Effect of PFOS and HBCD on the lipid profiles of developing rainbow trout (Onchorhynchus mykiss) analyzed with UHPLC/Q-TOF-MS

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Effect of PFOS and HBCD on the lipid profiles of

developing rainbow trout (Onchorhynchus mykiss)

analyzed with UHPLC/Q-TOF-MS

Vanja Stefanovic

Thesis for the Bachelor’s degree in chemistry at the University of Örebro

Supervisor: Prof. Tuulia Hyötyläinen, Ph.D.

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Abstract

Perfluorooctane sulfonate (PFOS) is widely used in industrial products and is potentially dangerous to the aquatic environment due to not being broken down whether by chemical or biological means, having a half-life of more than 41 years and disrupting hormones.

Hexabromocyclododecane (HBCD) is the third most used brominated flame retardant and is of environmental concern as it bioaccumulates and magnifies in the food chain and is highly toxic to aquatic organisms. The purpose of this study was to examine the effect of PFOS and HBCD on the embryos of rainbow trout (Onchorhynchus mykiss) by analyzing lipid profiles with UHPLC/Q-TOF-MS. The fish embryos were treated with various concentrations of PFOS and HBCD (0.058-58 µg/l and 0.014-14 µg/l respectively) with DMSO as carrier solvent and then extracted after homogenization with 0.9% NaCl-solution followed by

addition of ISTD mixture, methanol, methyl tert-butyl ether (MTBE) and MQ-water. The raw data was processed with MZmine-2.32. 153 lipids were identified with the main lipids

consisting of glycerophospholipids and triacylglycerols. A two-tailed t-test was used to study the impact of the chemical exposure on the embryos, where p-values below 0.05 were lipids considered as significant change. The HBCD exposure caused significant change in various triacylglycerols, whereas PFOS exposure caused significant change in triacylglycerols as well as in glycerophospholipids such as PC(O-38:5) and LPC(20:4). The results were in alignment with previous studies.

Keywords: Perfluorooctane sulfonate, hexabromocyclododecane, lipid metabolism, UHPLC/Q-TOF-MS

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Contents 1. Introduction……….. 1 1.1. Background theory………... 1 1.1.1. Lipid classification………. 1 1.1.2. PFOS……….. 3 1.1.3. HBCD………. 3 1.1.4. UHPLC/Q-TOF-MS………... 3 1.2 Purpose of study……… 3

1.3 PFOS and HBCD exposure………... 4

2. Materials and methods………. 5

2.1 Chemicals……….. 5

2.2 Extraction……….. 6

2.3 UHPLC/Q-TOF-MS analysis……… 7

2.4 Data processing………. 8

2.4.1. Statistical analysis……….. 9

3. Results and discussion………. 10

Acknowledgements……….. 21

References……… 22

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1

1. Introduction

1.1. Background theory

1.1.1. Lipid classification

Lipids are biomolecules that are hydrophobic and soluble in organic (nonpolar) solvents. They act as structural components of cell membranes, important signaling molecules and store energy. There are eight classes that lipids can be classified into based on their chemical structure, that are derived from two biochemical building blocks: ketoacyl and isoprene groups (Figure 1) (Lipidmaps.org, 2018).

Figure 1. The building blocks in which lipids are derived from. (Figure is adapted from Lipidmaps.org)

The lipids are divided into fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), saccharolipids (SL) and polyketides (PK) which are derived from condensation of ketoacyl subunits; and sterol lipids (ST) and prenol lipids (PR) which are derived from condensation of isoprene subunits. There are four categories relevant to this project: glycerolipids, glycerophospholipids, sphingolipids and sterol lipids.

Glycerolipids

Glycerolipids are mainly composed of mono-, di-, and tri-substituted glycerols with triglycerides being the most well-known which are fatty acid esters of glycerol

(Lipidmaps.org, 2018). This class of lipids constitute most of the storage fat in mammalian tissues and are most abundant in oils and fats of animal and plant origins. Examples are the sub classes triacylglycerols (TG) and diacylglycerols (DG) (Figure 2).

