µg/L, respectively.
Keywords Polycyclic aromatic hydrocarbons
Silicone rubber passive samplers
Mixture upconcentration
Passive dosing
1
Table of contents 1. Literature study .................................................................................................................... 13
1.1. Pollutants in the marine environment ............................................................................ 13
1.2. Polycyclic Aromatic Hydrocarbons (PAHs) ................................................................... 14
1.2.1. Accumulation in the environment ........................................................................... 14
1.2.2. Physicochemical properties ................................................................................... 15
1.2.3. Environmental concern .......................................................................................... 17
1.2.4. Monitoring of PAHs in the marine environment ...................................................... 18
1.2. Passive sampling .......................................................................................................... 21
1.2.1. Historical background ............................................................................................ 21
1.2.2. Principles of equilibrium passive sampling ............................................................. 21
1.2.3. Silicone rubber passive samplers .......................................................................... 22
1.3. Passive dosing ............................................................................................................. 23
1.3.1. Theoretical background ......................................................................................... 23
1.3.2. Passive dosing and risk assessment ..................................................................... 23
1.4. Gas chromatography-mass spectrometry ..................................................................... 24
1.4.1. Introduction ............................................................................................................ 24
1.4.2. Operating principle................................................................................................. 24
1.4.3. Quantification of PAHs by GC-MS ......................................................................... 25
1.5. Research goals ............................................................................................................. 26
2. Materials and methods ......................................................................................................... 28
2.1. The upconcentration experiment ...................................................................................... 28
2.1.1. Theoretical background ......................................................................................... 28
2.1.2. Choice of PAHs and concentration series ............................................................. 29
2.1.3. Partitioning calculations ........................................................................................ 29
2.1.4. Five concentration treatments ............................................................................... 31
2.1.5. Precleaning using Soxhlet extraction .................................................................... 32
2.1.6. Spiking of the bigger samplers .............................................................................. 32
2.1.7. Extraction and concentration of the spiked samplers ............................................. 33
2.1.8. Spiking of the smaller samplers............................................................................. 34
2.2. Biotesting ....................................................................................................................... 34
2.2.1. Theoretical background ......................................................................................... 34
2.2.2. Growth medium ..................................................................................................... 35
2
2.2.3. Algae culture and cell count .................................................................................. 36
2.2.4. Calculation of the growth inhibition ........................................................................ 36
2.3. Chemical analysis ............................................................................................................ 37
2.3.1. Analysis of the sampler extracts ........................................................................... 37
2.3.2. Analysis of the PAH concentration in the water phase after the growth inhibition
experiment ........................................................................................................................... 38
3. Results ................................................................................................................................ 39
3.1. The upconcentration experiment ................................................................................... 39
3.1.1. PAH concentration in stock solution ....................................................................... 39
3.1.2. PAH concentration on non-upconcentrated and upconcentrated samplers ............ 40
3.1.3. The upconcentration .............................................................................................. 42
3.1.4. Individual PAH concentration for each CT ............................................................. 43
3.2. Biotesting ...................................................................................................................... 46
3.2.1. GC-MS analysis of the water phase after growth inhibition .................................... 46
3.2.2. Calculation of the growth inhibition ........................................................................ 47
3.2.3. Growth inhibition curve .......................................................................................... 49
3.2.4. Results growth inhibition experiment 2................................................................... 49
4. Discussion ........................................................................................................................... 52
4.1. The upconcentration experiment ................................................................................... 52
4.1.1. PAH concentration in stock solution ....................................................................... 52
4.1.2. PAH concentration on non-upconcentrated and upconcentrated samplers ............ 52
4.1.3. Upconcentration factor 10 ...................................................................................... 53
4.1.4. Recovery ƩC 5 PAHs on samplers ........................................................................ 54
4.1.5. PAH recovery for each mixture component ............................................................ 55
4.1.6. PAH recovery as a function of log KOW ................................................................... 57
4.1.7. PAH recovery as function of volatility ..................................................................... 58
4.2. Biotesting ...................................................................................................................... 58
4.2.1. Validity of the growth inhibition experiments .......................................................... 58
4.2.2. PAH concentration in the water phase ................................................................... 59
4.2.3. Growth inhibition experiment 1 .............................................................................. 59
4.2.4. Growth inhibition experiment 2 .............................................................................. 60
5. Conclusion and future perspectives ..................................................................................... 61
References ................................................................................................................................. 63
Supporting information ................................................................................................................ 67
3
Attachment 1: PAH concentration on samplers before and after upconcentration ................... 67
Attachment 2: Processing results GC-MS for the non-upconcentrated samplers ..................... 69
Attachment 3: Processing results GC-MS for the upconcentrated samplers ............................ 81
Attachment 4: Processing results GC-MS for aqueous concentrations non-upconentated
samplers ................................................................................................................................. 92
Attachment 5: Processing results GC-MS for aqueous concentrations upconentated samplers
................................................................................................................................................ 96
Attachment 6: Data growth inhibition experiment 1 ................................................................ 101
Attachment 7: Data growth inhibition experiment 2 ................................................................ 103
Attachment 8: Statistical analysis of the PAH recovery on non-upconcentrated samplers ..... 105
Attachment 9: Statistical analysis of the PAH recovery on upconcentrated samplers ............ 107
4
List of abbreviations BELSPO Belgian Scientific Research Program
BRAIN-BE Belgian Research Action through Interdisciplinary Networks
EC European Commission
EC50 half maximal effective concentration
ERMCs environmentally realistic contaminant mixtures
GC-MS gas chromatography-mass spectrometry
GES good environmental status
HCHs hexachlorocyclohexanes
HOCs hydrophobic organic contaminants
IS internal standard
LDPE low-density polyethylene
LLE liquid liquid extraction
MSFD Marine Strategy Framework Directive
NewSTHEPS New Strategies for monitoring and risk assessment of Hazardous chemicals
in the marine Environment with Passive Samplers
NOEC no observed effect concentration
PAHs polycyclic aromatic hydrocarbons
PBDEs polybrominated diphenyl ethers
PCBs polychlorinated biphenyls
PDMS polydimethylsiloxane
POCIS polar organic chemical integrative sampler
PSDs passive sampling devices
rpm rotations per minute
SR recovery standard
SPMDs semipermeable membrane devices
SR silicone rubber
US EPA United States Environmental Protection Agency
WFD Water Framework Directive
5
List of figures
Figure 1: Input sources of pollutants found in the marine environment (Potters, 2013).
Figure 2: Passive sampling devices operate in two main regimes (kinetic and equilibrium regime) and
can be divided into three stages (linear, intermediate and equilibrium phase) (Vrana et al., 2005).
Figure 3: Boxplots for individual pesticides taken up by five different passive samplers: silicone
rubber (SR) (n = 86), polar organic chemical integrative sampler (POCIS-A) (n = 106), POCIS-B (n =
110), Chemcatcher® SDB-RPS (n = 65) and Chemcatcher® C18 (n = 54) in correlation to their octanol-
water partition coefficient (KOW) (Ahrens et al., 2015).
Figure 4: Schematic representation of the GC-MS setup (Crasto, 2014).
Figure 5: Schematic overview of the experimental setup followed for each of the 5 concentration
treatments (CT 1 – CT 5) and blanks to upconcentrate PAHs on AltecAlteSilTM silicone rubber passive
samplers.
Figure 6: Soxhlet extraction for precleaning.
Figure 7: Roller bank to spike the samplers.
Figure 8: Schematic overview of the experimental setup for each of the five concentration treatments
and blanks to test hypothesis 2.
Figure 9: ƩC 5 PAHs on non-upconcentrated big samplers (left) and upconcentrated small samplers
(right) for each CT compared to the theoretical expected concentration.
Figure 10: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 1 (above) and CT 5 (below).
Figure 11: ƩC 5 PAHs in the water phase after passive dosing from non-upconcentrated and
upconcentrated samplers.
Figure 12: Growth inhibition curve for P. tricornutum after 72 hours exposure to non-upconcentrated
and upconcentrated samplers.
Figure 13: Growth inhibition curve for P. tricornutum after 72 hours exposure to non-upconcentrated
and upconcentrated samplers.
Figure 14: Comparison growth inhibition curves of experiment 1 and 2 for P. tricornutum after 72
hours exposure to non-upconcentrated samplers (left) and upconcentrated samplers (right).
Figure 15: Upconcentration factor between non-upconcentrated and upconcentrated samplers
plotted for each concentration treatment.
6
Figure 16: PAH recovery on the non-upconcentrated samplers (left) and on the upconcentrated
samplers (right).
Figure 17: Recoveries of the five PAHs on the upconcentrated samplers.
Figure 18: Recovery of each of the PAHs in terms of log Kow before and after upconcentration for CT
1 and CT 5.
Figure 19: Recovery of each of the PAHs in terms of log vapor pressure at 25°C before and after
upconcentration for CT 1.
Figure S1 – part 1: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 2.
Figure S1 – part 2: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 3.
Figure S1 – part 3: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 4.
7
List of tables
Table 1: Physicochemical properties of five PAHs.
Table 2: Comparison between active grab sampling and passive sampling.
Table 3: Characteristic mass-to-charge ratio and retention time for five PAHs and their deuterated
analogues (EMIS, 2016).
Table 4: Average concentration of the five most common PAHs measured in the harbor of Zeebrugge
between March 4, 2015 and December 3, 2015.
Table 5: Desired exposure concentration for each PAH for CT 1 (1 µg/L).
Table 6: Loading conditions used for calculation of the concentrations on spiked sampler (Cp0).
Table 7: Exposure conditions used for calculation of the water concentrations in exposure (Cwe).
Table 8: Theoretical mass of each PAH required to reach the desired exposure concentrations.
Table 9: Concentration of the five PAHs in the five concentration treatments.
Table 10: Volumes of water added every 24 hours for spiking of the 1.0 g samplers.
Table 11: Composition synthetic sea water (ISO, 2006).
Table 12: Nutrient stock solutions (ISO, 2006).
Table 13: Composition internal standards (IS) and recovery standard (RS) for GC-MS analysis of the
sampler extracts.
Table 14: Dilution factor of each extract for GC-MS analysis.
Table 15: Dilution factor of each concentration treatment.
Table 16: Comparison between the nominal and actual PAH concentration in the stock solution with
the corresponding ratio between nominal and actual PAH concentration (efficiency).
Table 17: Comparison between the nominal and actual PAH concentration of the spiking solution for
each CT.
Table 18: Calculated Ʃ5 PAHs on samplers based on results GC-MS.
Table 19: Comparison of the total sum concentration on the big and small samplers.
Table 20: Concentration of the individual PAHs on the non-upconcentrated samplers (µg/g).
8
Table 21: Concentration of the individual PAHs on the upconcentrated samplers (µg/g).
Table 22: Comparison of the ƩC 5 PAHs in the water phase after growth inhibition experiment 1.
Table 23: Growth rate µ in growth inhibition experiment 1 with non-upconcentrated and
upconcentrated samplers.
Table 24: Growth inhibition Iµ in experiment 1 with non-upconcentrated and upconcentrated
samplers.
Table 25: Summary of the validity criteria for growth inhibition experiment 1 and 2.
Table 26: Growth rate µ in growth inhibition experiment 2 with non-upconcentrated and
upconcentrated samplers.
Table 27: Growth inhibition Iµ in experiment 2 with non-upconcentrated and upconcentrated
samplers.
Table S2 – part 1: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
Table S2 – part 2: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
Table S2 – part 3: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
Table S2 – part 4: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
Table S3 – part 1: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
Table S3 – part 2: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
Table S3 – part 3: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
Table S3 – part 4: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
Table S4 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with non-
upconcentrated samplers.
9
Table S4 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with non-
upconcentrated samplers.
Table S5 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with
upconcentrated samplers.
Table S5 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with
upconcentrated samplers.
Table S6 – part 1: Cell count growth inhibition experiment 1 with non-upconcentrated samplers.
Table S6 – part 2: Cell count growth inhibition experiment 1 with upconcentrated samplers.
Table S7 – part 1: Cell count growth inhibition experiment 2 with non-upconcentrated samplers.
Table S7 – part 2: Cell count growth inhibition experiment 2 with upconcentrated samplers.
10
Introduction The marine environment is a complex and dynamic system that is exposed to a wide range of
pollutants, including oils, plastics, chemicals and toxic compounds (Monteyne et al., 2013). Oceans
not only provide food resources for millions of people, but also play a major role in removing
atmospheric carbon dioxide and in providing atmospheric oxygen (Worm et al., 2006).
In order to protect these important ecosystems, international agreements such as the Water
Framework Directive (WFD, 2000/60/EC) and the Marine Strategy Framework Directive (MSFD,
2008/58/EC) were introduced by the European Union (Monteyne et al., 2013). These agreements
impose amongst others maximum concentration levels for organic priority chemicals such as
polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT),
hexachlorocyclohexanes (HCHs), phenol, polybrominated diphenyl ethers (PBDEs) and
polyaromatic hydrocarbons (PAHs) (Potters, 2013).
The most crucial step in assessing the concentration and distribution of organic compounds in the
marine environment is the implementation of accurate and reliable methods to sample the
pollutants in their complex matrices (Pintado-Herrera et al., 2016). Since the concentrations of most
priority chemicals are very low in the water phase (often below ng/L), it is a very challenging task
for the environmental scientist to get a clear image of the occurrence of the different chemicals in
specific areas. Moreover, concentration levels within the same area will also fluctuate in terms of
time due to temporary variable emissions such as currents, tides, river discharges and harbor
influences (Monteyne et al., 2013).
Marine sampling methods for organic contaminants include biomonitoring, active grab sampling
and passive sampling (Raub et al., 2015). Each method is characterized by a number of
advantages and disadvantages, but passive sampling is considered the most promising technique
(Raub et al., 2015; Monteyne et al., 2013; Ahrens et al., 2015). In combination with passive dosing,
realistic environmental conditions can be re-established in the laboratory without depletion of the
chemicals of interest. This is often required in ecotoxicity tests to evaluate the toxic effect of
chemicals on marine key organisms (Jahnke et al., 2016).
In time-integrative passive samplers, time-weighted average concentrations are integrated over the
period of deployment, while equilibrium passive samplers are based on the equilibration of organic
compounds in a receiving phase by a diffusion driven process (Górecki & Namieśnik, 2002). Such
passive samplers can be exposed in the field and used to recreate environmental concentrations
afterwards (passive dosing), without complicated pretreatments or depletion of the water phase or
sampler (Pintado-Herrera et al., 2016).
One disadvantage associated with passive dosing is that only one mixture concentration can be
tested. In certain ecotoxicity tests such as growth inhibition tests, it is required to have different test
concentrations to determine dose-response relationships. In case of field samples, there is only the
environmental concentration that can be re-established by passive dosing. To reach higher
equilibrium concentrations in the water phase, higher concentrations on the passive samplers need
11
to be reached since the sampler-water partitioning coefficient (KSW) is a constant for a given
compound and type of passive sampler. Furthermore, environmental risk assessment (ERA) works
with half maximal effective concentration (EC50) and no observed effect concentrations (NOECs),
which also require different concentration levels. However, no studies were found that deal with the
possibility to upconcentrate passive sampler.
This thesis responds to this need by investigating the possibility of upconcentrating environmentally
realistic contaminant mixtures (ERCMs) with passive samplers. This implies that the same amount
of components on a smaller sampler should theoretically give higher concentrations in the water
phase after dosing due to the constant sampler-water partitioning coefficient and higher
concentration on the sampler (KSW = Csampler/Cwater).
The second part of this thesis deals with the effect assessment of the upconcentrated smaller
samplers in comparison to the non-upconcentrated bigger samplers. The goal is to verify that the
ecotoxicological response is not influenced by the whole procedure of upconcentration. This is
done by a 72 hour growth inhibition experiment with the marine diatom Phaeodactylum tricornutum.
Altec AltsilTM silicone rubber samplers have been widely used as passive samplers for monitoring
studies and are suited to test the hypotheses (Monteyne et al., 2013; Claessens et al., 2015;
Jahnke et al., 2016; Ahrens et al., 2015). PAHs are considered model substances for silicone
rubber samplers and have been very well investigated in passive sampling and dosing (Rusina et
al., 2009; Monteyne et al., 2013; Claessens et al., 2015; Smith et al., 2013).
An important note is that this thesis deals with mixtures of PAHs rather than PAHs as single
substances. Many studies already investigated the effect of a single compound on marine species,
however only limited information concerning the effect of chemical mixtures on marine species is
available (Smith et al., 2013).
This thesis is framed within the BELSPO funded project called NewSTHEPS (New Strategies for
monitoring and risk assessment of Hazardous chemicals in the marine Environment by Passive
Samplers). The NewSTHEPS Project develops new approaches and techniques for monitoring
contaminants in the marine environment. One focus of the project is the use and applicability of
passive samplers. Its overall idea is to tackle scientific and methodological problems associated
with the implementation of the Good Environmental Status (GES) of the Marine Strategy
Framework Directive.
The GES is defined as the environmental status of marine waters where these provide ecologically
diverse and dynamic oceans and seas which are clean, healthy and productive (European
Commission, 2016a). It intends to assure that contamination levels do not give rise to pollution
effects in the marine environment (Flanders Marine Institute, 2016). It is a requirement for all
members of the European Union (Borja et al., 2013). The overall objective is to protect the marine
environment across Europe more efficiently (European Commission, 2016b). The project fits into
BRAIN-be, a framework that supports the scientific potential of Federal Scientific Institutions
(Science Policy PPS, 2016).
12
This thesis starts with a literature study. Hereby the properties and monitoring of PAHs are
discussed thoroughly and the connection is made with passive sampling and passive dosing. A
brief theoretical background of passive sampling and dosing is also included and the connection
with ecotoxicity testing is made. To finalize the literature study, a brief review of the gas
chromatography-mass spectrometry as an analytical tool for PAHs is included. The literature study
is followed by a description of the materials and methods used in this research, the results, a
discussion and a general conclusion.
13
1. Literature study
1.1. Pollutants in the marine environment Oceans and seas play a crucial role in maintaining a sustainable and livable planet. They cover
approximately 70% of the earth’s surface and over 90% of the planets living biomass can be found
in these ecosystems (National Geographic, 2017). Their enormous potential to store heat and
interact with gases in the atmosphere plays a key role in controlling the global temperature and the
distribution of pollutants. In this way oceans and seas have a direct influence on the earth’s weather
and long-term climate changes.
Despite the undeniable importance of the marine environment, oceans and seas have to deal with
a wide range of pollutants. There are thousands of toxic chemicals that are proven to cause harm
to different marine ecosystems and organisms (Kueh & Lam, 2008). The majority (44%) of these
pollutants originate form land-based activities such as domestic and industrial wastewater and
surface runoff (Kueh & Lam, 2008; Potters, 2013). Also airborne emissions from the land are a
major (33%) source of marine pollution. Remarkable is that only 12% of all pollution is due to
maritime activities and shipping accidents (Potters, 2013). The other fraction of the pollution
originates from the dumping of garbage and polluted water (10%) and offshore drilling and mining
(1%) (Figure 1) (Potters, 2013). In addition, there are economic activities, such as harbors and boat
traffic routes that contribute to the occurrence and complexity of the pollution of aquatic ecosystems
(Monteyne et al., 2013).
Figure 1: Input sources of pollutants found in the marine environment (Potters, 2013).
Pollution can be defined as “any form of contamination in an ecosystem with a harmful impact upon
the organisms in this ecosystem, by changing the growth rate and the reproduction of plant or
animal species, or by interfering with human amenities, comfort, health, or property values” or more
simplified “environmental contamination with man-made waste” (Kueh & Lam, 2008). Different
classes of pollutants tend to have different operating principles. Pollutants can be classified based
on their physicochemical constitution (organic – inorganic), physical state (solids – gasses –
14
solutes) or persistence (biodegradable – persistent) (Potters, 2013). Besides these classifications,
one can also consider compounds based on their ecotoxicity.
1.2. Polycyclic Aromatic Hydrocarbons (PAHs)
1.2.1. Accumulation in the environment PAHs are a class of nonpolar organic molecules composed of multiple benzene rings. They often
contain other additional rings, such as five-sided rings, but are exclusively composed of hydrogen
and carbon (Ravindra et al., 2008). Different configurations and numbers of rings result in different
properties (Agency for Toxic Substances and Disease Registry, 2012). PAHs are white/yellow
solids with relatively high molecular weights and low volatility at room temperature (Agency for
Toxic Substances and Disease Registry, 2012) (Table 1). Due to the low hydrogen-to-carbon ratio,
PAHs are the most stable form of hydrocarbons and usually occur in complex mixtures rather than
as single compounds (Ravindra et al., 2008). Most of them can be photo-oxidized and degraded to
simpler substances and because of their composition PAHs are very apolar and consequently
poorly soluble in water (Monteyne et al., 2013).
PAHs originate from natural as well as anthropogenic sources and are omnipresent in the
environment (Haritash & Kaushik, 2009). Natural sources include forest fires, volcanic eruptions
and exudates from trees and most fossil fuels (Haritash & Kaushik, 2009; Agency for Toxic
Substances and Disease Registry, 2012). Anthropogenic sources include domestic emissions (e.g.
burning of coal, gas and garbage), emission linked to transportation (e.g. aircrafts, ships, trains,
cars and machinery) and industrial emissions (e.g. aluminum and coke production) (Ravindra et
al., 2008). All above mentioned processes are characterized by incomplete combustion with oxygen
deficient conditions, high-pressure processes or high temperatures that lead to the formation of
PAHs from saturated hydrocarbons (Agency for Toxic Substances and Disease Registry, 2012;
Ravindra et al., 2008).
Most PAHs are emitted directly into the atmosphere (Ravindra et al., 2008). PAHs with less than
four rings tend to remain in the atmosphere until precipitation, while PAHs with more than four rings
will mostly absorb on fine particles (Skupinska et al., 2004). Because of the ability to be transported
in the gaseous phase or as particulate phase in aerosols, PAHs can travel long distances resulting
in a worldwide distribution with significant affection of coastal and surface waters (Manoli & Samara,
1999).
