upconcentration of realistic...

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


2.1.2. 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


2.2.2. 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






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


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.




samplers.

Table S2 – part 1: 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 S4 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with non-


9

Table S4 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with non-


Table S5 – part 1: Total PAH concentration in water phase after growth inhibtion experiment with


Table S5 – part 2: Total PAH concentration in water phase after growth inhibtion experiment with


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.


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).

https://pubchem.ncbi.nlm.nih.gov/

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)



Low analyte concentrations

(-)

Higher analyte concentrations (+) Kot et al. (2000)


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 -)


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)


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


CT 3


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


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



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


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



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


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


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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67

Supporting information

Attachment 1: PAH concentration on samplers before and after

upconcentration





68



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.



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


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



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


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


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


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


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


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



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-


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


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


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


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


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


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



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-


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


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


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


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


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.



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


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


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


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


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


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



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-


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


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


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


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


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


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



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



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-


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


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


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-


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


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


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


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


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-


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


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


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


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


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


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-


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


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


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


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


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



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.


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



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


Test Value = 100



Difference

Lower Upper

Recovery -2.371 2 .141 -8.14667 -22.9311 6.6378


Test Value = 100



Difference

Lower Upper

Recovery -.274 2 .810 -.97667 -16.3000 14.3467

106


Test Value = 100



Difference

Lower Upper

Recovery 1.226 2 .345 2.87000 -7.1997 12.9397


Test Value = 100



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%.


Test Value = 100



Difference

Lower Upper

Recovery -7.477 1 .045 -34.62000 -93.4497 24.2097


Test Value = 100



Difference

Lower Upper

Recovery -23.649 2 .002 -54.80333 -64.7741 -44.8325


Test Value = 100



Difference

Lower Upper

Recovery -10.494 2 .009 -43.42333 -61.2276 -25.6191

108


Test Value = 100



Difference

Lower Upper

Recovery -42.105 2 .001 -69.50000 -76.6022 -62.3978


Test Value = 100



Difference

Lower Upper

Recovery -124.290 1 .005 -85.76000 -94.5273 -76.9927

upconcentration of realistic...

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