Figure 2. Core structure representation of glycerolipids, here

DG(16:0/18:1(9Z)/0:0). (Figure is adapted from Lipidmaps.org)

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2 Glycerophospholipids

Glycerophospholipids are fundamental components of the lipid bilayer of cell membranes and are also involved in metabolism and signaling. Based on the character of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes, the glycerophospholipids are subdivided into specific classes (Lipidmaps.org, 2018). Examples of compounds found in biological membranes are phosphatidylethanolamine (PE), lyso-phosphatidylethanolamine (LysoPE, LPE), phosphatidylcholine (PC), lyso-phosphatidylcholine (LysoPC) and

phosphatidylinositol (PI) (Figure 3).

Figure 3. Core structure representation of

glycerophospholipids, here PC(16:0/18:1(9Z)). (Figure is adapted from Lipidmaps.org) Sphingolipids

Sphingolipids are a complex group of lipids and can be converted to many species. They do not contain glycerol as most other fats, but they are rather built up by the amino alcohol sphingosine. The basic common structural features are that they all contain a sphingoid base backbone and a long-chain fatty acyl-CoA (Figure 4) (Lipidmaps.org, 2018). When a fatty acid is attached to the sphingol through a peptide bond, a ceramide is formed. Ceramide (Cer) is the starting point for sphingomyelin (SM) and partly glycolipids and is present in high concentrations in cell membranes.

Figure 4. Core structure representation of sphingolipids, here Cer(d18:1/14:0). (Figure is adapted from Lipidmaps.org) Sterol lipids

Sterol lipids are crucial to cell membrane structures with cholesterol and its derivatives being the most common types. A fused four-ring structure is the core of the sterol lipids much like steroids, although steroids have different biological functions. An example of a sterol lipid is cholesteryl ester (CE) which is an ester of cholesterol (Figure 5).

Figure 5. Core structure representation of sterol lipids, here CE(12:0). (Figure is adapted from Lipidmaps.org)

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3 1.1.2. PFOS

Perfluorooctane sulfonate (PFOS) is an organic compound that is widely used in various industrial products such as detergents, waterproofing materials and firefighting foams (Kemi.se, 2017). It is not broken down in the environment either by chemical or biological means (Kemi.se, 2017) and is therefore potentially dangerous to the aquatic environment, especially considering its half-life being estimated at more than 41 years under experimental conditions (Baker and Hites, 2006), as well as its hormonal disruptive role (Du et al., 2012). PFOS has been found in diverse marine mammals, fishes and birds from the Baltic and the Mediterranean Seas (Kannan et al., 2002) as well as in liver samples from Greenland and the Faroe Islands with polar bears having the highest concentration (Bossi et al., 2005) and in fish from China (Shi et al. 2010).

1.1.3. HBCD

Hexabromocyclododecane (HBCD) is the third most used brominated flame retardant, it is used in the building and construction industry, and in consumer products (US EPA, 2017). It also seems to be a hormonal disrupter like PFOS and may disrupt endocrine function in animals and humans (van der Ven et al., 2006). HBCD is of environmental concern as it is persistent in the environment and bioaccumulates and magnifies in the food chain (US EPA, 2017). It is highly toxic to aquatic organisms and also presents concerns regarding human health as studies on animals show results that indicate potential reproductive, developmental and neurological effects (US EPA, 2017).

1.1.4. UHPLC/Q-TOF-MS

The basic components of an HPLC system consist of a pump, an injector, a column, one or more detectors in series and a Chromatography Data System (CDS).

The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) technique is a relatively new approach in chromatographic separations and provides higher sensitivity and resolution as well as a faster separation (Khan and Ali, 2015). A more accurate mass, reliable fragmentation and structure elucidation is provided with Q-TOF-MS (Khan and Ali, 2015) which uses a quadrupole, a collision cell and a time of flight unit to produce spectra, where the lighter ions will accelerate faster to the detector through the flight tube (Labcompare.com, 2018). This will determine the mass-to-charge ratios of the ions.

1.2. Purpose of this study

The purpose of this study was to examine the effects of PFOS and HBCD on the embryos of rainbow trout, Onchorhynchus mykiss, by analyzing lipid profiles in the treated samples with UHPLC/Q-TOF-MS.