Atmospheric precipitation of PAHs in the Mediterranean Sea only has been estimated on 35 to 70
tons/year (Lipiatou et al., 1997). Besides atmospheric inputs, also urban run-off, industrial effluents
and oil spillage and leakage are important PAH contributors for natural waters (Manoli & Samara,
1999). PAH pollution by urban run-off mainly consist of rainwater that came into contact with roads,
motorways, roofs, parking lots etc. Also rainwater itself is known to contain organic compounds
including PAHs (Manoli & Samara, 1999). Remarkable is that 89% of the emitted PAHs are found
in soils, 10% in sediments and only 0.5% in air and water (Skupinska et al., 2004).
15
A study of Wild and Jones (1995) mapped the production, storage, cycle and losses of PAHs in the
United Kingdom. In total 12 different compounds are included in the study and their total annual
emission was estimated around 1000 tons (Wild & Jones, 1995). 95% of this emission originates
from domestic coal combustion, vehicle emissions and unregulated fires, indicating the importance
of households regarding PAH emission (Wild & Jones, 1995).
A major part of the deposed PAHs will accumulate in hydrophobic sediments and organic materials,
including adipose tissues in aquatic organisms. PAHs accumulated in organisms can be transferred
along the food chain. However, a study of Broman et al. (1990) observed a decrease in PAH
concentration with increasing trophic levels. This apparent discrepancy can be explained by the
biotransformation of PAHs to intermediate metabolites. These metabolites often have a mutagenic
and carcinogenic potential, leading to a potential ecotoxicological risks for organisms of higher
trophic levels (Broman et al., 1990).
Degradation processes of PAHs in the marine environment are characterized by a low efficiency
and can be subdivided in biotic and abiotic degradation (Manoli & Samara, 1999). On the one hand,
biotic degradation includes the conversion of accumulated PAHs in marine organisms to potentially
toxic and carcinogenic metabolites. Also bacterial breakdown of aquatic PAHs can be considered
as biotic degradation. For example the three-ring PAH fluorene can be used as carbon source by
Arthrobacter sp., Brevibacterium sp., Mycobacterium sp. and Pseudomonas sp. (Seo et al., 2009).
However, the major part of these micro-organisms does not or only in very limited numbers occur
in the marine environment. On the other hand, PAHs can also undergo abiotic degradation
processes such as chemical degradation, photolysis, thermolysis and oxidation, but these effects
are mainly observed in the atmosphere rather than in aqueous systems because of lower
temperature and light intensity (Manoli & Samara, 1999).
1.2.2. Physicochemical properties PAHs are generally characterized by relatively high molecular weights, high melting and boiling
points, low volatility and poor solubility in aqueous solutions. They dissolve readily in organic and
lipophilic solvents (Skupinska et al., 2004). Despite the low volatility, the atmosphere provides a
major input of PAHs in aquatic ecosystems, especially by atmospheric precipitation. PAHs with the
same molecular mass and same amount of rings, but a different configuration may lead to
differences in compound properties (Skupinska et al., 2004). Also PAHs with substituted functional
groups such as -OH, -NO2, =O and -CH3 can be encountered in the environment (Skupinska et al.,
2004).
When PAHs are adsorbed to the surface of dust or other atmospheric particles, they are more
thermo- and photosensitive (Skupinska et al., 2004). Thermal degradation occurs at temperatures
of 50 °C and higher, while photo-oxidation is one of the major PAH removing processes in the
atmosphere. The latter particularly occurs under the influence of ultraviolet light and to a lower
extent also by exposure to visible light (Skupinska et al., 2004). Zakrzewski (1991) determined that
16
dust irradiation by ultraviolet light of about six hours resulted in 15-20% decomposition of adsorbed
PAHs.
Aqueous concentrations are rather low due to the low water solubility and high hydrophobicity. As
a result, PAHs tend to accumulate in less polar sediments and aquatic organisms to avoid the polar
water phase (Agency for Toxic Substances and Disease Registry, 2012).
The polarity of PAHs is usually expressed by the octanol water partitioning coefficient (KOW). This
coefficient is defined as the ratio of the PAH concentration in octanol vs. the concentration of the
PAH in the aqueous phase in an octanol/water system (Yamamoto, 2011). Log KOW has typical
values within the range of -3 to 7 for organic compounds (Yamamoto, 2011). The log KOW has
become a key parameter in assessing the environmental uptake of organic chemicals by aquatic
organisms, mainly because of its relation with water solubility, soil/sediment adsorption and bio-
accumulation (Yamamoto, 2011).
Most PAHs have log KOW values higher than 4 and are considered very hydrophobic, while organic
compounds with a log KOW lower than 3 are considered as rather hydrophilic (Yamamoto, 2011).
Once the distribution ratio of PAHs to octanol and water is known, the bio-accumulation in
(hydrophobic) organisms in aqueous environments can be estimated (Yamamoto, 2011). Table 1
shows that log KOW seems to increase as the molecular mass of the PAHs increases. This is similar
to the findings of Johnsen et al (2005), who demonstrated that as the molecular mass of the PAH
increases, the aqueous solubility decreases approximately logarithmically. Moreover, Skupinska et
al. (2004) proved that solubility of PAHs decreases with an increase in the number of aromatic
rings.
17
Table 1: Physicochemical properties of five PAHs.
PAH Acenaphthene Fluorene Phenanthrene Fluoranthene Pyrene
Structure
Chemical
formula1
C12H10 C13H10 C14H10 C16H10 C16H10
Molecular
weight (g/mol)1
154 166 178 202 202
Melting point
(°C)1
95.0 116 99.0 111 153
Boiling point
(°C)1
279 295 340 375 404
Vapor pressure
at 25 °C (Pa)1
0.287 0.043 0.016 0.001 0.006
Water solubility
at 20 °C (mg/L)2
3.47 1.99 1.60 0.27 0.14
Log KOW (L/kg)1
4.32
4.18
4.46
5.16
5.30
Log KSW (L/kg)3
Log KMW (L/kg)4
3.62
3.04
3.79
3.14
4.11
3.33
4.62
3.70
4.68
3.75
Log D at 20 °C
for Altesil SR
(m²/s)5
-10.0
-10.1
-10.2
-10.4
-10.4
1Source: PubChem (2017) 2Source: Skupinska et al. (2004) 3Source: Rusina et al. (2010b) 4Source: Smedes et al. (2009) 5Source: Rusina et al. (2010a)
1.2.3. Environmental concern At the end of the 18th century, a higher prevalence of skin cancer was observed among workers
who were exposed to soot and/or coal tar such as roofers (Skupinska et al., 2004). In the 20th
century, a relationship between gas industry/coal tar workers and lung cancer was described
(Skupinska et al., 2004). Further research revealed that the cancer was caused by the PAHs
present in the soot and coal tar (Skupinska et al., 2004). When PAHs enter the human body, they
are metabolically transformed to potential carcinogenic metabolites. These transformations are
mainly catalyzed by enzymes of the cytochrome P-450 family and epoxide hydrolases (Skupinska
et al., 2004).
18
Not all PAHs are characterized by the same toxicity. A study of Ravindra et al (2008) indicates that
as molecular weight increases, the carcinogenicity of PAHs increases and acute toxicity decreases.
Benzo(a)pyrene (five-ring), dibenz(b)fluoranthene (six-ring), benzo(b)fluoranthene (five-ring) and
indo(1,2,3-c,d)pyrene (six-ring) are classified as carcinogens, while phenanthrene (three-ring),
anthracene (three-ring), pyrene (four-ring) and benz(g,h,i)perylene (six-ring) are thought not to be
carcinogenic (Ravindra et al., 2008). The number of rings can be used to estimation the potential
risk, but does not give conclusive evidence. Besides the number of rings also the shape and
dimension of the chemicals may determine the biological activity of the PAHs (Skupinska et al.,
2004).
PAHs can easily disrupt various aquatic ecosystems, even at very low concentrations. PAHs are
endocrine disrupting chemicals that have, besides the carcinogenic effects, also a potential to
cause toxic and mutagenic effects on marine species (Haritash & Kaushik, 2009). Endocrine
disrupters can interfere with any system in an organism that is regulated by hormones. Their
influence is mainly observed at the first life stages of an organism, because of the intensive cell
growth and differentiation regulated by specified hormones in well-defined concentrations (National
Institutes of Health, 2017).
1.2.4. Monitoring of PAHs in the marine environment The International Agency for Research on Cancer recognized 30 PAHs as carcinogenic to humans
in 1983 (Nisbet & LaGoy, 1992). Fourteen years later, the United States Environmental Protection
Agency acknowledged 16 PAHs to be highly toxic and requested further investigation of these
compounds (Skupinska et al., 2004). PAHs became a point of major concern for human health and
increasing attention was given to environmental safety. The first publication dealing with monitoring
of PAHs in water dates back to 1994 (Eastwood et al., 1994).
Nowadays the monitoring of PAHs and other organic pollutants in seas and oceans is required by
the Water Framework Directive (WFD, 2000/60/EC) and the Marine Strategy Framework Directive
(MSFD, 2008/56/EC). The European legislations tries to ensure water quality standards are
achieved for six target PAHs (Mitina, 2015; Monteyne et al., 2013). These European target PAHs
are fluoranthene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene,
benzo(g,h,i)perylene and indeno(1,2,3-c,d)pyrene (Manoli & Samara, 1999).
Due to this broad range of PAHs and other organic components in the marine environment,
monitoring can be a real challenge for environmental chemists and requires suitable monitoring
techniques, effective communication and carefully considered data archiving systems.
Furthermore, monitoring these compounds is generally accompanied by a number of difficulties.
First of all, there are varying concentrations in the marine environment. Besides the main influences
such as tides, river discharges, harbor influences and boat traffic routes, the concentration of the
most volatile PAHs will also decrease with increasing water temperature. This gives the water
concentration a seasonal dimension as well (Prokeš et al., 2012). Moreover, monitoring of
expansive areas such as oceans and seas is a costly and time-consuming process.
19
Several methods for the estimation of freely dissolved water concentrations are reported in the
literature. A first sampling method is the classic active sampling or spot sampling, in which a sample
is taken at a particular place and time. This is a relatively easy way of collecting samples, but is
accompanied by a number of disadvantages (Vrana et al., 2005). It is for example not very
representative since the marine environment is a complex and dynamic system with constant
changes, as described above (Monteyne et al., 2013). Moreover, large volumes of water have to
be filtrated and extracted leading to higher costs (Raub et al., 2015).
A possible solution for the limitations of spot sampling is the installation of automatic sampling
systems to take numerous samples, but this also includes higher costs and more samples to be
processed. Active sampling furthermore involves high costs because of the analytical challenge
(low analyte concentrations, often below ng/L) and the presence of complex matrices which may
lead to interferences (Claessens et al, 2015). Monteyne et al. (2013) noticed that the water
concentrations of PAHs obtained by active sampling showed relatively high variations. These
variations were explained by the continuous interaction between discharges and flushing by the
seawater. There was added that the number of samples required to obtain the real time weighted
average concentrations would be unrealistically high (Monteyne et al, 2013). One way to overcome
these monitoring problems is the use of passive samplers (Table 2).
20
Table 2: Comparison between active grab sampling and passive sampling.
Active grab sampling Passive sampling Reference
Low tech (+)
Low tech (+) Claessens et al. (2015)
No detection of episodic
pollution/time-weighted average
concentrations (-)
Detection of episodic pollution/time-
weighted average concentrations
(+)
Górecki & Namieśnik (2002)
Claessens et al. (2015)
Rusina et al. (2010)
Ahrens et al. (2015) Vrana et al. (2005)
Short sampling time (+)
Long exposure time required (-) Claessens et al. (2015)
Kot et al. (2000)
Jahnke et al. (2016)
More complex sample preparation
(-)
Relatively simple sample
preparation (+)
Górecki & Namieśnik (2002)
Claessens et al. (2015)
Rusina et al. (2010)
Low analyte concentrations
(-)
Higher analyte concentrations (+) Kot et al. (2000)
Rusina et al. (2010)
Ahrens et al. (2015)
Vrana et al. (2005)
Analyte loss during transport and
storage (-)
Minimized analyte loss during
transport and storage (+)
Kot et al. (2000)
Determination of total
contaminant concentrations (+)
Determination of freely dissolved
contaminant concentrations (+ or -)
Claessens et al. (2015)
Higher analysis costs (-) Lower analysis costs (+) Górecki & Namieśnik (2002)
Vrana et al. (2005)
Less cost-efficient (-)
More cost-efficient (+) Claessens et al. (2015)
Kot et al. (2000)
Rusina et al. (2010)
Vrana et al. (2005)
A third marine sampling method for organic contaminants is biomonitoring (Raub et al., 2015).
Bivalves, fish and microalgae are commonly used organisms in biomonitoring studies (Stuer-
Lauridsen, 2005). This technique has the advantage that biomonitoring organisms can simply be
collected in the area of interest or they can be deployed in the marine environment for a
predetermined time (Raub et al., 2015). Just as passive samplers, biomonitoring organisms will
sample continuously during deployment. Organisms can be relocated to new sites where a new
steady state will be reached (Raub et al., 2015). However, the biomonitoring organisms have to be
healthy and have limited geographic ranges in which they can survive and grow (Lohmann & Muir,
2010). Within the same species, uptake rates can vary due to differences in sex, age, lipid content,
seasonal influences and the depth of deployment (Raub et al., 2015).
21
1.2. Passive sampling
1.2.1. Historical background The last three decades, alternatives to overcome the disadvantages of active grab samples have
been retought (Kot et al., 2000). Passive sampling devices have been available since the early
1970s, but were exclusively used for monitoring air quality, for example to measure toxic chemicals
in workspace air (Kot et al., 2000). Those principles of passive dosimetry for air sampling were then
taken over to monitor aquatic samples (Kot et al., 2000). The first peer reviewed paper with regard
to passive sampling for monitoring organic compounds in the aquatic environment dates back to
1987 (Kot et al., 2000).
With the development of semipermeable membrane devices (SPMD) by Huckins et al. in 1990,
passive sampling was introduced as an important tool for environmental monitoring of aquatic
organic pollutants (Claessens et al., 2015). Multiple new types of passive samplers were
introduced, including Diffusion Equilibrium in Thin films (DET) in 1991, Supported Liquid Membrane
(SLM) in 1992, Diffusive Gradient in Thin films (DGT) in 1994 (Vrana et al., 2005). Moreover, also
silicone rubber passive samplers are on the rise since last decade, mainly because of their
simplicity in use (Claessens et al., 2015). The accuracy and precision of passive samplers
increased and the detection of organic compounds at pg/L became a possibility in 1995 (Vrana et
al., 2005). The exposure of an organic polymer to water (passive sampling), has become a powerful
tool for detecting both environmental inorganic and organic pollutants (Rusina et al., 2010a).
1.2.2. Principles of equilibrium passive sampling Equilibrium passive sampling is based on the equilibration of freely dissolved contaminants
between the water phase and the collecting medium (Górecki & Namieśnik, 2002; Ahrens et al.,
2015). An important factor in the equilibration of the sampler are the octanol-water partitioning
coefficients (KOW’s) of the mixture components. The equilibration is based on a diffusion driven
process through a well-defined barrier, for example a membrane, and is caused by a difference in
chemical potential of the analyte in the water phase and the collecting medium. In equilibrium
passive samplers, partitioning of the compounds continues until an equilibrium is reached (Górecki
& Namieśnik, 2002). Besides their use for monitoring purposes, passive samplers have a potential
to be used in toxicity tests (Ahrens et al., 2015).
Most equilibrium passive sampling devices can be described by following uptake kinetics of Figure
2. The uptake profile of pollutants can be divided into three phases. The first phase is characterized
by a linear uptake and a negligible desorption from the receiving phase. The second phase initiates
when a half-saturation of the receiving phase is reached. The curve flattens and becomes
curvilinear in this intermediate phase. At the third and final phase, an equilibrium is reached: the
uptake rate equals the release rate and there is equilibrium partitioning between the medium and
the water phase (Ahrens et al., 2015). The first two stages are often considered as the kinetic
regime and the third stage as the equilibrium regime (Vrana et al., 2005).
22
Ahrens et al. (2015) characterized five different passive sampling devices under laboratory
conditions and studied amongst other things the log KOW ranges of these samplers. The results
indicate that all tested samplers were able to accumulate target compounds (pesticides) with a
wide range of log KOW values. Silicone rubber passive samplers were especially suited for
hydrophobic compounds with high log KOW values such as PAHs (Figure 3) (Ahrens et al., 2015).
Naturally occurring contaminant mixtures sampled with passive sampling have the potential to be
transferred into biotest systems.
Figure 2: Passive sampling devices operate in two main regimes (kinetic and equilibrium regime) and
can be divided into three stages (linear, intermediate and equilibrium phase) (Vrana et al., 2005).
Figure 3: Boxplots for individual pesticides taken up by five different passive samplers: silicone
rubber (SR) (n = 86), polar organic chemical integrative sampler (POCIS-A) (n = 106), POCIS-B (n =
110), Chemcatcher® SDB-RPS (n = 65) and Chemcatcher® C18 (n = 54) in correlation to their octanol-
water partition coefficient (KOW) (Ahrens et al., 2015).
1.2.3. Silicone rubber passive samplers Different polymers have the potential to equilibrate organic environmental micropollutants (Rusina
et al., 2010a). The most important polymer used for passive sampling these days is
polydimethylsiloxane (PDMS), also known as silicone rubber or low-density polyethylene (LDPE).
Silicone rubber passive samplers are available with the trade names AlteSilTM and Silastic® (Rusina
et al., 2010a). Currently, the most used time-integrative passive samplers are polar organic
chemical integrative sampler (POCIS) and Chemcatcher® (Ahrens et al., 2015).
Silicone rubber is frequently used as passive sampling device for hydrophobic organic chemicals
in the marine environment (Yates et al., 2007). Yates et al. (2007) found that partitioning into the
silicone rubber sheets is strongly determined by the hydrophobicity of the compounds.
Consequently, it can be stated that log KOW is a good predictor for log KSW with KSW the sampler-
water partitioning coefficient (Yates et al., 2007).
Silicone rubber polymers have high diffusion coefficients (D), making them a very suited polymer
for the uptake of hydrophobic organic compounds in equilibrium passive sampling (Rusina et al.,
23
2010a). With an average sampling rate (RS) of 0,86 L/day, silicone rubber passive samplers
showed the highest rate of the five tested samplers in the study of Ahrens et al. (2015).
1.3. Passive dosing
1.3.1. Theoretical background Passive dosing is characterized by a constant freely dissolved concentration provided by
continuous partitioning of hydrophobic organic compounds from a dominating reservoir (Smith et
al., 2010; Jahnke et al., 2016). This dominating reservoir is a biocompatible polymer such as
silicone rubber. While organic compounds are equilibrated on an adsorbing phase in passive
sampling, passive dosing can be described as the opposite process: continuous partitioning of
hydrophobic organic compounds from a biologically inert reservoir (such as silicone rubber) into
the water phase (Smith et al., 2010).
The polymer acts as a continuous source by replacing the removed component, providing constant
and defined exposure concentrations once steady state is reached (Claessens et al., 2015; Smith
et al., 2010). Combining passive field sampling and passive lab dosing for ecotoxicity tests with
hydrophobic contaminant mixture thus allows to mimic realistic environmental exposure
concentrations (Claessens et al., 2015).
Passive samplers can be seen as an infinite partitioning source because of the high KSW ratios of
the apolar organic compounds, where KSW represents the concentration of the compound on the
sampler divided by the concentration in the water phase (Birch et al., 2010) (Table 1). Loaded
samplers can be used several times as passive dosing devices without a significant reduction of
the analyte concentration in the polymer (Birch et al., 2010).
1.3.2. Passive dosing and risk assessment The combination of passive sampling with ecotoxicological risk assessment is an important but
often unrecognized factor (Jahnke et al., 2016). Risk assessment in aquatic toxicity tests is
especially challenging due to low aqueous solubilities and the sorption and eventual volatilization
losses that might occur while testing (Smith et al., 2010). Depletion of the test components during
the experiment, results in a weakly defined exposure concentration and a reduced test sensitivity
(Smith et al., 2010).
A feature of combining passive sampling in the marine environment with laboratory passive dosing
in toxicity experiments is the possibility to re-establish environmental concentration levels for a
broad range of components (a certain KOW range). This can be illustrated by the results of
Claessens et al. (2015). In this study, the combination of passive sampling and dosing was tested
with Phaeodactylum tricornutum. Environmental contaminant mixtures were loaded on the
samplers and passively dosed. The observed effects could not be explained by toxic components
measured in the sampling areas (Claessens et al., 2015). The most plausible explanation is the
presence of unmeasured compounds that are absorbed during field deployment of the passive
24
samplers and again released during passive dosing (Claessens et al., 2015). This shows the high
potential of this method, allowing to test naturally occurring contaminant mixtures of non-polar
compounds. Ecotoxicological effects can be assessed without chemical analysis.
Passive dosing is compatible with chemicals of a certain hydrophobicity range since the uptake
rate of very hydrophobic chemicals can be very slow. It is possible that the equilibrium state is not
reached within the sampling period. On the other hand, the depletion of more polar chemicals in
the water phase during passive dosing can result in non-negligible losses on the passive samplers
(Claessens et al., 2015).
1.4. Gas chromatography-mass spectrometry
1.4.1. Introduction The analytical technique gas chromatography-mass spectrometry (GC-MS) combines the
separation by gas chromatography and the detection by mass spectrometry for the analysis of
different compounds in a sample (Aebersold & Mann, 2003). The combined use of both techniques
was first introduced in 1952 by James et al. and has become one of the most applied techniques
for both identification and quantification of compounds in complex mixtures nowadays (Aebersold
& Mann, 2003; Bertrand, 1998).
GC-MS applications can be found in various sectors including:
- Food sector: food, beverage, flavor and fragrance analysis, quantification of compounds in
drinking water (Aebersold & Mann, 2003; Bertrand, 1998)
- Forensic and criminal cases: drugs detection, detection of metabolites in blood and urine,
explosives investigation (Aebersold & Mann, 2003)
- Environmental monitoring: quantification of pollutants in waste water (Aebersold & Mann, 2003;
Bertrand, 1998)
- Quality control of industrial products (Bertrand, 1998)
- Identification of the composition/molecular mass of unknown organic compounds (Bertrand,
1998)
1.4.2. Operating principle Before GC-MS analysis, a sample preparation has to be performed by either simply dissolving the
sample in a suited solvent or, in most cases, by an extraction of the analytes of interest using a
suited solvent (Bertrand, 1998; Columbia Analytical Services, 2008). Volatile organic solvents such
as hexane and dichloromethane are commonly used solvents for GC (Bertrand, 1998). After
sample preparation, 1 or maximum 2 µL of solved compound(s) is injected in the injection port of
the gas chromatograph (generally 1 to 100 pg per compound), after which the sample is volatilized
and mixed with an inert carrier gas such as helium or hydrogen in the capillary column (Bertrand,
1998).
The compounds in the mobile phase interact with the capillary column, also referred to as the
stationary phase. Different chemicals interact differently with the stationary phase, causing a
25
separation by a difference in retention time. Stronger interactions result in later exit into the mass
spectrometer and thus a higher retention time (Columbia Analytical Services, 2008). The analytes
that are separated in time exit the GC column and enter the ionization source of the mass
spectrometer, where they are ionized by electron bombarding at 70 eV, causing degradation into
positively charged molecular fragments or cations (Columbia Analytical Services, 2008; Bertrand,
1998).
The cations are accelerated to an electromagnetic field by lenses, where they are filtered according
to their mass to charge ratios (m/z). Uncharged and negatively charged ions are removed. The
separation is achieved by applying an alternating voltage between the opposite ends of the
quadrupole (Columbia Analytical Services, 2008). The detector amplifies the signal by an electron
multiplier and counts the signal of each mass-to-charge ratio, creating a mass spectrum. Detection
limits are generally around 1 to 100 pg for most compounds (Bertrand, 1998).
Figure 4: Schematic representation of the GC-MS setup (Crasto, 2014).
1.4.3. Quantification of PAHs by GC-MS The detection and quantification of PAHs and other organic environmental pollutants in complex
environmental samples can be performed by a number of analytical techniques, of which GC-MS
is one of the most important (James & Martin, 1952). For example the official environmental
protection agency (EPA) methods are based on GC-MS results for the quantification of pollutants
in drinking water, wastewater and surface waters (Bertrand, 1998). However, due to the low
concentration levels and the complexity of the samples, the analysis of PAHs can still present a
difficulty (James & Martin, 1952).
Table 3 represents the theoretically expected mass-to-charge ratio and retention time (RT) for the
five PAHs relevant for this research. Also the deuterated analogues are included.
26
Table 3: Characteristic mass-to-charge ratio and retention time for five PAHs and their deuterated
analogues (EMIS, 2016).
Component Characteristic mass-to-charge
ratio m/z for quantification
Retention time RT (minutes)
Acenaphthene 153+154 10,1
Acenaphthene-d10 164 10,0
Fluorene 166 10,9
Fluorene-d10 176 10,9
Phenanthrene 178 12,5
Phenanthrene-d10 188 12,4
Fluoranthene
Fluoranthene-d10
202
212
14,4
14,4
Pyrene 202 14,8
Pyrene-d10 212 14,7
An alternative to identify and quantify PAHs is by high performance liquid chromatography (HPLC)
with fluorescence detection or by high performance liquid chromatography–mass spectrometry
(HPLC-MS) as respectively described by Smith et al. (2013) and Booij et al. (2013).
1.5. Research goals This leads to the following hypotheses for this thesis:
Hypothesis 1: Realistic hydrophobic organic pollutant mixtures can be transferred
quantitatively from an equilibrium passive sampler to a smaller equilibrium passive sampler
without significant losses of the compounds. An upconcentration can be reached relatively
to the size difference of the samplers.
Hypothesis 1 implies that it is possible to upconcentrate samplers and thus reach higher
concentrations on the samplers due to the fact that KSW (= CS/CW) is a constant for a certain
compound and type of sampler. Depending on the used upconcentration factor, the final
concentration is expected to increase by the same factor.
Before upconcentration: CS = KSW * CW
After upconcentration: (Upconcentration factor * CS) = KSW * (Upconcentration factor * CW)
CS, upconcentrated = KSW * CW, upconcentrated
27
Hypothesis 2: The biological response exerted by the compounds on the samplers is not
influenced by the whole upconcentration procedure.
Hypothesis 2 implies that both upconcentrated (smaller) samplers and non-upconcentrated (bigger)
samplers exert the same ecotoxicological effect to marine test organisms. Similar effects are
expected for the same concentration levels, but with a shift in concentration at the upconcentrated
samplers as compared to the non-upconcentrated samplers.
28
2. Materials and methods
2.1. The upconcentration experiment
2.1.1. Theoretical background The first hypothesis states that PAHs on passive samplers can be upconcentrated relatively to the
difference in sampler size. To test this hypothesis, passive samplers were spiked with a mixture of
five PAHs in five different concentration treatments (CT 1 – CT 5). The extracts of the samplers
were used to spike smaller samplers to reach an upconcentration proportional to the sampler size
difference. The extracts of non-upconcentrated and upconcentrated samplers were analyzed by
GC-MS and a comparison was made.
As described earlier, silicone rubber passive samplers have been widely used for monitoring of
PAHs. In this study, AltecAlteSilTM silicone rubber sheets were used and will be referred to as
‘samplers’ in the further document. The samplers were manufactured from translucent, food grade
silicone rubber, with a hardness of 60 Shore A and were purchased form Altec Products Lts, St
Austell, United Kingdom.
The experimental design is displayed schematically in Figure 5. The represented steps were
followed for the 5 different concentration treatments, each in tenfold. Ten blanks were included,
they followed the same procedure but were spiked with pure methanol instead of a methanol-PAH
mixture.
Figure 5: Schematic overview of the experimental setup followed for each of the 5 concentration
treatments (CT 1 – CT 5) and blanks to upconcentrate PAHs on AltecAlteSilTM silicone rubber passive
samplers.
29
2.1.2. Choice of PAHs and concentration series PAHs have been well studied in passive sampling and have well known sampling rates and
diffusion coefficients (Rusina et al., 2009; Monteyne et al., 2013; Smedes et al., 2009). This makes
them appropriate test compounds for this research. The five PAHs used in this thesis were
acenaphthene, fluorene, phenanthrene, fluoranthene and pyrene (Table 1). The selection was
based on measured concentrations in the North Sea by Deschutter et al. (2015) (unpublished data).
PAH concentrations were monitored at different sampling locations along the Belgian coastal area
between March 4, 2015 and December 3, 2015. The highest concentrations were found for the five
above-mentioned PAHs. The average concentrations and concentration ratios of the five PAHs
measured in the harbor of Zeebrugge were calculated (Table 4). The same ratio was used in the
five concentration treatments to create realistic environmental contaminant mixtures. An important
note is that average water concentrations were used in this study.
Table 4: Average concentration of the five most common PAHs measured in the harbor of Zeebrugge
between March 4, 2015 and December 3, 2015.
PAH Average concentration (ng/L) Ratio
Acenaphthene 1.46 2.18
Fluorene 1.22 1.82
Phenanthrene 4.15 6.19
Fluoranthene 1.01 1.51
Pyrene 0.67 1.00
Velasquez (2015) studied the effect of a mixture of 8 PAHs (anthracene, benzo(a)anthracene,
benzo(b)fluoranthene, benzo(a)pyrene, chrysene, fluorene, phenanthrene, pyrene) on the growth
of Phaeodactylum tricornutum in a 72 hour growth inhibition experiment. A 50% effect concentration
(EC50) of 3.3 µg/L was found (Velasquez, 2015). This result was used as an indication for the order
of magnitude of the expected EC50 of the PAH mixture used in this thesis. Following five
concentration treatments for the PAH mixture were chosen:
1.00 µg/L → 3.20 µg/L → 10.2 µg/L → 32.8 µg/L → 105 µg/L (factor 3.2)
This series of concentration treatments covers a range of three log units and corresponds to the
desired exposure concentrations in the growth inhibition experiment with P. tricornutum.
2.1.3. Partitioning calculations Starting from the chosen final exposure conditions, the required spiking concentrations were
calculated for each concentration treatment based on the mass balance. Due to the different
partitioning coefficients, it was rather challenging to determine the required amount of each PAH.
30
Table 5 gives the desired exposure concentration for the five PAHs for the first concentration
treatment (CT 1; 1.00 µg/L) based on the naturally occurring concentration ratio. Multiplied by factor
3.2, the other concentration treatments (CT 2 – CT 5) were calculated.
The goal was to determine the mass of each of the five PAHs required to reach the final exposure
concentration. In the case of acenaphthene in the first concentration treatment (1 µg/L), a target
water exposure concentration (Cwe_t) of 0.172 µg/L was required (Table 5).
Table 5: Desired exposure concentration for each PAH for CT 1 (1 µg/L).
PAH Ratio Concentration (µg/L)
Acenaphthene 2.18 0.17
Fluorene 1.82 0.14
Phenanthrene 6.19 0.49
Fluoranthene 1.51 0.12
Pyrene 1.00 0.08
TOTAL CT 1 - 1.00
The required mass of each PAH for spiking was calculated using a number of intermediate steps.
In a first step, the concentration on the polymer after loading cp0 (µg/g) was calculated based on
the mass balance by Equation 1.
Cp0 = 𝑚𝑠∗(𝑚𝑝∗𝐾𝑀𝑊)
𝑉𝑀𝑊+𝑚𝑝∗𝐾𝑀𝑊 (Eq. 1)
The parameters used in this equation are given in Table 6.
Table 6: Loading conditions used for calculation of the concentrations on spiked sampler (Cp0).
Loading conditions Symbol Unit Value
Mass of substance mS µg -
Mass fraction methanol
WFm g/g 0.20
Methanol-water partitioning
coefficient
Log KMW L/kg 3.04; 3.14; 3.33; 3.70; 3.75
Volume methanol-water mix
VMW mL 100
Mass of polymer mp g 1.00
Subsequently, the exposure concentration of the polymer (Cwe) was calculated (Equation 2), based
on Cp0 and the parameters represented in Table 7.
31
Table 7: Exposure conditions used for calculation of the water concentrations in exposure (Cwe).
Exposure conditions Symbol Unit Value
Polymer water partitioning coefficient Log Kpw L/kg 3.6
Polymer in exposure me g 0.1
Volume water in exposure Ve mL 50
Mass of test organism mo g 0.001
Lipid content of test organism fL g/g 0.01
Cwe = 𝑚𝑒∗𝐶𝑝0
𝑚𝑒∗𝐾𝑃𝑊+𝑉𝑒+𝑚0∗𝑓𝐿∗𝐾𝑃𝑊∗1.1 (Eq. 2)
Cwe was calculated based on Cpo (and converted from µg/mL to µg/L). Subsequently, Cwe was
compared to the target water concentration Cwe_t. A solver was used to optimize the mass of
substance mS until Cwe_t equals Cwe.
Calculations for the five PAHs in the first concentration treatment were performed in this way.
Multiplied by factor 3.2, the masses for the other concentration treatments were calculated (Table
8).
Table 8: Theoretical mass of each PAH required to reach the desired exposure concentrations.
Desired
ΣCw
(µg/L)
mS for spiking using 100 mL MeOH-water mix (µg)
Acenaphth. Fluorene Phenanthrene Fluoranthene Pyrene Σ PAHs
CT 1 1.00 0.841 0.841 1.03 6.69 5.08 3.72
CT 2 3.20 1.03 2.69 3.30 21.4 16.3 11.9
CT 3 10.2 6.69 8.61 10.5 68.5 52.0 38.1
CT 4 32.8 5.08 27.6 33.8 219 166 122
CT 5 105 3.72 88.2 108 701 533 390
2.1.4. Five concentration treatments The mass of each PAH needed for spiking was pre-calculated based on mass balance calculations
(Table 8). Due to practical reasons (masses too low to weigh) a stock solution was made with the
same PAH ratio with a total concentration of 9.099 g/L. 20 mL of this stock solution were taken and
filled up with methanol to 200 mL total solution. This diluted stock solution was used to prepare the
five concentration treatments.
Table 9 gives the theoretical concentration of the five PAHs in the five concentration treatments
based on the theoretically required masses given in Table 8.
For example 0.042 mg/L of ancenaphthene equals (0.841 µg * 50)/1000. The numerator 50
converts 20 mL to 1 L and the denominator 1000 converts µg to mg.
32
Table 9: Concentration of the five PAHs in the five concentration treatments.
Cs (mg/L)
Acenaphthene Fluorene Phenanthrene Fluoranthene Pyrene Σ PAHs
CT 1 0.042 0.052 0.334 0.254 0.186 0.868
CT 2 0.135 0.165 1.07 0.813 0.595 2.78
CT 3 0.431 0.527 3.42 2.60 1.90 8.89
CT 4 1.38 1.69 11.0 8.32 6.09 28.4
CT 5 4.41 5.40 35.1 26.6 19.5 91.0
The five concentration treatments with total PAH concentrations of 0.868, 2.78, 8.89, 28.4 and 91.0
mg/L were made based on the stock solution.
2.1.5. Precleaning using Soxhlet extraction Altec AltesilTM silicone rubber passive samplers were pre-cleaning by Soxhlet extraction. This step
was needed to exclude any possible source of contamination on the sheets. The sheets were
extracted with 90 mL acetone/n-hexane (1:3 v/v) for 24 hours at 80°C. Aluminum foil was wrapped
around the extractors to avoid excessive heat loss that could result in condensation of the solvent
before reaching the cooler (Figure 6).
Figure 6: Soxhlet extraction for precleaning.
2.1.6. Spiking of the bigger samplers Precleaned samplers were dried with paper tissues and cut into pieces of 1.00 ± 0.01 g, referred
to as the bigger or 1.0 g samplers. The five concentration treatments were prepared based on the
stock solution. The calculated amount of stock solution was added to 200 mL amber bottles with a
33
precleaned sampler. Bottles were closed with aluminum foil and a plastic lid to avoid contact of the
spiking solution and any plastic phase.
Spiking was continued by adding a specified volume of water to each bottle every 24 hours to shift
the methanol-water ratio from 100 – 0 to 20 – 80 under continuous rolling of the bottles on a roller
bank (Table 10, Figure 7). The addition of water to the liquid fraction increases the affinity of the
sampler for the PAHs as described by Birch et al. (2010).
Table 10: Volumes of water added every 24 hours for spiking of the 1.0 g samplers.
Methanol
fraction (%)
Water
fraction (%)
Volume of
methanol (mL)
Volume of water
(mL)
Volume of water to
add each day (mL)
100 0 20,0 0 day 1: 0
80 20 20,0 5,00 day 2: 5,00
60 40 20,0 13,3 day 3: 8,30
40 60 20,0 30,0 day 4: 16,6
20 80 20,0 80,0 day 5: 50,0
Figure 7: Roller bank to spike the samplers.
2.1.7. Extraction and concentration of the spiked samplers Six samplers of each concentration treatment were extracted by Soxhlet extraction under the same
conditions as the precleaning. Extracts were concentrated to approximately 5 mL using a rotavapor
(Rotavapor R-100 Buchi) at a pressure of 450 ± 10 bar and a bath temperature of 40 °C. During
the concentration procedure, the pressure was lowered in steps of 20 mbar to accelerate the
process. Acetone and hexane evaporate at 556 and 335 mbar at 40°C, respectively (BUCHI – ISO
9001).
Extracts were transferred to glass test tubes and the round bottom flasks used for Soxhlet
extraction were rinsed three times with a small amount of hexane. Approximately 0.5 g sodium
sulfate was added and mixed with each extract to remove possible water residues. Once the
34
sodium sulfate had settled down, the extract was transferred to a new glass test tube and the tube
with the sodium sulfate was rinsed 3 times with a small volume of hexane. Extracts were brought
to dryness under a gentle nitrogen stream (nitrogen evaporation) to exchange the solvent to
methanol. 5 mL of methanol were added to each test tube and tubes were stored at 4°C until further
treatment.
2.1.8. Spiking of the smaller samplers The upconcentration was performed by spiking 0.10 ± 0.01 g samplers with the extracts of the 1.0
g samplers. The extracts (in 5 mL of methanol) were transferred to amber bottles and the tubes of
the extracts were rinsed three times with 5 mL of methanol. The spiking procedure of the 0.1 g
samplers was analogue to the spiking procedure of the 1.0 g samplers. Three replicates of each
concentration treatment were used for biotesting (hypothesis 2) and three replicates were used for
analysis with GC-MS (hypothesis 1).
2.2. Biotesting
2.2.1. Theoretical background The marine diatom Phaeodactylum tricornutum (Class Bacillariophyceae) is an eukaryotic,
photosynthetic organism that occurs naturally in marine ecosystems (Genome portal, 2017). P.
tricornutum is classified as phytoplankton and is located at the root of the food pyramid. It is typically
found in high abundance within the upper 50 m of the water column (Velasquez, 2015). P.
tricornutum is a model species that is frequently used in genome studies, but also in environmental
risk assessment with hydrophobic chemicals (Huysman et al., 2010; Claessens et al., 2013;
Everaert et al., 2016).
The ecotoxicological effect exerted by the different concentration treatments, both for
upconcentrated and non-upconcentrated mixtures was evaluated by passive dosing in a 72 hour
growth inhibition experiment with P. tricornutum. The experiment was based on the ISO guideline
10253. The growth inhibition experiment was repeated with the same samplers to confirm that the
PAHs on the samplers were not depleted.
The growth inhibition was calculated and dose-response curves were made. The PAHs in the water
phase after the experiment were extracted in dichloromethane using liquid liquid extraction (LLE).
The extracts were concentrated and diluted in hexane before GC-MS analysis. The results were
compared with the theoretical values. Figure 8 gives an overview of the experimental setup.
35
Figure 8: Schematic overview of the experimental setup for each of the five concentration treatments
and blanks to test hypothesis 2.
2.2.2. Growth medium Synthetic sea water with a salinity of approximately 33 g/L was prepared (Table 11) and filtered
through a 0.45 µm filter. The growth medium was prepared by adding 15 mL of nutrient stock
solution 1, 0.5 mL of nutrient stock solution 2 and 1 mL of stock solution 3 to 900 mL of synthetic
sea water and was diluted to 1 L with the same sea water (ISO, 2006) (Table 12). 50 mL of growth
medium were added to each erlenmeyer test flask together with a spiked sampler. The medium
was pre-equilibrated by passively dosing of the PAHs on a Heidolph Unimax 2010 rotary shaker
for 72 hours at 170 rpm.
Table 11: Composition synthetic sea water (ISO, 2006).
Salt Concentration of salt in synthetic sea water (g/L)
NaCl 22.0
MgCl2.6H2O 9.70
Na2SO4 3.70
CaCl2 1.00
KCl 0.65
NaHCO3 0.20
H3BO3 0.02
36
Table 12: Nutrient stock solutions (ISO, 2006).
Nutrient Concentration in stock
solution
Final concentration in test
solution
Stock solution 1
FeCl3.6H2O 48 mg/L 149 µg/L (Fe)
MgCl2.4H2O 144 mg/L 605 µg/L (Mn)
ZnSO4.7H2O 45 mg/L 150 µg/L (Zn)
CuSO4.5H2O 0.157 mg/L 0.6 µg/L (Cu)
CoCl2.6H2O 0.404 mg/L 1.5 µg/L (Co)
H3BO4 1140 mg/L 3.0 mg/L (B)
Na2EDTA 1000 mg/L 15.0 mg/L
Stock solution 2
Thiamin hydrochloride 50 mg/L 25 µg/L
Biotin 0.01 mg/L 0.005 µg/L
Vitamin B12
(cyanocobalamin)
0.10 mg/L 0.05 µg/L
Stock solution 3
K3PO4 3.0 g/L 3.0 mg/L; 0.438 mg/L P
NaNO3 50.0 g/L 50.0 mg/L; 8.24 mg/L N
Na2SiO3.5H2O 14.9 g/L 14.9 mg/L; 1.97 mg/L Si
2.2.3. Algae culture and cell count The marine diatom Phaeodactylum tricornutum Bohin strain 1052/1A was obtained from the Culture
Collection of Algae and Protozoa (Oban, United Kingdom) and maintained in the laboratory
according to the protocol described in ISO 10253. Four precultures were inoculated 72 hours before
the start of the experiment. Each test flask was inoculated with 1.0 * 104 cells/mL from the mixed
precultures and grown at 23 °C with a light intensity between 7000 and 9000 lx. The erlenmeyer
flasks were shaken manually once a day. An electronic particle counter (Coulter counter model DN,
Harpenden, Herts, UK) was used to count the algal cell density after 24, 48 and 72 hours. The cell
count is based on a change in impedance when the cells are pulled through an orifice together with
an electric current (Sowerby, 2007). The cells displace the electrolyte, resulting in a pulse in
impedance (Sowerby, 2007).
The pH was measured each day for one replicate of each concentration treatment.
2.2.4. Calculation of the growth inhibition The specific growth rate (µ) was calculated by Equation 3.
µ = ln(𝑁𝐿)−ln(𝑁0)
𝑡𝐿−𝑡0 (Eq. 3)
37
Hereby t0 and tL represent the time of inoculation (day 0) and the moment of measuring the cell
density (day 1, 2 or 3), respectively. N0 was the inoculated initial cell density at t0 (1.0 * 104 cells/mL)
and NL the measured cell density at time tL (cells/mL).
Subsequently, the growth inhibition Iµ was calculated by Equation 4.
Iµ = (µ𝑎𝑣𝑒𝑟𝑎𝑔𝑒𝑏𝑙𝑎𝑛𝑘𝑠−µ𝑖µ𝑎𝑣𝑒𝑟𝑎𝑔𝑒𝑏𝑙𝑎𝑛𝑘𝑠
) ∗ 100 (Eq. 4)
With µi the specific growth rate of test flask i.
2.3. Chemical analysis
2.3.1. Analysis of the sampler extracts Analysis of the stock solution, the 1.0 g sampler extracts and the 0.1 g sampler extracts were
performed by GC-MS. Samplers were extracted with internal standard (IS) composed of the
deuterated analogues of the used PAHs (Table 13). The internal standard was used to check the
efficiency of the extraction. Concentration of the extracts was performed equally to the other
extracts (rotary evaporation and nitrogen evaporation). The concentrated extracts were diluted 0x,
5x, 10x or 50x in hexane depending on the expected concentration of the five PAHs in each
concentration treatment (Table 14). 10 µL recovery standard (acenaphtene-d10) were added to
each extract before transferring them to GC vials for analysis.
Table 13: Composition internal standards (IS) and recovery standard (RS) for GC-MS analysis of the sampler extracts.