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4 1.3 PFOS and HBCD Exposure

The fish embryos used in this project were acquired from another not yet published project done at the Finnish Environment Institute SYKE (“Toxicity of HBCD and PFOS for Rainbow Trout (Onchorhynchus mykiss), Heli Ratia et al.”). The purpose of the study was to modify standardized toxicity tests for boreal species and to test alternative methods for animal testing to test wastewater toxicity. “OECD Test No. 210: Fish, Early–life Stage Toxicity Test” was used to test the toxicity. 10 µg/l of dimethyl sulfoxide solvent (DMSO) was added to every treatment (except in the control) to be used as carrier solvent. The concentrations tested for HBCD was 0.014, 0.14, 1.4 and 14 µg/l, and for PFOS 0.058, 0.58, 5.8 and 58 µg/l. The results from the study showed that the yolk sac size and fish weight were smaller in the HBCD exposures compared to the control, that the fish were significantly longer in the two highest PFOS exposures and that the yolk sac size was slightly bigger compared to the control. The study concluded that HBCD and PFOS were not acutely toxic to the early stage of the rainbow trout at concentrations which could be measured from Finnish wastewaters.

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2. Materials and methods

2.1 Chemicals

Methanol 99.9% – HPLC grade from Fisher Scientific (Loughborough, Leics. U.K.). Methyl tert–butyl ether (MTBE) 99.8% from Sigma Aldrich (Darmstadt, Germany). MQ water – HPLC grade from Honeywell Riedel-de Haën (Germany).

Formic acid 99.5+%: LC/MS grade from Fisher Scientific. Ammonium acetate ≥98% from Sigma Aldrich. Lipid standards from Avanti Polar Lipids Inc. (Alabaster, AL, United States) and Larodan (Solna, Sweden). 0.9% NaCl–solution.

For the internal standard mixture (in CHCl3:MeOH (2:1, v/v)), the following lipids were used:

• PE (17:0/17:0) • SM (d18:1/17:0) • Cer (d18:1/17:0) • PC (17:0/17:0) • LysoPC (17:0) • PC (16.0/d31/18:1) • TG (17:0/17:0/17:0) • CE (17:0)

For calibration curve (in CHCl3:MeOH (2:1, v/v)):

• LysoPC (18:0) • CE (18:1(9Z)) • Cer (d18:1/24:0) (24:0 Ceramide) • Cer (d18:0/18:1(9Z)) (N–18:1 sphinganine) • TG (16:0/16:0/16:0) • PC (16:0/16:0) • TG (18:0/18:0/18:0) • CE (18:0) • LysoPC (18:1) • LysoPE (18:1) • PC (16:0/18:1) • Cer (d18:1/18:1(9Z)) • PC (18:0/18:0) • PE (16:0/18:1) • CE (18:2(9Z, 12Z)) • CE (16:0) • PI (18:0–20:4) • PI (16:0–18:1) • DG (18:1) • CE (18:2)

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6 2.2 Extraction

The samples and solvents were always kept on cold ice plates during sample preparation and stored in either –20°C or –80°C freezers.

There should have been a total of 90 fish embryo samples, but the lowest concentrations of PFOS (0.058 µg/l) was missing (9 samples) so there was a total of 81 fish samples.

All of the 81 Eppendorf tubes to be used were weighed separately, and then with the frozen fish added so that the weight of the fish could be calculated. The fish that were not yet weighed were kept on dry ice as the fish needed to be frozen when weighed. After weighing, the fish were put on ice plates to thaw before homogenizing. When the weight of the fish was acquired, the double amount of 0.9% NaCl–solution was added. Example of sample 1:

Weight of E. tube E. tube + fish Fish NaCl

1.0930 g 1.1842 g 1.1842–1.0930 = 0.0912 g = 91.2 mg 182.4 µl

The fish were homogenized with a pestle mixer and 20 µl was transferred to new Eppendorf tubes. The two blanks (one for each batch) contained 20 µl NaCl–solution.

The extraction protocol was as follows:

1. 80 µl of ISTD mixture, c =17.5 ppm, was added in all samples and blanks. 2. 240 µl methanol was added to all samples and then the samples were vortexed.

3. 800 µl Methyl tert–butyl ether (MTBE) was added and then the samples were incubated on a shaker at room temperature for 60 minutes. The speed on the shaker was around 385 rpm.

4. 200 µl MQ–water was added and then the samples were incubated for 10 minutes before being centrifuged at 9000 RCF for 10 minutes.