Component IS high (µg/mL) IS low (µg/mL)
Acenaphtene-d10 950 (RS) 95 (RS)
Fluorene-d10 1030 103
Phenanthrene-d10 4010 401
Fluoranthene-d10 3910 391
Pyrene-d10 4080 408
Table 14: Dilution factor of each extract for GC-MS analysis.
Internal Standard added (µL) (low or high)
Dilution factor End volume extract (mL)
CT 1
10 (IS low) 0x and 5x 2
CT 2
20 (IS low) 0x and 10x 2
CT 3
100 (IS low) 0x and 5x 10
CT 4
20 (IS high) 5x and 10x 10
CT 5
100 (IS high) 10x and 50x 10
Blanks 10 (IS low) 0x and 5x 2
38
Analysis was performed by GC-MS (HP 6890-HP 5972 from Agilent) in full scan mode using
electron impact ionization. The gas chromatograph had a fused silica DB-5MS column of 30 m and
an internal diameter of 0.25 mm. The column was coated with a 0.250 µm stationary phase (5%
phenyl groups and 95% alkyl groups). Helium was used as carrier gas. 1 µL extract was injected
per sample (split injection). The Xcalibur™ Software was used for processing the results.
2.3.2. Analysis of the PAH concentration in the water phase after the growth
inhibition experiment Besides the analysis of the stock solution and the extracts of the non-upconcentrated and
upconcentrated samplers, also the water phase after the first growth inhibition experiment was
analyzed.
The algae were separated from the aqueous solution by vacuum filtration over a 2.7 µm glass
microfiber filter. The mass of the water samples was determined gravimetrically by weighing the
bottles with and without the water phase. The weight was corrected with the density of sea water
(1027 kg/m³). Internal standard was added and the water phases were extracted with 30 mL
dichloromethane (liquid liquid extraction of LLE). The lower organic layer was percolated over 3 g
Na2SO4 to remove possible remaining water. The upper water layer was extracted two more times
with 20 mL dichloromethane and the organic layer was likewise percolated and added to the
organic phase. 5 mL of hexane were added to the organic phase and this phase was concentrated
to ca. 5 mL with the rotary evaporator. Extracts were further concentrated with a gentle nitrogen
stream until the specified end volume represented in Table 15. The extract were diluted in hexane
for analysis by GC-MS. A ‘spike’ was included in twofold in the LLE as reference material, to
demonstrate that the method was suitable for the extraction/analysis of the PAH mixture.
Table 15: Dilution factor of each concentration treatment.
Internal Standard
added (µL) (low or
high)
Dilution factor End volume extract
(mL)
CT 1 25 (IS low) 0x 0.5
CT 2 25 (IS low) 0x 0.5
CT 3 50 (IS low) 0x 0.5
CT 4 20 (IS high) 0x and 4x 2.0
CT 5 50 (IS high) 0x and 4x 5.0
Blanks (samplers without PAHs) 25 (IS low) 0x 0.5
Blanks (no samplers) 25 (IS low) 0x 0.5
Spike 50 (IS low) 0x 0.5
39
3. Results
3.1. The upconcentration experiment The stock solution and the extracts of the non-upconcentrated and upconcentrated samplers were
analyzed by GC-MS as described in materials and methods. For each sample, the obtained peaks
for the five PAHs were verified. This was done by comparing the retention times of the peaks with
the theoretical expected retention times, given in Table 3. The surface area below the peak was
determined automatically by the Xcalibur™ Software, but was checked manually and corrected
where necessary.
The actual PAH concentration in the stock solution was calculated taking into account the dilution
factor of the GC-MS samples (3.1.1. PAH concentration in stock solution). The PAH concentration
on the samplers was calculated based on the measured concentrations in the extracts, taking into
account the volume and dilution factor of the extract, the internal standard recovery and the exact
sampler weight (3.1.2. PAH concentration on non-upconcentrated and upconcentrated samplers).
3.1.1. PAH concentration in stock solution For analysis, the stock solution (consisting of acenaphthene, fluorene, phenanthrene, fluoranthene
and pyrene) with a nominal sum concentration of PAHs of 909.9 mg/L was diluted 250x. The
concentrations of the five PAHs in the extracts were obtained using the Xcalibur™ Software after
analysis by GC-MS. Also the concentration of the internal standards (deuterated PAHs) were
determined. Based on these results, the amount of compounds in the stock solution were calculated
(µg/mL). In the calculations, the dilution factor was taken into account by multiplying the measured
PAH concentration in extract (µg/mL) with the dilution factor (250x). The theoretical concentration
of each component in the stock solution was compared to the measured concentration of each
component in the stock solution (Table 16). The efficiency (%) represents the ratio of the
compounds in the actual stock solution to the nominal concentration in the stock solution.
Table 16: Comparison between the nominal and actual PAH concentration in the stock solution with
the corresponding ratio between nominal and actual PAH concentration (efficiency).
Nominal ƩC 5 PAHs in stock
solution (mg/L)
Measured ƩC 5 PAHs in
stock solution (mg/L)
Efficiency (%)
Acenaphthene 44.1 39.4 89.4
Fluorene 54.0 52.3 96.8
Phenanthrene 351 335 95.6
Fluoranthene 266 242 90.9
Pyrene 195 181 93.1
TOTAL 910 850 93.5
40
3.1.2. PAH concentration on non-upconcentrated and upconcentrated
samplers The stock solution was used to make a series of five concentration treatments (CT 1 - CT 5) to
spike the 1.0 g samplers (Table 17). The nominal ƩC 5 PAHs for the spiking solutions were
calculated for the five concentration treatments, based on the nominal ƩC 5 PAHs in the stock
solution. The actual ƩC 5 PAHs were calculated based on the measured ƩC 5 PAHs in the stock
solution.
Table 17: Comparison between the nominal and actual PAH concentration of the spiking solution for
each CT.
Nominal ƩC 5 PAHs (mg/L) Actual ƩC 5 PAHs (mg/L) Efficiency (%)
CT 1
0.868 0.811 93.5
CT 2
2.78 2.60 93.5
CT 3
8.89 8.30 93.5
CT 4
28.4 26.6 93.5
CT 5 91.0 85.0 93.5
Spiked samplers were extracted with IS and the extracts were transferred to hexane and diluted
for analysis by GC-MS. The results of the GC-MS report include the concentrations of
acenaphthene, fluorene, phenanthrene, fluoranthene and pyrene (µg/mL) and the concentration of
their deuterated analogues, respectively acenaphthene-d10, fluorene-d10, phenanthrene-d10,
fluoranthene-d10, pyrene-d10 (µg/mL). These obtained extract concentrations were converted step
by step to determine the concentration on the samplers (in µg/g). Hereby taking into account the
dilution factor, the amount of IS added, the theoretical and real concentration of the internal
standard and the mass of the sampler. The below mentioned steps were followed.
Step 1: Measured concentrations for the five PAHs and their deuterated analogues (IS, RS) in each
extract (in µg/mL) were obtained from the GC-MS report after the analysis using the Xcalibur™
Software.
Step 2: The theoretical IS-concentrations in the extracts (CIS extract, theoretical, in µg/mL) were calculated
based on the volume of IS-working solution added to the sample (VIS added, in µL), the theoretical
concentration of the internal standard (CIS stock, theoretical, in µg/mL), the dilution factor (D) and the final
volume of the extract (Vextract, in mL).
CIS extract, theoretical = (𝑉𝐼𝑆𝑎𝑑𝑑𝑒𝑑/1000)∗𝐶𝐼𝑆𝑠𝑡𝑜𝑐𝑘,𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙
𝐷∗𝑉𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (Eq. 5)
41
Step 3: The percentage recovery of the IS in the extract was calculated based on the measured
IS-concentration in the extract (CIS extract, measured, in µg/mL) and CIS extract, theoretical.
RecoveryIS extract = 𝐶𝐼𝑆𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑
𝐶𝐼𝑆𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙∗ 100 (Eq. 6)
Step 4: The amount of compound in the extract (µg) was calculated taking into account the volume
of the extract and the dilution factor.
mextract, step 1 = Cextract, measured * Vextract * D (Eq. 7)
Step 5: The amount of compound in the extract (µg) was calculated taking into account the volume
of the extract, the dilution factor and the IS recovery.
mextract, step 2 = mextract, step 1 * 𝐶𝐼𝑆𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙
𝐶𝐼𝑆𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 (Eq. 8)
Step 6: The PAH concentration on the sampler (µg/g) was calculated taking into account the
sampler weight (msampler, in g).
msampler = 𝑚𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑠𝑡𝑒𝑝2
𝑚𝑠𝑎𝑚𝑝𝑙𝑒𝑟 (Eq. 9)
Step 7: The PAH concentration on the sampler was calculated for the five PAHs and summed to
get the total PAH concentration on the sampler. This was repeated for all extracts.
Different dilutions of each extract were analyzed. In case different dilution factors for the same
extract gave a reliable result, the dilution that gave the best recovery for the IS and RS was used
for further data analysis. The raw data from the GC-MS report all intermediate steps and results
are included in Attachment 2 and 3.
The extracts of the (non-upconcentrated) samplers that were not analyzed were used to spike
smaller samplers. After spiking, the upconcentrated samplers were extracted by Soxhlet extraction
with internal standard and the PAH concentration in these extracts was determined analogous to
the non-upconcentrated samplers (analysis by GC-MS and step 1 – 7). A summary of the calculated
results for both non-upconcentrated (big) and upconcentrated (small) samplers is given in Table
18. Replicate 3 of CT 1 and replicate 2 of CT 5 were not included because the extracts were lost
during spiking/upconcentrating.
42
Table 18: Calculated Ʃ5 PAHs on samplers based on results GC-MS.
Mixture treatment ƩC 5 PAHs big
sampler (µg/g)
Average ƩC 5
PAHs big sampler
(µg/g)
ƩC 5 PAHs
small sampler
(µg/g)
Average ƩC 5 PAHs
small sampler
(µg/g)
CT 1 (replicate 1)
75.4 75.9 ± 2.8 548 512 ± 36
(replicate 2)
72.2 475
(replicate 3)
80.1 -
CT 2 (replicate 1)
221 230 ± 11.5 1102 1132 ± 75
(replicate 2)
222 1049
(replicate 3)
247 1244
CT 3 (replicate 1)
767 794 ± 38 3952 4534 ± 388
(replicate 2)
763 5100
(replicate 3)
851 4550
CT 4 (replicate 1)
2682 2638 ± 79 7377 7821 ± 564
(replicate 2)
2712 7419
(replicate 3)
2519 8668
CT 5 (replicate 1)
8043 7771 ± 181 12254 11685 ± 569
(replicate 2)
7519 -
(replicate 3)
7753 11116
Blank (replicate 1)
14.5 19.3 ± 3.3 19.8 33.3 ± 9.0
(replicate 2)
24.2 39.5
(replicate 3)
19.2 40.5
3.1.3. The upconcentration The obtained data from GC-MS analysis were converted to the concentrations on the samplers,
taking into account the exact weight of the sampler, the dilution factor and end volume of the extract
and the internal standard. Table 19 gives the average calculated ƩC 5 PAHs for each concentration
treatment, compared to the theoretical expected concentrations. This data is presented graphically
in Figure 9 by plotting the logarithm of the ƩC 5 PAHs on the sampler for each CT compared to the
theoretical ƩC 5 PAHs. The theoretical ƩC 5 PAHs on the samplers after spiking were calculated
43
by Equation 1. Hereby the mass of substance (mS) for each PAH in each concentration treatment
was calculated based on the measurement of the stock solution.
Table 19: Comparison of the total sum concentration on the big and small samplers.
ƩC 5 PAHs on big samplers
(µg/g)
ƩC 5 PAHs on small samplers
(µg/g)
Average
upconcentra-
tion factor
Theo-
retical
Calculated Recovery
(%)
Theo-
retical
Calculated Recovery
(%)
Theo-
retical
Calcu-
lated
CT 1
78.3
75.9 ± 2.8
97.0
783
512 ± 36.3
65.4
10
6.9
CT 2
250 230 ± 12 91.9 2504 1132 ± 75 45.2 10 4.9
CT 3
801 794 ± 38 99.0 8014 4523 ± 388 56.4 10 5.7
CT 4
2564 2638 ± 79 103 25644 7822 ± 564 30.5 10 3.0
CT 5
8206 7771 ± 181 94.7 82060 11685 ± 569 14.2 10 1.5
Blank 0 19.3 ± 3.3 - 0 33.3 ± 9.0 - - -
Figure 9: ƩC 5 PAHs on non-upconcentrated big samplers (left) and upconcentrated small samplers
(right) for each CT compared to the theoretical expected concentration.
3.1.4. Individual PAH concentration for each CT In the previous section, the total PAH concentrations were considered. However, for each
concentration treatment, a subdivision can be made for acenaphthene, fluorene, phenanthrene,
fluoranthene and pyrene. This subdivision for the non-upconcentrated samplers is given in Table
44
20 and for the upconcentrated samplers in Table 21. The theoretical concentrations were again
calculated by Equation 1 and were based on the measured PAH concentration in the stock solution.
Table 20: Concentration of the individual PAHs on the non-upconcentrated samplers (µg/g).
CT 1 CT 2 CT 3 CT 4 CT 5
theo mea theo mea theo mea theo mea theo mea
Acenaphthene 3.44 3.16 11.0 9.12 35.3 29.4 113 89.6 361 295
Fluorene 4.65 4.34 14.9 13.2 47.6 46.0 152 138 487 442
Phenanthrene 30.5 29.6 97.8 92.6 313 317 1000 999 3200 3130
Fluoranthene 22.6 22.7 72.4 66.5 232 241 741 797 2370 2330
Pyrene 17.0 16.1 54.4 48.6 174 161 557 614 1780 1570
TOTAL 78.3 75.9 250 230 801 794 2564 2640 8210 7770
theo = theoretical concentration mea = measured concentration Table 21: Concentration of the individual PAHs on the upconcentrated samplers (µg/g).
CT 1 CT 2 CT 3 CT 4 CT 5
theo mea theo mea theo mea theo mea theo mea
Acenaphthene 34.4 10.9 110 27.8 353 98.4 1130 291 3610 1030 Fluorene 46.5 26.5 149 53.8 476 178 1520 467 4870 1210 Phenanthrene 306 190 978 413 3130 1630 10000 3380 32000 5180 Fluoranthene 226 168 724 378 2320 1520 7410 2210 23700 2680 Pyrene 170 116 544 259 1740 1100 5570 1480 17800 1580 TOTAL 783 512 2500 1130 8010 4520 25600 7830 82000 11700
theo = theoretical concentration mea = measured concentration
In Figure 10 a graphical comparison of the five PAHs before and after upconcentration was made
and compared to the theoretical concentrations. The logarithm of the concentration on the sampler
was plotted for the five PAHs. A good correspondence between the actual and theoretical
concentrations on the samplers was observed before upconcentration for all concentration
treatments. After upconcentration, greater differences were observed and the observed differences
varied for the five PAHs. The graphs for CT 2, CT 3 and CT 4 can be found in the supplementary
information (Attachment 1).
45
Figure 10: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 1 (above) and CT 5 (below).
46
3.2. Biotesting
3.2.1. GC-MS analysis of the water phase after growth inhibition PAHs in the filtered water phase after the first growth inhibition experiment were extracted by LLE
as described in material and methods. The results of the GC-MS report include the concentration
of acenaphthene, fluorene, phenanthrene, fluoranthene and pyrene (µg/mL) and the concentration
of their deuterated analogues, respectively acenaphthene-d10, fluorene-d10, phenanthrene-d10,
fluoranthene-d10, pyrene-d10 (µg/mL). These extract concentrations were converted step by step
to determine the concentration in the water phase (µg/L). The steps followed to calculate these
concentration in the water phase were performed similar to the calculation of the PAH
concentrations on the samplers. However, instead of calculating the PAH concentration on the
samplers (step 6), the PAH concentration in the water phase was calculated:
Cwater phase = 𝑚𝑒𝑥𝑡𝑟𝑎𝑐𝑡,𝑠𝑡𝑒𝑝2
𝑉𝑠𝑎𝑚𝑝𝑙𝑒 (Eq. 10)
The raw data from the GC-MS report and the results of all intermediate steps are included in
Attachment 4 and 5. A summary of the calculated results is presented in Table 22. The calculated
concentrations are averages of three replicates for each CT. For the water phase in the experiment
with the small samplers, only two replicates were included for CT 2, 3 and 4. The missing samples
were lost during the upconcentration procedure because of a pipetting error, a broken bottle during
spiking and a cracked vial during nitrogen evaporation. The upconcentration factor is the ratio of
the calculated concentrations for the water phase and the upconcentrated (big) and non-
upconcentrated (small) samplers, respectively. It should be noted that the ƩC 5 PAHs on the blanks
were very high. The explanation could be found at the recoveries of the internal standards. The
blanks showed 30 times higher recoveries than theoretically expected, probably due to the addition
of a wrong IS volume. For the other samples, no abnormal IS recoveries were found.
47
Table 22: Comparison of the ƩC 5 PAHs in the water phase after growth inhibition experiment 1.
ƩC 5 PAHs water phase big
samplers (µg/L)
ƩC 5 PAHs water phase
small samplers (µg/L)
Upconcentration factor
Theoretical Calculated Theoretical Calculated Theoretical Calculated
CT 1
1.00
1.72 ± 0.10
10.0
10.11 ± 2.14
10
5.9
CT 2
3.20 8.95 ± 1.29 32.0 38.22 ± 0.70 10 4.3
CT 3
10.24 22.08 ± 1.38 102.4 80.93 ± 29.33 10 3.7
CT 4
32.77 80.10 ± 4.55 327.7 205.33 ± 26.53 10 2.6
CT 5
104.9 224.60 ± 3.43 1049 267.09 ± 8.87 10 1.2
Blank 0 0.97 ± 0.34 0 1.69 ± 0.41 - -
Figure 11: ƩC 5 PAHs in the water phase after passive dosing from non-upconcentrated and
upconcentrated samplers.
3.2.2. Calculation of the growth inhibition Based on the measured cell density, the growth inhibition of P. tricornutum after 72 hours exposure
to the big samplers was calculated by Equation 3 and 4. Tables 23 and 24 respectively represent
the average growth inhibition for three replicates at each CT. The measured cell densities with the
non-upconcentrated and upconcentrated samplers in the two growth inhibition experiments are
given in Attachment 6 and 7.
48
Table 23: Growth rate µ in growth inhibition experiment 1 with non-upconcentrated and
upconcentrated samplers.
Average growth rate µ day 0 – day 3 (d-1)
(±std dev)
Measured CW
Non-upconcentrated
samplers
Upconcentrated
samplers
Non-upconcentrated
samplers
Upconcentrated
samplers
CT 1
1.28 ± 0.01
1.21 ± 0.00
1.72 ± 0.10 10.11 ± 2.14
CT 2
1.28 ± 0.01
1.18 ± 0.00
8.95 ± 1.29 38.22 ± 0.70
CT 3
1.26 ± 0.02
0.88 ± 0.18
22.08 ± 1.38 80.93 ± 29.33
CT 4
1.04 ± 0.01
0.11 ± 0.07
80.10 ± 4.55 205.33 ± 26.53
CT 5 0.01 ± 0.06
-0.06 ± 0.05
224.60 ± 3.43 267.09 ± 8.87
Blanks 1.30 ± 0.01 1.25 ± 0.02 0.97 ± 0.34 1.69 ± 0.41
Table 24: Growth inhibition Iµ in experiment 1 with non-upconcentrated and upconcentrated
samplers.
Average growth inhibition Iµ day 0 –
day 3 (d-1) (±std dev)
Measured CW
Non-
upconcentrated
samplers (%)
Upconcentrated
samplers (%)
Non-upconcentrated
samplers
Upconcentrated
samplers
CT 1 1.64 ± 0.82
5.44 ± 0.12
1.72 ± 0.10 10.11 ± 2.14
CT 2 1.74 ± 0.67
7.66 ± 0.26
8.95 ± 1.29 38.22 ± 0.70
CT 3 3.01 ± 1.18
31.13 ± 13.80
22.08 ± 1.38 80.93 ± 29.33
CT 4 20.56 ± 1.03
91.57 ± 5.73
80.10 ± 4.55 205.33 ± 26.53
CT 5 98.68 ± 4.47
104.51 ± 4.48
224.60 ± 3.43 267.09 ± 8.87
Blanks 0.22 ± 0.65 2.35 ± 1.86 0.97 ± 0.34 1.69 ± 0.41
49
Table 25: Summary of the validity criteria for growth inhibition experiment 1 and 2.
Experiment 1 Experiment 2
2 days
3 days 2 days 3 days
Factor increase cells 16.3 49.9 11.5 32.3
CV growth rate (%) 1.2 1.1 2.9 3.8
Mean sectional CV (%) 8.3 14.0 6.2 11.5
ΔpH 0.43 0.43 0.28 0.28
3.2.3. Growth inhibition curve By plotting the percentage growth inhibition (Table 24) in terms of the logarithm of the total PAH
concentration in the water phase (ƩCW) for each concentration treatment (Table 22), a growth
inhibition curve was drawn up. The non-upconcentrated and upconcentrated samplers were plotted
on the same graph to facilitate the comparison (Figure 12).
Figure 12: Growth inhibition curve for P. tricornutum after 72 hours exposure to a mixture of PAHs
using passive dosing for upconcentrated and non-upconcentrated samplers.
The freely dissolved aqueous PAH concentrations causing 50% growth inhibition (EC50) were 131.8
µg/L and 109.6 µg/L for the experiment with the non-upconcentrated and upconcentrated samplers,
respectively.
3.2.4. Results growth inhibition experiment 2 The growth inhibition experiment was repeated to see if the response would stay identical and use
a biotest to check for depletion of the PAHs on the samplers. In other words, we expected the same
growth inhibitions and thus the same growth inhibition curve.
50
Table 26: Growth rate µ in growth inhibition experiment 2 with non-upconcentrated and
upconcentrated samplers.
Average growth rate µ day 0 – day 3 (d-1) (±SD)
Non-upconcentrated samplers
Upconcentrated samplers
CT 1
1.26 ± 0.09
1.03 ± 0.04
CT 2
1.23 ± 0.07
0.99 ± 0.02
CT 3
1.28 ± 0.02
0.65 ± 0.24
CT 4
0.99 ± 0.02
0.05 ± 0.06
CT 5 -0.01 ± 0.04
0.03 ± 0.06
Blanks 1.26 ± 0.03 1.10 ± 0.02
Table 27: Growth inhibition Iµ in experiment 2 with non-upconcentrated and upconcentrated
samplers.
Average growth inhibition day 0 – day 3 (d-1) (±SD)
Non-upconcentrated samplers
Upconcentrated samplers
CT 1 -8.56 ± 7.63
10.59 ± 3.45
CT 2 -6.58 ± 6.08
14.69 ± 2.09
CT 3 -10,99 ± 1.68
43.96 ± 20.78
CT 4 2.10 ± 2.10
95.98 ± 5.36
CT 5 3.13 ± 3.13
97.80 ± 5.08
Blank -9.05 ± 2.33 4.50 ± 1.85
In CT 3 of the upconcentrated sampler, the measured cell number was 205980 cells/mL on day
three compared to 27680 cells/mL and 62100 cells/mL for the other two replicates. The most
obvious explanation for the outlier is a mistake in the spiking procedure resulting in reduced PAH
loading of this sampler. The outlier was removed from the dataset.
51
Figure 13: Growth inhibition curve for P. tricornutum after 72 hours exposure to a mixture of PAHs
using passive dosing for upconcentrated and non-upconcentrated samplers.
The freely dissolved aqueous PAH concentrations causing 50% growth inhibition (EC50) in the
second test were 93.3 µg/L and 74.1 µg/L for the non-upconcentrated and upconcentrated
samplers, respectively.
The growth inhibition after exposure to the non-upconcentrated samplers was compared between
experiment 1 and experiment 2 and the same was done for the upconcentrated samplers (Figure
14).
Figure 14: Comparison growth inhibition curves of experiment 1 and 2 for P. tricornutum after 72
hours exposure to non-upconcentrated samplers (left) and upconcentrated samplers (right).
52
4. Discussion
4.1. The upconcentration experiment
4.1.1. PAH concentration in stock solution The total PAH concentration measured in the stock solution corresponds with 93.5% of the
theoretical concentration that was expected in the stock solution. The theoretical concentration was
based on the actual weighted masses of each PAH (Table 16). This means that there was a
reduction of 6.5% of the initially added PAHs to the stock solution. The highest reductions were
observed for acenaphthene (10.6%) and fluoranthene (9.1%), while pyrene, phenanthrene and
fluorene have the highest recovery (respectively 93.1%, 95.6% and 96.8%).
4.1.2. PAH concentration on non-upconcentrated and upconcentrated
samplers Based on the stock solution, five dilutions or concentration treatments were made for spiking of the
sampler, with each 93.5% of the theoretical PAH concentration (Table 17). The same recovery was
assumed for each CT, because the spiking solutions were dilutions of this stock solution. Further
calculation were based on the actual concentrations measured in the stock solution instead of the
theoretical concentrations. This way, a more accurate view on the upconcentration experiment was
possible.
Table 18 represents the calculated PAH concentration on non-upconcentrated and upconcentrated
samplers for each CT (in triplicate). Standard deviations were lower for CT’s with a lower total PAH
concentration and increased with increasing total concentration.
On the one hand, the theoretical and calculated ƩC 5 PAHs on the non-upconcentrated samplers
were compared. Hereby a good resemblance could be observed (Table 19). Recoveries after
spiking range from 91.9% up to 102.8% for the non-upconcentrated samplers. The recovery
represents the ratio of compound found on the samplers to the theoretical expected concentration
on the sampler. A recovery higher than 100%, as observed for CT 4 can be explained by the
standard deviation (theoretical: 2564 µg/g vs. calculated 2638 ± 79) or more likely by the possibility
that the spiking procedure occurred more efficiently than the theoretical estimation. In general,
spiking of the big samplers occurred as expected, which confirms that the used spiking procedure
was effective and pre-calculations based on the mass balance were correct.
On the other hand, the theoretical and calculated total PAH concentration on the upconcentrated
smaller samplers have lower recoveries. In the theoretical concentration, an upconcentration by a
factor 10 was presumed. The recovery of PAHs on the small samplers ranged from 14.2% for CT
5 to 65.4% for CT 1. There was a clear trend of increasing upconcentration with decreasing sum
PAH mixture concentration.
53
4.1.3. Upconcentration factor 10 The objective was to prove that the concentration on the samplers was 10x higher after
upconcentration on 10x smaller samplers. However, an upconcentration factor of 10 was not
reached for any of the five CT’s. The upconcentration factors were 6.9, 4.9, 5.7, 3.0 and 1.5 for CT
1 to CT 5, respectively. This shows that the upconcentration process was more efficient for the
concentration treatments with lower ƩC 5 PAHs. The upconcentration factors were plotted versus
the three replicates of each concentration treatment (Figure 15). Only CT 3 seems to be rather
deviant in the trend that the upconcentration factor decreases with increasing concentration
treatment.
Figure 15: Upconcentration factor between non-upconcentrated and upconcentrated samplers
plotted for each concentration treatment.
Possible explanations for the lower upconcentration factors are compound losses due to
volatilization or reaching the maximum capacity of the 0.1 g samplers. These possible explanations
will be discussed in more detail.
4.1.3.1. Compound losses due to volatilization
Despite the fact that PAHs are solids with a rather low volatility at room temperature, compound
losses due to volatilization have to be taken into account. The five PAHs in order of decreasing
volatility are acenaphthene, fluorene, phenanthrene, pyrene and fluoranthene (Table 1). In the
experimental procedure of upconcentration, the extracts of the non-upconcentrated samplers were
concentrated by a rotavapor at a temperature of 40°C. This temperature in combination with the
pressure of 450 mbar and lower can contribute to significant losses of the most volatile PAHs
(acenaphthene, fluorene and possibly phenanthrene). Furthermore, the extracts were brought to
dryness afterwards to exchange the solvent to methanol. This step was in all likelihood responsible
for the highest volatilization losses in the procedure.
54
By comparing the actual ratio of each of the five PAHs with the initial ratio of the stock solution, it
is striking that the biggest losses were observed for acenaphthene and fluorene, the two most
volatile PAHs. This suggests that volatilization could be the responsible process for a big part of
the PAH losses.
4.1.3.2. Capacity of the small samplers
Another explanation for not reaching an upconcentration by a factor 10 might be that the loading
capacity of the small samplers could have been reached. The 10x upconcentrated samplers have
a mass of 0.1 g and a PAH concentration of 512 µg/g, 1132 µg/g, 4523 µg/g, 7822 µg/g, 11685
µg/g for CT 1, CT 2, CT 3, CT 4 and CT 5, respectively. Especially for the last concentration
treatments, these sum PAH concentrations were very high. For CT 5 there were 11685 µg or 11.69
mg of PAHs loaded on a 0.1 g sampler. This equals 11.7% of the mass of the sampler, which
makes it possible that the maximum loading capacity was reached or almost reached.
It also has to be taken into account that the total mixture concentrations used in these experiments
were much higher than the concentrations observed in the marine environment. By comparing the
used concentrations with the concentrations measured in the marine environment by Deschutter et
al. (2015), it can be deducted that actual concentrations in the water phase are a factor 400 000
lower in the North Sea.
4.1.4. Recovery ƩC 5 PAHs on samplers The recovery for the ƩC 5 PAHs on the non-upconcentrated and upconcentrated samplers was
calculated. This recovery corresponds to the ratio of the actual (calculated) concentration on the
sampler to the theoretical expected concentration and is expressed as a percentage (Figure 16).
Recovery on the spiked samplers that were not upconcentrated was close to 100%. The variations
however were relatively high and three replicates of different concentration treatments (CT 1, CT 3
and CT4) showed a recovery higher than 100%. This means that the spiking of these samplers has
continued more efficient than theoretically expected. The theoretical expected concentration was
calculated by Equation 1 and Equation 2. For CT 2 and CT 3 no replicates with over 100% recovery
were found.
The total PAH concentration on the upconcentrated samplers was lower than the theoretical
expected concentration, resulting in lower recoveries. The findings for the recoveries result in lower
upconcentration factors for the five concentration treatments.
55
Figure 16: PAH recovery on the non-upconcentrated samplers (left) and on the upconcentrated
samplers (right).
Statistical analysis by means of five One-Sample t-tests confirmed that for the non-upconcentrated
samplers, there was no statistically significant difference between the actual recoveries and the
theoretical recovery of 100% for CT 1 to CT 5 (Attachment 8). This indicates that the spiking
procedure was very efficient and also that Soxhlet extraction was highly efficient.
One-Sample t-tests for the recoveries of the upconcentrated samplers significantly differed from
the theoretical recovery of 100% for all CTs (Attachment 9). Possible explanations for this
observation have been described earlier in this document.
4.1.5. PAH recovery for each mixture component For each concentration treatment, a subdivision can be made for the recovery of acenaphthene,
fluorene, phenanthrene, fluoranthene and pyrene. In Figure 17, the recovery of the five PAHs in
each concentration treatment was compared for the non-upconcentrated and upconcentrated
samplers. This visual presentation of the recoveries gives an overview of the PAHs that contribute
the most to the lower recovery after upconcentration. For CT 1, CT 2 and CT 3 it becomes
immediately clear that the biggest losses occurred for the three most volatile PAHs, namely
acenaphthene, fluorene and phenanthrene with a vapor pressures of respectively 0.287, 0.043 and
0.016 Pa at 25°C. Acenaphthene is the most volatile and also has the lowest recoveries, followed
by fluorene and phenanthrene respectively. This suggests that volatilization was an important factor
for PAH losses. The reason that these losses were observed for the upconcentrated samplers and
not for the non-upconcentrated samplers can be explained by the working procedure of the
upconcentration process. The major issue with the currently used procedure is that the extracts
were brought to dryness to exchange the solvent. Bringing the extracts to dryness was not an
optimal process step, but was necessary to exchange the solvent to methanol. This step was
required to have an equal spiking procedure for both types of samplers. Methanol was used
because of its well-known partitioning coefficients. Since this is probably the step in the
56
upconcentration procedure that caused the biggest losses, alternatives such as extracting with the
spiking solvent (methanol) should be considered in any possible further studies.
However, the trend that recovery decreases with increasing volatility was not observed for CT 4
and CT 5 at the first sight. In CT 4, the recoveries for the five PAHs were in close proximity of each
other and in CT 5 this trend was not present at all. CT 5 even showed an opposite trend; the
partitioning of acenaphthene was the highest and the partitioning of pyrene was the lowest. This
could be explained by the partitioning coefficients of the PAHs. Acenaphthene partitions faster as
compared to the more heavy PAHs such as pyrene, which means that these faster partitioning
PAHs might have equilibrated more than the slow partitioning PAHs before the capacity limit of the
samplers was reached. By comparing the concentration of the five PAHs on the samplers before
and after upconcentration (Figure 10), the same conclusions could be made for CT 1, CT 2 and CT
3, while in CT 4 and CT 5 no higher compound losses were found for the most volatile PAHs.
57
Figure 17: Recoveries of the five PAHs on the upconcentrated samplers.
4.1.6. PAH recovery as a function of log KOW Recoveries of the five PAHs were plotted in terms of their log KOW values for both the non-
upconcentrated and upconcentrated samplers in CT 1 (Figure 16). CT 1 was selected because the
recoveries/upconcentration increase with decreasing mixture concentration. No linear relationships
were found.
Figure 18: Recovery of each of the PAHs in terms of log Kow before and after upconcentration for CT
1 and CT 5.
58
4.1.7. PAH recovery as function of volatility It was already shown that the PAH recovery was lower for the most volatile compounds. By plotting
the PAH recovery in terms of the logarithm of the vapor pressure, a linear relationship can be found
for the upconcentrated samplers: the PAH recovery decreases with increasing vapor pressure
(Figure 19). For the non-upconcentrated samplers, this relationship was less clear since the
recoveries were around 100% for each PAH. For the upconcentrated samplers, a clear trend could
be observed. This confirms the preliminary conclusion that major compound losses during the
upconcentration procedure were a consequence of volatilization.
Figure 19: Recovery of each of the PAHs in terms of log vapor pressure at 25°C before and after
upconcentration for CT 1.
4.2. Biotesting
4.2.1. Validity of the growth inhibition experiments The second hypothesis was tested by performing a growth inhibition experiment with the big and
small samplers. The experiment was repeated with the same samplers as a control and to
ecotoxicologically check for depletion of the PAHs from the samplers. The validity criteria are based
on the protocol ISO 10253. The first criterion was the factor for cell increase. This factor was
calculated by dividing the average cell density (cells/mL) by the initial cell density (10000 cells/mL).
This factor must be higher than 16 to comply with the norm. Secondly, the coefficient of variation
of the growth rate (CV, in %) should not exceed 7% of the controls. The average sectional CV was
obtained by calculating the average sectional growth rates. The change in temperature and pH
during the experiment should not exceed 1°C and 1 pH-unit, respectively.
For the first growth inhibition experiment, all criteria were fulfilled after 48 and 72 hours. For the
second experiment, the factor for increase in cells was 11.5 after 48 hours, thus lower than 16.
Anyhow, after 72 hours, the factor for increase in cells was fulfilled (32.3). All other parameters met
59
the criteria. Since only the growth rate and inhibition after 72 hours were used, the data could be
used without any restrictions.
4.2.2. PAH concentration in the water phase The PAH concentrations in the aqueous phase after the first growth inhibition experiment were
analyzed by GC-MS. The results are given in Table 22 and compared to the theoretical
concentrations that were determined before the start of the experiments. However, the measured
concentrations did not match with these concentrations. The reason for these deviations can be
found in the estimation of several parameters in the calculation of final aqueous concentrations
after growth inhibition. For example the mass of the test organism P. tricornutum and its C-load or
lipid content were parameters that could not be determined exactly, but they have an influence on
the final exposure concentration. From Table 22, it can be deduced that the actual (calculated)
concentrations in the aqueous phase were a factor 1.7 to 2.4 higher than theoretically expected
with the non-upconcentrated samplers. For the upconcentrated samplers, lower aqueous
concentrations than expected were found for CT 3, CT 4 and CT 5. CT 1 and CT 2 more or less
matched with the expected concentrations. Remarkable was that the calculated concentrations for
CT 5 were considerably lower than expected (268 µg/L instead of 1049 µg/L). This can be related
to the recovery and upconcentration factor that was the lowest for CT 5. A low recovery means a
low upconcentration factor and a low upconcentration factor results in lower aqueous
concentrations because there were less PAHs on the samplers, resulting in a lower equilibrium
concentration. None of the five PAHs in the highest concentration treatment exceeded the solubility
limit in the water phase (Table 1).
Summarized, table 22 indicates that the measured concentrations in the water phase did not match
with the initially determined concentration series (theoretical concentrations). The concentrations
for the non-upconcentrated samplers were approximately a factor two higher than expected, while
the concentrations in the water phase for the upconcentrated samplers were lower than expected,
except for CT 1 and CT 2. The concentrations seem to decrease relatively to the theoretical
concentrations with increasing concentration treatment for the water phase with the upconcentrated
samplers.
However, the fact that the actual concentrations do not equal the initial expectations does not
necessarily have a negative influence on the experiment since we analyzed the PAH
concentrations in the water to be able to work with the actual rather than the nominal water
concentrations.
4.2.3. Growth inhibition experiment 1 The results are expressed as a growth inhibition curve with on the y-axis the percentage growth
inhibition and on the x-axis the logarithm of the actual total aqueous sum PAH concentration in the
water phase (Figure 12). The curves present the inhibition caused by the PAHs provided via
passive dosing from non-upconcentrated and upconcentrated samplers. It can be concluded that
60
both curves coincide very well. The EC50-value for the experiment with the big samplers was 131.8
µg/L, while the EC50 for the small samplers was 109.6 µg/L. When taking into account the natural
variation of the data, it can be stated that both curves were very similar.
It can be concluded that despite the fact that the upconcentration factor 10 was not reached, the
ecotoxicological effect after the upconcentration procedure was still the same, as expected.
4.2.4. Growth inhibition experiment 2 The growth inhibition experiment was repeated under the same conditions, with the same samplers
but with other precultures. This repetition was done to confirm that no significant PAH depletion
occurred after the first experiment and that the same aqueous concentrations and thus inhibition
effect could be obtained.
The growth inhibition curve (Figure 13) does not exactly match the expectations at first glance.
Remarkable was that a negative growth inhibition, or growth stimulation, was observed for CT 1,
CT 2 and CT 3. Because of this, the curve for the non-upconcentrated samplers was shifted
downwards. The EC50-value for the experiment with the big samplers was 93.3 µg/L, while the EC50
for the small samplers was 74.1 µg/L. These value were lower than the first growth inhibition
experiment (respectively 131.8 µg/L and 109.6 µg/L). Since lower EC50 concentrations mean
higher effects, this would presume that the concentrations on the samplers increased after the first
growth inhibition experiment or that the concentrations in the aqueous phase were higher in the
second experiment.
The most plausible explanation for the growth stimulation rather than inhibition for the first
concentration treatments was that the second experiment was not completely randomized. It is
possible that the test flasks were not properly randomized before placing them on the light rack.
The light intensity on the edges can be lower than the intensity in the center. Concentration
treatments with more test flasks in the center can have a higher growth rate because of the more
optimal conditions. Furthermore, the test conditions in the second experiment were not as good as
the first experiment due to the lower factor for cell increase. Measurement of the cell density with
the Coulter Counter was always completely ramdomized.
The fact that both curves do not completely coincide as expected was most probably a
consequence of the surrounding factors and insufficient randomization rather than of depletion of
the PAHs from the samplers.
61
5. Conclusion and future perspectives According to hypothesis 1, was expected that the sampler concentration after the upconcentration
procedure was 10 times higher due to the spiking of the extracts on 10 times smaller samplers
(upconcentration relatively to the size difference of the samplers). The data showed a general trend
for the tested concentration treatments: the upconcentration factor increases with decreasing
mixture concentrations. For the initial ƩC 5 PAHs of 1.72 µg/L (CT 1), a concentration of 10.11 µg/L
was found after upconcentration. This corresponds to an upconcentration factor of 5.9. The
upconcentration factors for the higher concentration treatments were respectively 4.3, 3.7, 2.6 and
1.2. Zooming in on the individual mixture components revealed that the most volatile PAHs resulted
in the lowest recoveries. A linear relationship between the recovery and volatility of the compounds
was found, while no obvious relationship with the log KOW was found. In summary, hypothesis 1 is
not fully achieved, but showed good potential. The limitations can be mostly explained by
volatilization and capacity limits. In general, the experiments showed potential for upconcentrating
environmentally realistic contaminant mixtures becaused the natural concentrations are much
lower than the concentrations used in this research and thus avoiding any capacity limits on the
silicone rubber passive samplers.
Hypothesis 2, that states that the biological response is not influenced by the whole
upconcentration procedure, can be accepted. The growth inhibition curves for the experiment with
non-upconcentrated and upconcentrated samplers were not significantly different from each other.
The EC50-values were respectively 131.8 µg/L and 109.6 µg/L. However, by repeating the growth
inhibition experiment, growth stimulations were observed for the CT 1, CT 2 and CT 3 for the
experiment with the non-upconcentrated samplers. This resulted in a lower accordance between
the two curves and respective EC50-values of 93.3 µg/L and 74.1 µg/L. Because the samplers
reached (or were close to reaching) their maximum loading capacity, it was very unlikely that the
lower EC50-values in the repeated growth inhibition test were caused by depletion of the samplers.
A possible explanation can be found in the not completely randomized test conditions.
Possible further research in upconcentration of passive samplers can take into account some
findings of this thesis. First of all, it is recommended to work with mixture concentrations lower than
the concentrations used in this thesis to avoid that the samplers reach their maximum carrying
capacity. Secondly, it should be taken into account that the most volatile PAHs showed lower
recoveries than the less volatile PAHs. This could be partly explained by the upconcentration
procedure where the extracts were brought to dryness to exchange the solvent. For future
applications it is highly recommended to partially reconsider the upconcentration procedure when
working with rather volatile compounds e.g. by extracting immediately with methanol, the actual
solvent used for spiking the samplers.
In this thesis, an upconcentration factor 10 was tested. A suggestion for future research can be to
perform the upconcentration experiment for different upconcentration factors. For example by
testing with an upconcentration factor 3.2, 10, 32, 100 … (factor 3.2) by varying the size of the
62
silicone rubber samplers. This information could give more insight in the possibilities and limitations
of upconcentrating passive samplers. Another interesting approach could be to work directly with
passive samplers that were deployed in the environment to work with realistic contaminant mixture
concentrations.
63
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Supporting information
Attachment 1: PAH concentration on samplers before and after
upconcentration
Figure S1 – part 1: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 2.
Figure S1 – part 2: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 3.
68
Figure S1 – part 3: Comparison of the concentration of the five PAHs on the sampler before and after
upconcentration for CT 4.
69
Attachment 2: Processing results GC-MS for the non-upconcentrated
samplers The results of the GC-MS report for the extracts of the non-upconcentrated samplers for the five
concentration treatments and their dilutions is given in Table S2 – part 1 - 4. These results were
used to calculate the concentration on the samplers in µg/g as described in the results section.
The labeling of the extracts was done as follows: concentration treatment_replicate_dilution factor.
For example CT1_3_d10 represents replicate three of concentration treatment 1 and was diluted
10 times. IS 1-5 the internal standards acenaphtene-d10, fluorene-d10, phenanthrene-d10,
fluoranthene-d10, pyrene-d10, respectively.
Table S2 – part 1: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
CT1_1
_d5
CT1_1
_d5
CT1_1_
d10
CT1_2
_d5
CT1_2_
d10
CT1_3
_d5
CT1_3_
d10
CT2_1_
d10
CT2_1_
d20
concentrat
ion in
extract
(result
GC-report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphth
ene 0.242 0.258 0.107 0.261 0.107 0.290 0.100 0.262 0.129
fluorene 0.416 0.413 0.149 0.421 0.143 0.467 0.160 0.443 0.199
phenanthr
ene 2.784 2.687 1.094 2.558 1.016 3.035 1.030 3.146 1.320
fluoranthe
ne 2.162 2.022 0.825 2.032 0.834 2.399 0.868 2.389 1.068
pyrene 1.569 1.484 0.567 1.463 0.586 1.726 0.627 1.681 0.791
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.054 0.061 0.027 0.057 0.022 0.053 0.022 0.040 0.017
IS 2 0.105 0.096 0.037 0.098 0.038 0.102 0.028 0.063 0.030
IS 3 0.369 0.361 0.145 0.373 0.160 0.374 0.129 0.289 0.124
IS 4 0.363 0.363 0.160 0.354 0.145 0.399 0.155 0.296 0.132
IS 5 0.391 0.403 0.141 0.369 0.163 0.427 0.157 0.305 0.146
parameter
s needed
to
calculate
results
- - - - - - - - -
weight of
sampler
(g) 1.02 1.02 1.02 1.02 1.02 1.01 1.01 0.99 0.99
70
volume of
extract
(mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
dilution 5.0 5.0 10.0 5.0 10.0 5.0 10.0 10.0 20.0
volume of
IS-working
solution
added to
sample
(µL) 10 10 10 10 10 10 10 20 20
concentrat
ion of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 66.5 66.5 66.50 66.5 66.50 66.5 66.50 66.5 66.50
IS 2 103.0 103.0 103.00 103.0 103.00 103.0 103.00 103.0 103.00
IS 3 401.0 401.0 401.00 401.0 401.00 401.0 401.00 401.0 401.00
IS 4 391.0 391.0 391.00 391.0 391.00 391.0 391.00 391.0 391.00
IS 5 408.0 408.0 408.00 408.0 408.00 408.0 408.00 408.0 408.00
theoretical
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.067 0.067 0.033 0.067 0.033 0.067 0.033 0.067 0.033
IS 2 0.103 0.103 0.052 0.103 0.052 0.103 0.052 0.103 0.052
IS 3 0.401 0.401 0.201 0.401 0.201 0.401 0.201 0.401 0.201
IS 4 0.391 0.391 0.196 0.391 0.196 0.391 0.196 0.391 391.00
IS 5 0.408 0.408 0.204 0.408 0.204 0.408 0.204 0.408 408.00
recovery
IS in
extract (%) % % % % % % % % %
IS 1 82 92 82 85 67 80 65 61 52
IS 2 102 93 72 95 74 99 54 62 59
IS 3 92 90 73 93 80 93 64 72 62
IS 4 93 93 82 91 74 102 79 76 67
IS 5 96 99 69 90 80 105 77 75 72
amount of
compound
in extract
taking into
account
the
volume
and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenaphth
ene 2.423 2.584 2.138 2.608 2.150 2.901 2.006 5.250 5.178
71
fluorene 4.163 4.128 2.981 4.206 2.853 4.674 3.193 8.870 7.957
phenanthr
ene 27.844 26.872 21.877 25.585 20.328 30.352 20.605 62.929 52.801
fluoranthe
ne 21.624 20.224 16.500 20.315 16.681 23.988 17.358 47.774 42.733
pyrene 15.689 14.838 11.342 14.626 11.719 17.264 12.545 33.618 31.640
amount of
compound
in extract
taking into
account
the IS µg µg µg µg µg µg µg µg µg
acenaphth
ene 2.97 2.80 2.61 3.06 3.23 3.61 3.07 8.65 9.97
fluorene 4.08 4.43 4.13 4.42 3.85 4.73 5.88 14.42 13.47
phenanthr
ene 30.26 29.89 30.16 27.54 25.54 32.57 31.97 87.40 85.55
fluoranthe
ne 23.26 21.79 20.22 22.41 22.43 23.53 21.92 63.21 63.38
pyrene 16.36 15.04 16.43 16.16 14.65 16.50 16.28 44.90 44.25
sum 76.93 73.94 73.55 73.60 69.70 80.94 79.12 218.57 216.62
concentrat
ion on
sheet
taking into
account
the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenapht
hene 2.9 2.7 2.6 3.0 3.2 3.6 3.0 8.7 10.1
fluorene 4.0 4.3 4.0 4.3 3.8 4.7 5.8 14.6 13.6
phenanth
rene 29.7 29.3 29.6 27.0 25.0 32.2 31.7 88.3 86.4
fluoranth
ene 22.8 21.4 19.8 22.0 22.0 23.3 21.7 63.8 64.0
pyrene 16.0 14.7 16.1 15.8 14.4 16.3 16.1 45.4 44.7
TOTAL 75.4 72.5 72.1 72.2 68.3 80.1 78.3 220.8 218.8
IS / RS OK OK OK OK OK OK ±
± (OK)
±
72
Table S2 – part 2: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
CT2_2_
d10
CT2_2_
d40
CT2_3_
d10
CT2_3_
d20
CT3_1
_d5
CT3_1_
d20
CT3_2
_d5
CT3_2_
d20
CT3_3
_d5
concentrat
ion in
extract
(result
GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphth
ene 0.469 0.071 0.328 0.145 0.527 0.115 0.643 0.127 0.610
fluorene 0.762 0.102 0.463 0.210 0.788 0.143 1.097 0.183 0.872
phenanthr
ene 5.495 0.789 3.481 1.387 5.884 1.174 7.334 1.187 6.086
fluoranthe
ne 3.946 0.123 2.689 1.213 4.586 0.888 4.527 1.016 4.695
pyrene 3.097 0.100 1.931 0.848 3.329 0.663 3.363 0.825 3.396
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.065 0.012 0.048 0.017 0.131 0.023 0.117 0.031 0.131
IS 2 0.131 0.016 0.073 0.031 0.189 0.038 0.238 0.043 0.205
IS 3 0.480 0.068 0.286 0.116 0.782 0.148 0.961 0.158 0.766
IS 4 0.503 0.013 0.283 0.122 0.783 0.165 0.756 0.167 0.758
IS 5 0.534 0.019 0.296 0.139 0.854 0.166 0.920 0.223 0.835
parameter
s needed
to
calculate
results
- - - - - - - - -
weight of
sampler
(g) 1.00 1.00 1.00 1.00 0.99 0.99 1.00 1.00 1.00
volume of
extract
(mL) 2.0 2.0 2.0 2.0 10.0 10.0 10.0 10.0 10.0
dilution 10.0 40.0 10.0 20.0 5.0 20.0 5.0 20.0 5.0
volume of
IS-working
solution
added to
sample
(µL) 20 20 20 20 100 100 100 100 100
concentrat
ion of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 66.5 66.50 66.5 66.50 66.5 66.50 66.5 66.50 66.5
73
IS 2 103.0 103.00 103.0 103.00 103.0 103.00 103.0 103.00 103.0
IS 3 401.0 401.00 401.0 401.00 401.0 401.00 401.0 401.00 401.0
IS 4 391.0 391.00 391.0 391.00 391.0 391.00 391.0 391.00 391.0
IS 5 408.0 408.00 408.0 408.00 408.0 408.00 408.0 408.00 408.0
theoretical
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.067 0.017 0.067 0.033 0.133 0.033 0.133 0.033 0.133
IS 2 0.103 0.026 0.103 0.052 0.206 0.052 0.206 0.052 0.206
IS 3 0.401 0.100 0.401 0.201 0.802 0.201 0.802 0.201 0.802
IS 4 0.391 0.098 0.391 0.196 0.782 0.196 0.782 0.196 0.782
IS 5 0.408 0.102 0.408 0.204 0.816 0.204 0.816 0.204 0.816
recovery
IS in
extract
(%) % % % % % % % % %
IS 1 98 70 72 52 99 69 88 93 98
IS 2 127 60 71 61 92 75 115 83 99
IS 3 120 68 71 58 97 74 120 79 95
IS 4 129 13 72 63 100 84 97 85 97
IS 5 131 19 73 68 105 81 113 109 102
amount of
compound
in extract
taking into
account
the
volume
and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenaphth
ene 9.379 5.649 6.551 5.803 26.331 23.098 32.164 25.474 30.476
fluorene 15.243 8.135 9.251 8.391 39.380 28.616 54.853 36.554 43.597
phenanthr
ene 109.902 63.124 69.613 55.471
294.20
4 234.894
366.69
0 237.408
304.31
5
fluoranthe
ne 78.916 9.859 53.785 48.504
229.28
9 177.680
226.36
0 203.209
234.75
2
pyrene 61.934 7.975 38.622 33.933
166.43
9 132.617
168.15
9 165.054
169.78
6
amount of
compound
in extract
taking into
account
the IS µg µg µg µg µg µg µg µg µg
74
acenaphth
ene 9.57 8.02 9.04 11.25 26.64 33.67 36.54 27.52 31.01
fluorene 11.98 13.49 13.09 13.85 42.96 38.34 47.54 44.27 43.85
phenanthr
ene 91.78 92.74 97.67 95.48 301.77 318.08 306.12 302.16 318.68
fluoranthe
ne 61.32 74.20 74.23 77.57 229.02 210.87 234.27 237.95 242.09
pyrene 47.31 42.50 53.22 49.96 159.03 162.99 149.08 151.07 166.02
sum 221.97 230.95 247.25 248.11 759.42 763.94 773.56 762.97 801.65
concentrat
ion on
sheet
taking into
account
the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenapht
hene 9.6 8.0 9.0 11.2 26.9 34.0 36.5 27.5 31.0
fluorene 12.0 13.5 13.1 13.9 43.4 38.7 47.5 44.3 43.9
phenanth
rene 91.8 92.7 97.7 95.5 304.8 321.3 306.1 302.2 318.7
fluoranth
ene 61.3 74.2 74.2 77.6 231.3 213.0 234.3 238.0 242.1
pyrene 47.3 42.5 53.2 50.0 160.6 164.6 149.1 151.1 166.0
TOTAL 222.0 230.9 247.2 248.1 767.1 771.7 773.6 763.0 801.7
IS / RS ± (OK) NOK ± (OK) ± (OK) OK OK NOK OK OK
75
Table S2 – part 3: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
CT3_3
_d20
CT4_1
_d10
CT4_1_
d100
CT4_2
_d10
CT4_2_
d100
CT4_3
_d10
CT4_3
_d10
CT5_1
_d50
CT5_1_
d200
concentra
tion in
extract
(result
GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenapht
hene 0.125 0.887 0.067 1.126 0.106 1.363 0.067 0.581 0.250
fluorene 0.190 1.362 0.090 1.687 0.158 2.162 0.097 0.842 0.352
phenanth
rene 1.313 9.328 0.695 11.377 1.094 8.522 0.751 5.363 2.296
fluoranthe
ne 1.007 7.369 0.578 8.803 0.891 12.627 0.622 4.893 1.678
pyrene 0.748 5.253 0.424 6.580 0.638 9.435 0.427 3.494 1.249
IS-
concentra
tion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.025 0.112 0.009 0.160 0.016 0.182 0.011 0.125 0.058
IS 2 0.039 0.190 0.016 0.242 0.022 0.322 0.014 0.198 0.075
IS 3 0.153 0.767 0.055 0.892 0.087 0.739 0.063 0.666 0.289
IS 4 0.156 0.766 0.056 0.890 0.087 1.273 0.064 0.795 0.272
IS 5 0.179 0.846 0.056 0.954 0.081 1.408 0.062 0.902 0.270
paramete
rs needed
to
calculate
results
- - - - - - - - -
weight of
sampler
(g) 1.00 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99
volume of
extract
(mL) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
dilution 20.0 10.0 100.0 10.0 100.0 10.0 100.0 50.0 200.0
volume of
IS-
working
solution
added to
sample
(µL) 100 20 20 20 20 20 20 100 100
concentra
tion of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
76
IS 1 66.50 665 665.00 665 665.00 665 665.00 665 665.00
IS 2 103.00 1030 1030.00 1030 1030.00 1030
1030.0
0 1030 1030.00
IS 3 401.00 4010 4010.00 4010 4010.00 4010
4010.0
0 4010 4010.00
IS 4 391.00 3910 3910.00 3910 3910.00 3910
3910.0
0 3910 3910.00
IS 5 408.00 4080 4080.00 4080 4080.00 4080
4080.0
0 4080 4080.00
theoretica
l IS-
concentra
tion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.033 0.133 0.013 0.133 0.013 0.133 0.013 0.133 0.033
IS 2 0.052 0.206 0.021 0.206 0.021 0.206 0.021 0.206 0.052
IS 3 0.201 0.802 0.080 0.802 0.080 0.802 0.080 0.802 0.201
IS 4 0.196 0.782 0.078 0.782 0.078 0.782 0.078 0.782 0.196
IS 5 0.204 0.816 0.082 0.816 0.082 0.816 0.082 0.816 0.204
recovery
IS in
extract
(%) % % % % % % % % %
IS 1 74 84 70 120 117 137 83 94 173
IS 2 76 92 77 117 105 156 68 96 146
IS 3 76 96 69 111 109 92 79 83 144
IS 4 80 98 71 114 112 163 82 102 139
IS 5 88 104 69 117 99 173 76 111 132
amount of
compoun
d in
extract
taking
into
account
the
volume
and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenapht
hene 25.05 88.70 66.83 112.58 105.67 136.26 67.10 290.4 500.8
fluorene 37.95 136.23 90.01 168.73 157.79 216.21 97.20 421.0 703.4
phenanth
rene 262.68 932.83 695.31
1137.6
5 1093.97 852.18 750.80 2681.7 4592.9
fluoranthe
ne 201.31 736.92 577.70 880.32 891.16
1262.6
7 622.26 2446.6 3355.9
pyrene 149.53 525.31 423.86 657.98 638.08 943.51 426.50 1746.9 2497.9
77
amount of
compoun
d in
extract
taking
into
account
the IS µg µg µg µg µg µg µg µg µg
acenapht
hene 33.86 104.98 94.82 93.78 90.46 99.68 80.89 308.7 289.07
fluorene 50.21 147.32 117.09 143.76 149.67 138.44 143.25 438.7 480.16
phenanth
rene 343.61 975.58 1011.93
1022.6
0 1003.63 925.18 952.24 3230.0 3187.44
fluoranthe
ne 252.57 752.63 812.72 773.86 796.49 775.76 757.96 2405.4 2409.68
pyrene 170.34 506.69 618.21 562.83 644.98 546.76 559.23 1580.1 1888.45
sum 850.58
concentra
tion on
sheet
taking
into
account
the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenapht
hene 33.9 106.0 95.8 94.7 91.4 100.7 81.7 311.8 292.0
fluorene 50.2 148.8 118.3 145.2 151.2 139.8 144.7 443.1 485.0
phenant
hrene 343.6 985.4 1022.2 1032.9 1013.8 934.5 961.9 3262.6 3219.6
fluoranth
ene 252.6 760.2 820.9 781.7 804.5 783.6 765.6 2429.7 2434.0
pyrene 170.3 511.8 624.5 568.5 651.5 552.3 564.9 1596.0 1907.5
TOTAL 850.6 2512.3 2681.6 2623.1 2712.3 2510.9 2518.8 8043.2 8338.2
IS / RS OK OK OK OK OK NOK OK OK NOK
78
Table S2 – part 4: Calculation of the PAH concentration on the non-upconcentrated samplers based
on the measured extract concentrations.
CT5_2_
d50
CT5_2_d
200
CT5_3_
d50
CT5_3_d
200
Bl_1_
d0
Bl_1_
d5
Bl_2_
d0
Bl_2_
d5
Bl_3_
d0
concentrati
on in
extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphthe
ne 0.494 0.216 0.575 0.244 0.202 0.054 0.661 0.109 0.411
fluorene 0.707 0.290 0.833 0.371 0.328 0.095 0.826 0.158 0.563
phenanthre
ne 4.864 2.061 5.717 2.500 2.178 0.573 4.741 1.000 3.222
fluoranthen
e 3.797 1.617 4.553 2.241 1.568 0.459 2.899 0.761 2.354
pyrene 2.718 1.188 3.345 1.660 1.203 0.330 1.968 0.484 1.642
IS-
concentrati
on in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.115 0.046 0.135 0.053 0.312 0.047 0.466 0.078 0.384
IS 2 0.166 0.086 0.196 0.091 0.465 0.081 0.611 0.112 0.505
IS 3 0.640 0.273 0.749 0.344 1.691 0.318 2.030 0.415 1.714
IS 4 0.670 0.263 0.763 0.359 1.471 0.340 1.594 0.417 1.655
IS 5 0.723 0.298 0.874 0.401 1.512 0.324 1.645 0.410 1.659
parameters
needed to
calculate
results
- - - - - - - - -
weight of
sampler (g) 1.00 1.00 0.99 0.99 0.95 0.99 0.99 0.99 0.99
volume of
extract
(mL) 10.0 10.0 10.0 10.0 2.0 2.0 2.0 2.0 2.0
dilution 50.0 200.0 50.0 200.0 1.0 5.0 1.0 5.0 1.0
volume of
IS-working
solution
added to
sample (µL) 100 100 100 100 10 10 10 10 10
concentrati
on of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 665 665 665 665 66.5 66.50 66.5 66.5 66.50
IS 2 1030 1030 1030 1030 103.0 103.0 103.0 103.0 103.0
IS 3 4010 4010 4010 4010 401.0 401.0 401.0 401.0 401.0
IS 4 3910 3910 3910 3910 391.0 391.0 391.0 391.0 391.0
79
IS 5 4080 4080 4080 4080 408.0 408.0 408.0 408.0 408.0
theoretical
IS-
concentrati
on in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.133 0.033 0.133 0.033 0.333 0.067 0.333 0.067 0.333
IS 2 0.206 0.052 0.206 0.052 0.515 0.103 0.515 0.103 0.515
IS 3 0.802 0.201 0.802 0.201 2.005 0.401 2.005 0.401 2.005
IS 4 0.782 0.196 0.782 0.196 1.955 0.391 1.955 0.391 1.955
IS 5 0.816 0.204 0.816 0.204 2.040 0.408 2.040 0.408 2.040
recovery IS
in extract
(%) % % % % % % % % %
IS 1 86 140 102 159 94 71 140 117 115
IS 2 81 167 95 177 90 78 119 109 98
IS 3 80 136 93 172 84 79 101 104 86
IS 4 86 134 98 184 75 87 82 107 85
IS 5 89 146 107 197 74 79 81 100 81
amount of
compound
in extract
taking into
account the
volume and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenaphthe
ne 246.863 432.526 287.545 487.279 0.404 0.542 1.323 1.091 0.822
fluorene 353.344 579.933 416.694 741.214 0.656 0.945 1.653 1.580 1.125
phenanthre
ne
2432.23
1 4121.997
2858.50
8 5000.999 4.356 5.728 9.482
10.00
0 6.443
fluoranthen
e
1898.65
8 3233.713
2276.37
9 4482.984 3.137 4.593 5.798 7.609 4.709
pyrene
1359.12
1 2375.989
1672.48
6 3319.227 2.405 3.295 3.936 4.844 3.283
amount of
compound
in extract
taking into
account the
IS µg µg µg µg µg µg µg µg µg
acenaphthe
ne 285.71 309.43 283.02 306.98 0.43 0.77 0.94 0.93 0.71
fluorene 438.77 347.37 438.41 419.04 0.73 1.21 1.39 1.45 1.15
phenanthre
ne 3045.93 3023.61 3060.60 2913.19 5.16 7.23 9.36 9.66 7.54
fluoranthen
e 2214.50 2407.56 2331.77 2442.99 4.17 5.28 7.11 7.13 5.56
80
pyrene 1533.58 1626.52 1561.21 1689.07 3.25 4.15 4.88 4.82 4.04
sum 7518.49 7714.51 7675.00 7771.27 13.74 18.63 23.70 24.00 18.99
concentrati
on on sheet
taking into
account the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenaphth
ene 285.7 309.4 285.9 310.1 0.5 0.8 1.0 0.9 0.7
fluorene 438.8 347.4 442.8 423.3 0.8 1.2 1.4 1.5 1.2
phenanthr
ene 3045.9 3023.6 3091.5 2942.6 5.4 7.3 9.5 9.8 7.6
fluoranthe
ne 2214.5 2407.6 2355.3 2467.7 4.4 5.3 7.2 7.2 5.6
pyrene 1533.6 1626.5 1577.0 1706.1 3.4 4.2 4.9 4.9 4.1
TOTAL 7518.5 7714.5 7752.5 7849.8 14.5 18.8 23.9 24.2 19.2
IS / RS OK NOK OK NOK OK OK
±
(OK) OK OK
81
Attachment 3: Processing results GC-MS for the upconcentrated
samplers The results of the GC-MS report for the extracts of the upconcentrated samplers for the five
concentration treatments and their dilutions is given in Table S3 – part 1 - 4. These results were
used to calculate the concentration on the samplers in µg/g as described in the results section.
The labeling of the extracts was done as follows: concentration treatment_replicate_dilution factor.
Internal standard (IS 1-5) represent acenaphtene-d10, fluorene-d10, phenanthrene-d10,
fluoranthene-d10, pyrene-d10 respectively.
Table S3 – part 1: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
CT1_1
_d5
CT1_2
_d5
CT2_1_
d10
CT2_2_
d10
CT2_3_
d10
CT2_3_
d20
CT3_1
_d5
CT3_1
_d5
CT3_1_
d10
concentrat
ion in
extract
(result
GC-report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphth
ene 0.139 0.096 0.102 0.115 0.151 0.130 0.188 0.205 0.091
fluorene 0.282 0.286 0.200 0.221 0.241 0.226 0.321 0.333 0.178
phenanthr
ene 1.858 2.262 1.619 1.564 1.942 1.662 2.799 2.742 1.387
fluoranthe
ne 1.740 2.129 1.560 1.460 1.771 1.621 2.555 2.386 1.398
pyrene 1.255 1.563 1.113 1.051 1.326 1.210 1.859 1.742 0.976
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.078 0.065 0.054 0.056 0.064 0.042 0.131 0.127 0.063
IS 2 0.100 0.122 0.087 0.085 0.082 0.069 0.208 0.212 0.099
IS 3 0.371 0.504 0.320 0.318 0.354 0.283 0.761 0.719 0.401
IS 4 0.372 0.545 0.327 0.337 0.328 0.331 0.775 0.714 0.408
IS 5 0.414 0.588 0.362 0.364 0.373 0.333 0.843 0.794 0.423
parameter
s needed
to
calculate
results
- - - - - - - - -
weight of
sampler
(g) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
volume of
extract
(mL) 2.0 2.0 2.0 2.0 2.0 2.0 10.0 10.0 10.0
dilution 5.0 5.0 10.0 10.0 10.0 20.0 5.0 5.0 10.0
82
volume of
IS-working
solution
added to
sample
(µL) 10 10 20 20 20 20 100 100 100
concentrat
ion of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 66.5 66.5 66.5 66.5 66.5 66.50 66.5 66.5 66.50
IS 2 103.0 103.0 103.0 103.0 103.0 103.00 103.0 103.0 103.00
IS 3 401.0 401.0 401.0 401.0 401.0 401.00 401.0 401.0 401.00
IS 4 391.0 391.0 391.0 391.0 391.0 391.00 391.0 391.0 391.00
IS 5 408.0 408.0 408.0 408.0 408.0 408.00 408.0 408.0 408.00
theoretical
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.067 0.067 0.067 0.067 0.067 0.033 0.133 0.133 0.067
IS 2 0.103 0.103 0.103 0.103 0.103 0.052 0.206 0.206 0.103
IS 3 0.401 0.401 0.401 0.401 0.401 0.201 0.802 0.802 0.401
IS 4 0.391 0.391 0.391 0.391 0.391 0.196 0.782 0.782 0.391
IS 5 0.408 0.408 0.408 0.408 0.408 0.204 0.816 0.816 0.408
recovery
IS in
extract (%) % % % % % % % % %
IS 1 117 97 82 85 96 127 99 95 95
IS 2 98 118 84 82 80 135 101 103 96
IS 3 93 126 80 79 88 141 95 90 100
IS 4 95 139 84 86 84 169 99 91 104
IS 5 102 144 89 89 91 163 103 97 104
amount of
compound
in extract
taking into
account
the
volume
and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenaphth
ene 1.386 0.961 2.041 2.295 3.011 5.191 9.380 10.233 9.091
fluorene 2.816 2.863 4.006 4.427 4.818 9.039 16.072 16.661 17.846
phenanthr
ene 18.577 22.624 32.377 31.275 38.838 66.471
139.95
1
137.10
4 138.736
83
fluoranthe
ne 17.398 21.294 31.196 29.203 35.419 64.855
127.73
9
119.30
9 139.839
pyrene 12.550 15.626 22.255 21.028 26.522 48.416 92.967 87.090 97.589
amount of
compound
in extract
taking into
account
the IS µg µg µg µg µg µg µg µg µg
acenaphth
ene 1.19 0.99 2.49 2.71 3.13 4.09 9.51 10.72 9.62
fluorene 2.89 2.42 4.76 5.37 6.02 6.70 15.91 16.23 18.49
phenanthr
ene 20.07 18.00 40.56 39.38 43.98 47.09 147.41 152.90 138.87
fluoranthe
ne 18.28 15.28 37.32 33.93 42.22 38.34 128.90 130.65 134.12
pyrene 12.36 10.85 25.08 23.55 29.03 29.64 89.96 89.47 94.05
sum 54.79 47.54 110.21 104.94 124.38 125.86 391.69 399.97 395.15
concentrat
ion on
sheet
taking into
account
the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenapht
hene 11.9 9.9 24.9 27.1 31.3 40.9 95.1 107.2 96.2
fluorene 28.9 24.2 47.6 53.7 60.2 67.0 159.1 162.3 184.9
phenanth
rene 200.7 180.0 405.6 393.8 439.8 470.9 1474.1 1529.0 1388.7
fluoranth
ene 182.8 152.8 373.2 339.3 422.2 383.4 1289.0 1306.5 1341.2
pyrene 123.6 108.5 250.8 235.5 290.3 296.4 899.6 894.7 940.5
TOTAL 547.9 475.4 1102.1 1049.4 1243.8 1258.6 3916.9 3999.7 3951.5
IS / RS OK ± OK OK OK ± OK OK OK
84
Table S3 – part 2: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
CT3_2
_d5
CT3_2_
d10
CT3_3
_d5
CT3_3_
d10
CT4_1_
d10
CT4_1_
d20
CT4_2_
d10
CT4_2_
d20
CT4_3_
d10
concentrat
ion in
extract
(result
GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphth
ene 0.220 0.111 0.205 0.112 0.313 0.120 0.255 0.106 0.331
fluorene 0.426 0.213 0.367 0.176 0.496 0.182 0.394 0.159 0.501
phenanthr
ene 3.573 1.791 3.246 1.365 3.397 1.422 3.037 1.263 3.487
fluoranthe
ne 3.438 1.857 3.081 1.561 2.126 0.854 2.241 0.750 2.498
pyrene 2.665 1.408 2.351 1.159 1.350 0.540 1.475 0.540 1.763
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.141 0.077 0.142 0.057 0.143 0.050 0.135 0.065 0.133
IS 2 0.221 0.110 0.215 0.119 0.210 0.082 0.195 0.071 0.207
IS 3 0.788 0.398 0.815 0.341 0.817 0.346 0.770 0.321 0.766
IS 4 0.788 0.419 0.774 0.358 0.808 0.337 0.806 0.285 0.812
IS 5 0.854 0.440 0.853 0.418 0.896 0.332 0.852 0.302 0.807
parameter
s needed
to
calculate
results
- - - - - - - - -
weight of
sampler
(g) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
volume of
extract
(mL) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
dilution 5.0 10.0 5.0 10.0 10.0 20.0 10.0 20.0 10.0
volume of
IS-
working
solution
added to
sample
(µL) 100 100 100 100 20 20 20 20 20
concentrat
ion of IS-
working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
85
IS 1 66.5 66.50 66.5 66.50 665 665.00 665 665.00 665
IS 2 103.0 103.00 103.0 103.00 1030 1030.00 1030 1030.00 1030
IS 3 401.0 401.00 401.0 401.00 4010 4010.00 4010 4010.00 4010
IS 4 391.0 391.00 391.0 391.00 3910 3910.00 3910 3910.00 3910
IS 5 408.0 408.00 408.0 408.00 4080 4080.00 4080 4080.00 4080
theoretical
IS-
concentrat
ion in
extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.133 0.067 0.133 0.067 0.133 0.067 0.133 0.067 0.133
IS 2 0.206 0.103 0.206 0.103 0.206 0.103 0.206 0.103 0.206
IS 3 0.802 0.401 0.802 0.401 0.802 0.401 0.802 0.401 0.802
IS 4 0.782 0.391 0.782 0.391 0.782 0.391 0.782 0.391 0.782
IS 5 0.816 0.408 0.816 0.408 0.816 0.408 0.816 0.408 0.816
recovery
IS in
extract
(%) % % % % % % % % %
IS 1 106 116 107 85 108 76 101 97 100
IS 2 107 107 104 116 102 80 95 69 100
IS 3 98 99 102 85 102 86 96 80 96
IS 4 101 107 99 91 103 86 103 73 104
IS 5 105 108 104 102 110 81 104 74 99
amount of
compound
in extract
taking into
account
the
volume
and
dilution of
extract µg µg µg µg µg µg µg µg µg
acenaphth
ene 10.994 11.131 10.269 11.160 31.253 24.003 25.520 21.176 33.096
fluorene 21.296 21.311 18.329 17.606 49.644 36.449 39.404 31.773 50.114
phenanthr
ene
178.64
7 179.079
162.28
1 136.454 339.668 284.455 303.675 252.606 348.729
fluoranthe
ne
171.90
2 185.678
154.04
8 156.121 212.647 170.822 224.113 150.089 249.794
pyrene
133.27
2 140.789
117.56
9 115.877 135.050 107.957 147.518 107.911 176.275
amount of
compound
in extract
taking into
account
the IS µg µg µg µg µg µg µg µg µg
86
acenaphth
ene 10.36 9.60 9.64 13.07 29.06 31.70 25.17 21.78 33.15
fluorene 19.82 19.98 17.58 15.23 48.61 45.69 41.56 46.19 49.88
phenanthr
ene 181.93 180.41 159.62 160.41 333.30 329.41 316.39 315.12 365.09
fluoranthe
ne 170.57 173.16 155.66 170.68 205.76 198.10 217.47 206.19 240.48
pyrene 127.36 130.49 112.53 113.13 122.99 132.79 141.33 145.97 178.17
sum 510.03 513.64 455.03 472.52 739.73 737.69 741.92 735.24 866.78
concentrat
ion on
sheet
taking into
account
the
sampler
weight
(µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenapht
hene 103.6 96.0 96.4 130.7 290.6 317.0 251.7 217.8 331.5
fluorene 198.2 199.8 175.8 152.3 486.1 456.9 415.6 461.9 498.8
phenanth
rene 1819.3 1804.1 1596.2 1604.1 3333.0 3294.1 3163.9 3151.2 3650.9
fluoranth
ene 1705.7 1731.6 1556.6 1706.8 2057.6 1981.0 2174.7 2061.9 2404.8
pyrene 1273.6 1304.9 1125.3 1131.3 1229.9 1327.9 1413.3 1459.7 1781.7
TOTAL 5100.3 5136.4 4550.3 4725.2 7397.3 7376.9 7419.2 7352.4 8667.8
IS / RS OK OK OK OK OK OK OK OK OK
87
Table S3 – part 3: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
CT4_3_d20 CT5_1_d50 CT5_1_d10 CT5_3_d50 CT5_3_10
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml
acenaphthene 0.122 0.177 1.705 0.207 2.253
fluorene 0.185 0.223 1.681 0.212 2.309
phenanthrene 1.414 0.976 7.767 0.867 8.078
fluoranthene 1.058 0.499 4.501 0.465 4.759
pyrene 0.786
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.055 0.121 1.217 0.128 1.290
IS 2 0.081 0.195 1.592 0.175 1.977
IS 3 0.309 0.712 6.695 0.715 7.126
IS 4 0.316 0.677 6.845 0.735 7.716
IS 5 0.346 0.722 7.254 0.798 8.078
parameters
needed to
calculate
results
- - - - -
weight of
sampler (g) 0.10 0.10 0.10 0.10 0.10
volume of
extract (mL) 10.0 10.0 10.0 10.0 10.0
dilution 20.0 50.0 10.0 50.0 10.0
volume of IS-
working
solution added
to sample (µL) 20 100 100 100 100
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 665.00 665 665.00 665 665.00
IS 2 1030.00 1030 1030.00 1030 1030.00
IS 3 4010.00 4010 4010.00 4010 4010.00
IS 4 3910.00 3910 3910.00 3910 3910.00
IS 5 4080.00 4080 4080.00 4080 4080.00
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.067 0.133 0.665 0.133 0.665
IS 2 0.103 0.206 1.030 0.206 1.030
IS 3 0.401 0.802 4.010 0.802 4.010
IS 4 0.391 0.782 3.910 0.782 3.910
88
IS 5 0.408 0.816 4.080 0.816 4.080
recovery IS in
extract (%) % % % % %
IS 1 82 91 183 96 194
IS 2 78 94 155 85 192
IS 3 77 89 167 89 178
IS 4 81 87 175 94 197
IS 5 85 88 178 98 198
amount of
compound in
extract taking
into account
the volume
and dilution of
extract µg µg µg µg µg
acenaphthene 24.427 88.417 170.493 103.520 225.257
fluorene 37.052 111.638 168.084 105.990 230.895
phenanthrene 282.895 488.076 776.742 433.652 807.826
fluoranthene 211.565 249.557 450.058 232.598 475.881
pyrene 157.257 151.784 287.211 142.067 297.490
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg
acenaphthene 29.69 118.16 108.76 124.61 120.31
fluorene 47.23 550.00 465.22 486.57 454.55
phenanthrene 367.53 288.26 257.08 247.41 241.14
fluoranthene 261.81 171.57 161.53 145.22 150.25
pyrene 185.46 97.41 93.15 107.80 116.13
sum 891.73 1225.39 1085.74 1111.61 1082.39
concentration
on sheet
taking into
account the
sampler
weight (µg/g) µg/g µg/g µg/g µg/g µg/g
acenaphthene 296,9 974,1 931,5 1078,0 1161,3
fluorene 472,3 1181,6 1087,6 1246,1 1203,1
phenanthrene 3675,3 5500,0 4652,2 4865,7 4545,5
fluoranthene 2618,1 2882,6 2570,8 2474,1 2411,4
pyrene 1854,6 1715,7 1615,3 1452,2 1502,5
TOTAL 8917,3 12253,9 10857,4 11116,1 10823,9
IS / RS OK OK NOK OK ±
89
Table S3 – part 4: Calculation of the PAH concentration on the upconcentrated samplers based on
the measured extract concentrations.
Bl_1_d
0
Bl_1_d
5
Bl_1_d
0
Bl_2_d
0
Bl_2_d
5
Bl_2_d
0
Bl_3_d
0
Bl_3_d
5
Bl_3_d
0
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphthen
e 0.031 0.019 0.033 0.109 0.032 0.116 0.040 0.024 0.054
fluorene 0.060 0.027 0.052 0.145 0.058 0.162 0.087 0.033 0.083
phenanthrene 0.240 0.142 0.220 0.677 0.359 0.679 0.491 0.231 0.488
fluoranthene 0.183 0.112 0.154 0.453 0.231 0.443 0.480 0.221 0.556
pyrene 0.133 0.073 0.126 0.319 0.172 0.340 0.360 0.161 0.418
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.227 0.083 0.213 0.379 0.075 0.381 0.338 0.064 0.327
IS 2 0.345 0.138 0.338 0.512 0.099 0.597 0.446 0.085 0.474
IS 3 1.259 0.463 1.185 1.799 0.351 1.768 1.646 0.339 1.602
IS 4 1.254 0.478 1.140 1.526 0.349 1.582 1.295 0.362 1.513
IS 5 1.435 0.504 1.254 1.640 0.364 1.698 1.311 0.393 1.533
parameters
needed to
calculate
results
- - - - - - - - -
weight of
sampler (g) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
volume of
extract (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
dilution 1.0 5.0 1.0 1.0 5.0 1.0 1.0 5.0 1.0
volume of IS-
working
solution
added to
sample (µL) 10 10 10 10 10 10 10 10 10
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 66.5 66.5 66.50 66.5 66.5 66.50 66.5 66.5 66.50
IS 2 103.0 103.0 103.00 103.0 103.0 103.00 103.0 103.0 103.00
IS 3 401.0 401.0 401.00 401.0 401.0 401.00 401.0 401.0 401.00
IS 4 391.0 391.0 391.00 391.0 391.0 391.00 391.0 391.0 391.00
IS 5 408.0 408.0 408.00 408.0 408.0 408.00 408.0 408.0 408.00
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.333 0.067 0.333 0.333 0.067 0.333 0.333 0.067 0.333
90
IS 2 0.515 0.103 0.515 0.515 0.103 0.515 0.515 0.103 0.515
IS 3 2.005 0.401 2.005 2.005 0.401 2.005 2.005 0.401 2.005
IS 4 1.955 0.391 1.955 1.955 0.391 1.955 1.955 0.391 1.955
IS 5 2.040 0.408 2.040 2.040 0.408 2.040 2.040 0.408 2.040
recovery IS in
extract (%) % % % % % % % % %
IS 1 68 125 64 114 112 115 102 96 98
IS 2 67 134 66 99 96 116 87 83 92
IS 3 63 116 59 90 88 88 82 85 80
IS 4 64 122 58 78 89 81 66 92 77
IS 5 70 124 61 80 89 83 64 96 75
amount of
compound in
extract taking
into account
the volume
and dilution
of extract µg µg µg µg µg µg µg µg µg
acenaphthen
e 0.063 0.191 0.065 0.219 0.320 0.232 0.081 0.236 0.108
fluorene 0.119 0.272 0.105 0.290 0.578 0.324 0.175 0.332 0.166
phenanthrene 0.481 1.421 0.439 1.354 3.590 1.358 0.983 2.308 0.976
fluoranthene 0.366 1.116 0.308 0.905 2.310 0.886 0.960 2.208 1.112
pyrene 0.266 0.726 0.253 0.639 1.725 0.680 0.719 1.607 0.836
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg µg µg µg µg
acenaphthen
e 0.09 0.15 0.10 0.19 0.28 0.20 0.08 0.25 0.11
fluorene 0.18 0.20 0.16 0.29 0.60 0.28 0.20 0.40 0.18
phenanthrene 0.77 1.23 0.74 1.51 4.10 1.54 1.20 2.73 1.22
fluoranthene 0.57 0.91 0.53 1.16 2.59 1.10 1.45 2.39 1.44
pyrene 0.38 0.59 0.41 0.79 1.93 0.82 1.12 1.67 1.11
sum 1.98 3.09 1.94 3.95 9.51 3.93 4.05 7.43 4.06
concentration
on sheet
taking into
account the
sampler
weight (µg/g) µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g
acenaphthen
e 0.9 1.5 1.0 1.9 2.8 2.0 0.8 2.5 1.1
fluorene 1.8 2.0 1.6 2.9 6.0 2.8 2.0 4.0 1.8
phenanthren
e 7.7 12.3 7.4 15.1 41.0 15.4 12.0 27.3 12.2
fluoranthene 5.7 9.1 5.3 11.6 25.9 11.0 14.5 23.9 14.4
pyrene 3.8 5.9 4.1 7.9 19.3 8.2 11.2 16.7 11.1
91
TOTAL 19.8 30.9 19.4 39.5 95.1 39.3 40.5 74.3 40.6
IS / RS ± (OK) ± (OK) OK OK OK OK OK OK OK
92
Attachment 4: Processing results GC-MS for aqueous concentrations
non-upconentated samplers Table S4 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with non-upconcentrated samplers.
CT1_1 CT1_2 CT1_3 CT2_1 CT2_2 CT2_3 CT3_1 CT3_2 CT3_3
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphthene 0.006 0.004 0.005 0.069 0.012 0.049 0.049 0.047 0.036
fluorene 0.010 0.008 0.006 0.128 0.026 0.086 0.099 0.101 0.075
phenanthrene 0.077 0.060 0.059 0.915 0.181 0.628 0.797 0.826 0.627
fluoranthene 0.037 0.029 0.029 0.610 0.079 0.417 0.320 0.362 0.275
pyrene 0.024 0.023 0.020 0.388 0.056 0.313 0.199 0.246 0.175
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.046 0.035 0.028 0.090 0.021 0.034 0.065 0.064 0.061
IS 2 0.058 0.040 0.036 0.111 0.028 0.045 0.071 0.074 0.076
IS 3 0.060 0.046 0.038 0.158 0.026 0.085 0.080 0.080 0.075
IS 4 0.074 0.058 0.041 0.173 0.036 0.097 0.087 0.096 0.088
IS 5 0.070 0.057 0.051 0.181 0.034 0.106 0.086 0.096 0.084
parameters
needed to
calculate
results
- - - - - - - - -
weight of
extract (g) 36.080 41.009 41.345 40.392 42.335 42.412 40.686 41.243 38.881
final volume of
extract (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
dilution 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
volume of IS-
working
solution added
to extract (µL) 25 25 25 25 25 25 50 50 50
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
IS 2 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04
IS 3 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03
IS 4 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01
IS 5 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.050 0.050 0.050 0.050 0.050 0.050 0.100 0.100 0.100
93
IS 2 0.052 0.052 0.052 0.052 0.052 0.052 0.104 0.104 0.104
IS 3 0.052 0.052 0.052 0.052 0.052 0.052 0.103 0.103 0.103
IS 4 0.051 0.051 0.051 0.051 0.051 0.051 0.101 0.101 0.101
IS 5 0.053 0.053 0.053 0.053 0.053 0.053 0.105 0.105 0.105
recovery IS in
extract (%) % % % % % % % % %
IS 1 92.5 70.2 56.6 180.9 41.7 69.1 65.6 64.3 61.4
IS 2 111.8 77.1 68.8 214.1 54.7 85.7 68.6 71.6 73.0
IS 3 117.3 89.3 73.3 307.2 50.5 164.7 77.7 78.0 72.9
IS 4 146.1 114.4 80.5 342.3 71.8 192.9 85.7 94.9 87.4
IS 5 133.2 108.6 97.7 344.5 65.2 202.6 81.5 91.4 79.9
amount of
compound in
extract taking
into account
the volume and
dilution of the
extract µg µg µg µg µg µg µg µg µg
acenaphthene 0.003 0.002 0.002 0.034 0.006 0.025 0.024 0.024 0.018
fluorene 0.005 0.004 0.003 0.064 0.013 0.043 0.049 0.050 0.038
phenanthrene 0.039 0.030 0.029 0.457 0.091 0.314 0.399 0.413 0.313
fluoranthene 0.018 0.015 0.015 0.305 0.040 0.208 0.160 0.181 0.137
pyrene 0.012 0.011 0.010 0.194 0.028 0.156 0.100 0.123 0.088
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg µg µg µg µg
acenaphthene 0.003 0.003 0.004 0.019 0.014 0.036 0.037 0.037 0.030
fluorene 0.004 0.005 0.005 0.030 0.024 0.050 0.072 0.070 0.052
phenanthrene 0.033 0.034 0.040 0.149 0.180 0.191 0.513 0.530 0.430
fluoranthene 0.013 0.013 0.018 0.089 0.055 0.108 0.186 0.191 0.157
pyrene 0.009 0.010 0.010 0.056 0.043 0.077 0.122 0.135 0.110
concentration
in extract
taking into
account the
sample weight µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L
acenaphthene 0.095 0.074 0.104 0.470 0.338 0.844 0.910 0.889 0.761
fluorene 0.122 0.125 0.114 0.742 0.561 1.181 1.772 1.705 1.326
phenanthrene 0.910 0.819 0.966 3.687 4.241 4.497 12.618 12.841 11.063
fluoranthene 0.348 0.310 0.437 2.205 1.302 2.546 4.582 4.624 4.042
pyrene 0.251 0.253 0.247 1.395 1.015 1.820 3.009 3.271 2.818
TOTAL 1.726 1.580 1.867 8.499 7.457 10.887 22.891 23.331 20.011
94
Table S4 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with non-upconcentrated samplers.
CT4_1 CT4_2 CT4_3 CT5_1 CT5_2 CT5_3 Bl_1 Bl_2 Bl_3
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphthene 0.031 0.035 0.050 0.033 0.030 0.022 0.001 0.004 0.002
fluorene 0.071 0.071 0.110 0.078 0.071 0.048 0.002 0.002 0.002
phenanthrene 0.577 0.582 0.809 0.670 0.693 0.525 0.026 0.027 0.039
fluoranthene 0.263 0.302 0.454 0.269 0.273 0.238 0.159 0.016 0.044
pyrene 0.177 0.202 0.322 0.163 0.178 0.150 0.086 0.009 0.024
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.045 0.045 0.052 0.044 0.050 0.046 0.028 0.045 0.031
IS 2 0.058 0.063 0.074 0.054 0.062 0.063 0.035 0.052 0.041
IS 3 0.067 0.078 0.101 0.066 0.067 0.059 0.049 0.059 0.039
IS 4 0.084 0.087 0.109 0.069 0.068 0.071 0.343 0.071 0.055
IS 5 0.078 0.087 0.106 0.075 0.078 0.066 0.339 0.078 0.051
parameters
needed to
calculate
results
- - - - - - - - -
weight of
extract (g) 41.232 42.497 41.620 41.767 41.309 36.905 41.530 40.407 41.487
final volume of
extract (mL) 2.0 2.0 2.0 5.0 5.0 5.0 0.5 0.5 0.5
dilution 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
volume of IS-
working
solution added
to extract (µL) 20 20 20 50 50 50 25 25 25
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 9.98 9.98 9.98 9.98 9.98 9.98 1.00 1.00 1.00
IS 2 10.40 10.40 10.40 10.40 10.40 10.40 1.04 1.04 1.04
IS 3 10.30 10.30 10.30 10.30 10.30 10.30 1.03 1.03 1.03
IS 4 10.10 10.10 10.10 10.10 10.10 10.10 1.01 1.01 1.01
IS 5 10.50 10.50 10.50 10.50 10.50 10.50 1.05 1.05 1.05
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.100 0.100 0.100 0.100 0.100 0.100 0.050 0.050 0.050
IS 2 0.104 0.104 0.104 0.104 0.104 0.104 0.052 0.052 0.052
IS 3 0.103 0.103 0.103 0.103 0.103 0.103 0.052 0.052 0.052
IS 4 0.101 0.101 0.101 0.101 0.101 0.101 0.051 0.051 0.051
95
IS 5 0.105 0.105 0.105 0.105 0.105 0.105 0.053 0.053 0.053
recovery IS in
extract (%) % % % % % % % % %
IS 1 44.9 44.7 52.1 44.0 50.3 46.5 55.4 91.1 62.5
IS 2 55.7 60.5 70.9 52.1 59.9 60.2 67.4 99.4 78.4
IS 3 64.6 75.3 98.5 64.0 65.3 57.4 95.7 114.3 76.4
IS 4 83.1 85.8 108.3 68.6 67.7 70.4 679.8 140.7 108.2
IS 5 74.1 82.5 101.3 71.0 74.2 63.0 645.2 149.3 97.8
amount of
compound in
extract taking
into account
the volume and
dilution of the
extract µg µg µg µg µg µg µg µg µg
acenaphthene 0.061 0.070 0.100 0.166 0.148 0.111 0.001 0.002 0.001
fluorene 0.141 0.142 0.220 0.388 0.357 0.238 0.001 0.001 0.001
phenanthrene 1.154 1.164 1.618 3.349 3.464 2.626 0.013 0.014 0.020
fluoranthene 0.526 0.604 0.907 1.343 1.365 1.192 0.080 0.008 0.022
pyrene 0.355 0.403 0.645 0.817 0.888 0.752 0.043 0.005 0.012
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg µg µg µg µg
acenaphthene 0.136 0.158 0.191 0.376 0.293 0.240 0.001 0.002 0.002
fluorene 0.253 0.235 0.310 0.746 0.596 0.396 0.001 0.001 0.001
phenanthrene 1.785 1.546 1.643 5.236 5.302 4.578 0.014 0.012 0.026
fluoranthene 0.633 0.704 0.838 1.959 2.017 1.693 0.012 0.006 0.020
pyrene 0.479 0.489 0.636 1.150 1.196 1.192 0.007 0.003 0.012
concentration
in extract
taking into
account the
sample weight µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L
acenaphthene 3.302 3.708 4.594 9.011 7.104 6.490 0.028 0.048 0.038
fluorene 6.145 5.520 7.456 17.864 14.437 10.734 0.032 0.030 0.031
phenanthrene 43.297 36.373 39.470 125.357 128.349 124.052 0.330 0.294 0.617
fluoranthene 15.353 16.571 20.124 46.898 48.831 45.871 0.282 0.143 0.493
pyrene 11.615 11.497 15.291 27.537 28.964 32.306 0.161 0.078 0.301
TOTAL 79.712 73.668 86.935 226.666 227.685 219.453 0.833 0.594 1.481
96
Attachment 5: Processing results GC-MS for aqueous concentrations
upconentated samplers Table S5 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with upconcentrated samplers.
CT1_1 CT1_2 CT1_3 CT2_1 CT2_2 CT2_3 CT3_1 CT3_2 Spike_1
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
acenaphthene 0.015 0.009 0.017 0.023 0.025 0.081 0.032 0.056 0.037
fluorene 0.036 0.019 0.043 0.075 0.093 0.276 0.110 0.247 0.054
phenanthrene 0.388 0.253 0.458 0.945 1.165 3.736 1.367 3.358 0.143
fluoranthene 0.311 0.163 0.291 0.570 0.726 2.603 0.902 2.027 0.056
pyrene 0.193 0.107 0.196 0.386 0.498 1.821 0.641 1.395 0.038
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.025 0.023 0.026 0.024 0.026 0.055 0.047 0.062 0.029
IS 2 0.037 0.029 0.033 0.030 0.038 0.074 0.063 0.073 0.034
IS 3 0.043 0.038 0.040 0.030 0.037 0.077 0.064 0.075 0.035
IS 4 0.225 0.062 0.053 0.033 0.042 0.100 0.081 0.085 0.045
IS 5 0.203 0.057 0.056 0.040 0.047 0.097 0.081 0.089 0.050
parameters
needed to
calculate
results
- - - - - - - - -
weight of
extract (g) 41.618 35.051 43.025 42.421 41.738 41.396 42.601 41.367 40.000
final volume of
extract (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
dilution 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
volume of IS-
working
solution added
to extract (µL) 25 25 25 25 25 50 50 50 25
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
IS 2 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04
IS 3 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03
IS 4 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01
IS 5 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
IS 1 0.050 0.050 0.050 0.050 0.050 0.100 0.100 0.100 0.050
97
IS 2 0.052 0.052 0.052 0.052 0.052 0.104 0.104 0.104 0.052
IS 3 0.052 0.052 0.052 0.052 0.052 0.103 0.103 0.103 0.052
IS 4 0.051 0.051 0.051 0.051 0.051 0.101 0.101 0.101 0.051
IS 5 0.053 0.053 0.053 0.053 0.053 0.105 0.105 0.105 0.053
recovery IS in
extract (%) % % % % % % % % %
IS 1 49.8 45.7 53.0 48.2 53.0 55.6 46.6 62.0 57.2
IS 2 70.2 56.4 62.8 58.6 72.6 71.6 60.3 69.9 66.2
IS 3 83.2 73.3 78.2 57.8 71.1 74.7 62.3 72.8 68.9
IS 4 445.0 122.8 104.8 65.6 82.3 99.2 80.6 84.2 88.2
IS 5 387.4 108.4 107.6 76.4 89.8 92.5 77.4 84.3 95.9
amount of
compound in
extract taking
into account
the volume
and dilution of
the extract µg µg µg µg µg µg µg µg µg
acenaphthene 0.008 0.004 0.009 0.011 0.012 0.040 0.016 0.028 0.019
fluorene 0.018 0.010 0.021 0.038 0.046 0.138 0.055 0.124 0.027
phenanthrene 0.194 0.127 0.229 0.472 0.582 1.868 0.684 1.679 0.071
fluoranthene 0.155 0.082 0.146 0.285 0.363 1.301 0.451 1.014 0.028
pyrene 0.096 0.053 0.098 0.193 0.249 0.911 0.321 0.698 0.019
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg µg µg µg µg
acenaphthene 0.015 0.009 0.016 0.023 0.023 0.073 0.035 0.045 0.033
fluorene 0.026 0.017 0.034 0.064 0.064 0.193 0.091 0.177 0.041
phenanthrene 0.233 0.173 0.293 0.817 0.819 2.499 1.098 2.308 0.103
fluoranthene 0.035 0.067 0.139 0.434 0.441 1.312 0.560 1.204 0.032
pyrene 0.025 0.049 0.091 0.253 0.277 0.984 0.414 0.827 0.020
concentration
in extract
taking into
account the
sample weight µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L
acenaphthene 0.364 0.267 0.383 0.552 0.554 1.756 0.816 1.097 0.818
fluorene 0.621 0.483 0.787 1.511 1.533 4.664 2.133 4.277 1.022
phenanthrene 5.604 4.931 6.799 19.259 19.634 60.380 25.771 55.782 2.587
fluoranthene 0.839 1.898 3.225 10.238 10.567 31.705 13.147 29.101 0.800
pyrene 0.597 1.402 2.120 5.961 6.637 23.779 9.729 19.997 0.498
TOTAL 8.026 8.980 13.314 37.520 38.925 122.284 51.596 110.254
5.724
98
Table S5 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with upconcentrated samplers.
CT4_1 CT4_3
CT5_1
a
CT5_1
b
CT5_2
a
CT5_2
b
CT5_3
a
CT5_3
b
Spike_
2
concentration
in extract
(result GC-
report) µg/mL µg/mL µg/mL µg/ml µg/mL µg/mL µg/mL µg/mL
µg/mL
acenaphthen
e 0.014 0.009 0.037 0.008 0.044 0.007 0.046 0.008 0.034
fluorene 0.046 0.039 0.090 0.018 0.103 0.020 0.107 0.017 0.051
phenanthrene 0.490 0.365 0.610 0.141 0.816 0.156 0.776 0.137 0.142
fluoranthene 0.244 0.153 0.231 0.045 0.295 0.054 0.286 0.050 0.048
pyrene 0.154 0.092 0.127 0.025 0.160 0.032 0.157 0.029 0.037
IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
µg/mL
IS 1 0.014 0.011 0.047 0.011 0.052 0.011 0.049 0.009 0.027
IS 2 0.017 0.012 0.055 0.012 0.063 0.015 0.060 0.010 0.030
IS 3 0.024 0.013 0.057 0.012 0.061 0.012 0.060 0.010 0.035
IS 4 0.035 0.017 0.067 0.013 0.066 0.012 0.061 0.010 0.043
IS 5 0.032 0.015 0.062 0.014 0.068 0.016 0.061 0.012 0.047
parameters
needed to
calculate
results
- - - - - - - - -
weight of
extract (g) 41.189 42.269 38.062 38.062 41.942 41.942 42.989 42.989 40.000
final volume
of extract
(mL) 2.0 2.0 5.0 5.0 5.0 5.0 5.0 5.0 0.5
dilution 4.0 4.0 1.0 4.0 1.0 4.0 1.0 4.0 1.0
volume of IS-
working
solution
added to
extract (µL) 20 20 50 50 50 50 50 50
25
concentration
of IS-working
solution
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
IS 1 9.98 9.98 9.98 9.98 9.98 9.98 9.98 9.98 1.00
IS 2 10.40 10.40 10.40 10.40 10.40 10.40 10.40 10.40 1.04
IS 3 10.30 10.30 10.30 10.30 10.30 10.30 10.30 10.30 1.03
IS 4 10.10 10.10 10.10 10.10 10.10 10.10 10.10 10.10 1.01
IS 5 10.50 10.50 10.50 10.50 10.50 10.50 10.50 10.50 1.05
theoretical IS-
concentration
in extract µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
µg/mL
99
IS 1 0.025 0.025 0.100 0.025 0.100 0.025 0.100 0.025 0.050
IS 2 0.026 0.026 0.104 0.026 0.104 0.026 0.104 0.026 0.052
IS 3 0.026 0.026 0.103 0.026 0.103 0.026 0.103 0.026 0.052
IS 4 0.025 0.025 0.101 0.025 0.101 0.025 0.101 0.025 0.051
IS 5 0.026 0.026 0.105 0.026 0.105 0.026 0.105 0.026 0.053
recovery IS in
extract (%) % % % % % % % %
%
IS 1 57.8 42.1 46.6 45.1 51.8 45.6 49.4 36.0 54.2
IS 2 67.0 46.5 52.7 44.4 61.0 58.6 57.9 38.0 57.4
IS 3 92.9 49.6 55.5 46.2 59.3 48.4 58.0 39.7 68.7
IS 4 138.4 67.5 66.1 50.3 65.5 46.1 60.0 41.4 85.7
IS 5 123.7 59.0 58.9 51.6 64.4 59.5 58.2 47.1 90.4
amount of
compound in
extract taking
into account
the volume
and dilution of
the extract µg µg µg µg µg µg µg µg
µg
acenaphthen
e 0.110 0.075 0.184 0.163 0.219 0.147 0.232 0.160 0.017
fluorene 0.370 0.311 0.451 0.366 0.517 0.401 0.534 0.337 0.026
phenanthrene 3.921 2.923 3.049 2.828 4.081 3.115 3.880 2.735 0.071
fluoranthene 1.950 1.223 1.154 0.899 1.476 1.081 1.429 0.992 0.024
pyrene 1.230 0.736 0.636 0.499 0.799 0.633 0.785 0.582 0.019
amount of
compound in
extract taking
into account
the IS µg µg µg µg µg µg µg µg
µg
acenaphthen
e 0.190 0.179 0.394 0.361 0.422 0.323 0.469 0.443 0.032
fluorene 0.552 0.669 0.855 0.824 0.847 0.683 0.922 0.887 0.045
phenanthrene 4.219 5.895 5.491 6.126 6.877 6.442 6.686 6.893 0.103
fluoranthene 1.409 1.810 1.747 1.788 2.253 2.343 2.381 2.393 0.028
pyrene 0.994 1.247 1.080 0.968 1.240 1.064 1.349 1.236 0.021
concentration
in extract
taking into
account the
sample
weight µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L µg/L
acenaphthen
e 4.625 4.238 10.357 9.493 10.066 7.710 10.918 10.316 0.793
fluorene 13.394 15.819 22.466 21.646 20.199 16.293 21.449 20.639 1.121
phenanthren
e
102.43
5
139.47
3
144.25
7
160.94
7
163.96
5
153.58
5
155.52
5
160.34
6 2.580
100
fluoranthene 34.204 42.822 45.888 46.963 53.708 55.861 55.388 55.676 0.694
pyrene 24.142 29.501 28.380 25.437 29.556 25.365 31.388 28.762 0.518
TOTAL
178.79
9
231.85
3
251.34
8
264.48
7
277.49
4
258.81
5
274.66
8
275.73
7
5.707
101
Attachment 6: Data growth inhibition experiment 1 Table S6 – part 1: Cell count growth inhibition experiment 1 with non-upconcentrated samplers.
cell counts (cells/mL)
day 1 day 2 day 3
blank 1 (no sampler) 42668 164320 490280
blank 2 (no sampler) 44152 156760 482040
blank 3 (no sampler) 44944 167620 540420
blank 4 (unspiked
sampler)
43048 155980 486660
blank 5 (unspiked
sampler)
43880 167980 509920
blank 6 (unspiked
sampler)
44376 166600 482320
CT1_1 43740 147260 454700
CT1_2 43600 158420 478040
CT1_3 43432 153340 486960
CT2_1 43012 149680 481080
CT2_2 43236 155780 446880
CT2_3 41820 160000 469660
CT3_1 34036 137600 448820
CT3_2 37852 134420 411040
CT3_3 39876 143580 452420
CT4_1 26972 75680 209320
CT4_2 29192 83780 229220
CT4_3 29732 86020 229860
CT5_1 11052 13620 11900
CT5_2 12588 13500 11500
CT5_3 12584 11820 8080
102
Table S6 – part 2: Cell count growth inhibition experiment 1 with upconcentrated samplers.
cell counts (cells/mL)
day 1 day 2 day 3
blank 1 (no sampler) 42668 164320 490280
blank 2 (no sampler) 44152 156760 482040
blank 3 (no sampler) 44944 167620 540420
blank 4 (unspiked
sampler)
50592 172280 473500
blank 5 (unspiked
sampler)
47352 161380 376460
blank 6 (unspiked
sampler)
44256 153020 425320
CT1_1 45088 139080 386240
CT1_2 8912 22480 --
CT1_3 42692 132640 381360
CT2_1 36632 121840 344740
CT2_2 58136 169600 352380
CT2_3
CT3_1 23940 47020 89020
CT3_2 38392 113780 314840
CT3_3 23492 54760 98520
CT4_1 14056 23000 15500
CT4_2
CT4_3 13800 13720 11240
CT5_1 13500 10940 7680
CT5_2 15156 11220 7900
CT5_3 15148 10740 12320
103
Attachment 7: Data growth inhibition experiment 2 Table S7 – part 1: Cell count growth inhibition experiment 2 with non-upconcentrated samplers.
cell counts (cells/mL)
day 1 day 2 day 3
blank 1 (no sampler) 33852 108600 282220
blank 2 (no sampler) 38340 115680 322960
blank 3 (no sampler) 35344 116360 332520
blank 4 (no sampler) 33248 103620 267540
blank 5 (no sampler) 38588 124960 397300
blank 6 (no sampler) 35632 122120 332880
blank 7 (unspiked
sampler)
43356 142580 439000
blank 8 (unspiked
sampler)
45104 157780 504420
blank 9 (unspiked
sampler)
40028 132920 402120
CT1_1 37620 118200 288320
CT1_2 50576 170680 542940
CT1_3 50872 170140 550500
CT2_1 41260 118860 338180
CT2_2 - - -
CT2_3 43152 156680 500400
CT3_1 58952 185060 542700
CT3_2 38932 134920 435400
CT3_3 55156 178740 484060
CT4_1 26160 66260 178920
CT4_2 33832 81820 225480
CT4_3 29704 71660 194620
CT5_1 12124 10480 12040
CT5_2 9744 6900 9320
CT5_3 11600 11300 9280
104
Table S7 – part 2: Cell count growth inhibition experiment 2 with upconcentrated samplers.
cell counts (cells/mL)
day 1 day 2 day 3
blank 1 (no sampler) 33852 108600 282220
blank 2 (no sampler) 38340 115680 322960
blank 3 (no sampler) 35344 116360 332520
blank 4 (no sampler) 33248 103620 267540
blank 5 (no sampler) 38588 124960 397300
blank 6 (no sampler) 35632 122120 332880
blank 7 (unspiked
sampler)
36512 106920 294220
blank 8 (unspiked
sampler)
34636 99200 293340
blank 9 (unspiked
sampler)
34528 98960 251100
CT1_1 38632 95820 212240
CT1_2 31692 84760 202440
CT1_3 31772 97660 263540
CT2_1 35288 91500 211460
CT2_2 33248 82340 182760
CT2_3 - - -
CT3_1 18524 24440 27680
CT3_2 30844 84640 205980
CT3_3 22484 38840 62100
CT4_1 13892 14120 14280
CT4_2 - - -
CT4_3 11084 10460 9680
CT5_1 11476 11400 14260
CT5_2 10344 13200 7480
CT5_3 10552 10100 11340
105
Attachment 8: Statistical analysis of the PAH recovery on non-
upconcentrated samplers All statistical analyses in this thesis were performed using the statistical software package SPSS.
In order to prove that the PAH recovery of each CT is not significantly different from 100 % for the
non-upconcentrated samplers, 5 one-sample t-tests were performed. For each concentration
treatment, the average recovery is compared to the theoretical recovery of 100 %. The hypotheses
are formulated as follows:
H0,a: µrecovery, CT1 = 100% H0,b: µrecovery, CT2 = 100% H0,c: µrecovery, CT3 = 100%
H1,a: µrecovery, CT1 ≠ 100% H1,b: µrecovery, CT2 ≠ 100% H1,c: µrecovery, CT3 ≠ 100%
H0,d: µrecovery, CT4 = 100% H0,e: µrecovery, CT5 = 100%
H1,d: µrecovery, C4 ≠ 100% H1,e: µrecovery, CT5 ≠ 100%
Results of the statistical analyses are given below. The values of the test quantity ‘t’ are -1.028, -
2.371, 0.274, 1.226 and -2.861 with respective p-values of 0.412, 0.141, 0.810, 0.345 and 0.104.
Based on the 5 % significance level, H0 is accepted for each CT. It can be concluded with 95 %
certainty that the average recoveries on the non-upconcentrated samplers do not differ significantly
from 100 %.
One-Sample Test CT 1
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -1.028 2 .412 -3.01333 -15.6204 9.5938
One-Sample Test CT 2
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -2.371 2 .141 -8.14667 -22.9311 6.6378
One-Sample Test CT 3
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -.274 2 .810 -.97667 -16.3000 14.3467
106
One-Sample Test CT 4
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery 1.226 2 .345 2.87000 -7.1997 12.9397
One-Sample Test CT 5
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -2.861 2 .104 -5.29667 -13.2617 2.6684
107
Attachment 9: Statistical analysis of the PAH recovery on
upconcentrated samplers In order to confirm the suspicion that the PAH recovery of the upconcentrated samplers is
significantly different form 100% for each concentration treatment, 5 one-sample t-tests were
performed. The average recovery is compared to the theoretical recovery of 100% for each
concentration treatment. The hypotheses are formulated as follows:
Hypotheses small samplers:
H0,a: µrecovery, CT1 = 100% H0,b: µrecovery, CT2 = 100% H0,c: µrecovery, CT3 = 100%
H1,a: µrecovery, CT1 ≠ 100% H1,b: µrecovery, CT2 ≠ 100% H1,c: µrecovery, CT3 ≠ 100%
H0,d: µrecovery, CT4 = 100% H0,e: µrecovery, CT5 = 100%
H1,d: µrecovery, C4 ≠ 100% H1,e: µrecovery, CT5 ≠ 100%
Results of the statistical analyses are given below. The values of the test quantity ‘t’ are -7.477, -
23.649, -10.494, -42.105 and -124.290 with respective p-values of 0.045, 0.002, 0.009, 0.001 and
0.005. Based on the 5% significance level, H0 is rejected for each CT. It can be concluded with
95% certainty that the average recoveries on the non-upconcentrated samplers differ significantly
from 100%.
One-Sample Test CT 1
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -7.477 1 .045 -34.62000 -93.4497 24.2097
One-Sample Test CT 2
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -23.649 2 .002 -54.80333 -64.7741 -44.8325
One-Sample Test CT 3
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -10.494 2 .009 -43.42333 -61.2276 -25.6191
108
One-Sample Test CT 4
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -42.105 2 .001 -69.50000 -76.6022 -62.3978
One-Sample Test CT 5
Test Value = 100
t df Sig. (2-tailed) Mean Difference
95% Confidence Interval of the
Difference
Lower Upper
Recovery -124.290 1 .005 -85.76000 -94.5273 -76.9927