5. 750 µl of the organic phase was collected to LC vials.

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7 2.3 UHPLC/Q-TOF-MS analysis

UHPLC

The UHPLC system used was 1290 Infinity II from Agilent Technologies (Santa Clara, CA, USA).

Multisampler: The UHPLC system was equipped with a multisampler (maintained at 10°C)

using 10% DCM in MeOHand ACN:MeOH:IPA:H2O (1:1:1:1, v/v/v/v) + 0.1% HCOOH as

needle wash solutions after each injection for 7.5 s each, a quaternary solvent manager and a column thermostat (maintained at 50 °C).

Column: Separations were performed in an ACQUITY UPLC® BEH C18 column (2.1 mm ×

100 mm, particle size 1.7 µm) by Waters (Milford, USA). The flow rate was 0.4 mL min−1_and

the injection volume was 1 µL.

Solvents: (A) Aqueous phase contained 800 ml H2O, 800 µl formic acid (0.1 %) and 0.6166 g

(1%) NH4Ac (1M). (B) Organic phase contained ACN:IPA (1:1, 250 ml in each), 500 µl formic

acid (0.1%) and 0.3854 g (1%) NH4Ac.

The gradient is as follows: from 0 to 2 min 35-80% B, from 2 to 7 min 80-100% B and from 7 to 14 min 100% B. Each run was followed by a 7 min re-equilibration period under initial conditions (35% B).

Gases: Nitrogen generated by a nitrogen generator is used as the nebulizing gas at a pressure

of 21 psi, drying gas at a flow rate of 14 L min−1 _{(at 193°C) and sheath gas at a flow rate of}

11 L min-1_{(at 379°C). Pure nitrogen (6.0) from Praxair (Fredericia, Denmark) is used as the}

collision gas. Q-TOF

The Q-TOF instrument was 6545 Q-TOF LC/MS from Agilent Technologies interfaced with a dual jet stream electrospray (dual ESI) ion source.

MS conditions: The capillary voltage and the nozzle voltage are kept at 3643 V and 1500 V, respectively.

The reference mass solution including ions at m/z 121.0509 and 922.0098 is prepared according to instructions by Agilent and it is introduced to the mass spectrometer through the other nebulizer in the dual ESI ion source.

The acquisition mass range is m/z 100-1700 and the instrument was run using the extended dynamic range with an approximate resolution of 30,000 FWHM measured at m/z 1521.9715. Sample analysis cycle

The calibration standards were run first and then the lids were changed on them before running the rest of the samples for approximately 40 hours. One blank was analyzed per batch, and there were two batches.

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8 2.4 Data analysis

The raw data was processed with MZmine–2.32, and the analysis was done through Excel. Data processing procedure:

• Raw data methods → Raw data import

• Mass detection: Raw data methods → Peak detection → Mass detection

• Chromatogram builder: Raw data methods → Peak detection → Chromatogram builder Parameters:

– Min time span (min): 0.08 – Min height: 1800

– m/z tolerance: 0.006 m/z or 2.0 ppm

• Chromatogram deconvolution: Peak list methods → Peak detection → Chromatogram deconvolution

Parameters [Algorithm]:

– Chromatographic threshold: 70.0% – Search minimum in RT range (min): 0.06 – Minimum relative height: 0.01%

– Minimum absolute height: 1800 – Min ratio of peak top/edge: 1.6 – Peak duration range (min): 0.08 – 5.00

• Isotopic peaks grouper: Peak list methods → Isotopes → Isotopic peaks grouper Parameters:

– m/z tolerance: 0.005 m/z or 5.0 ppm

– Retention time tolerance: 0.05 absolute (min) – Maximum charge: 2

– Representative isotope: Most intense

• Alignment: Peak list methods → Alignment → Join aligner Parameters:

– m/z tolerance: 0.008 m/z or 10.0 ppm – Weight for m/z: 2

– Retention time tolerance: 0.08 absolute (min) – Weight for RT: 1

• Filtering: Peak list methods → Filtering → Peak list rows filter Parameters:

– Minimum peaks in a row: 3 – m/z: 350.0000 – 1100.0000 – Retention time: 2.00 – 11.00

• Gap filling: Peak list methods → Gap filling → Peak finder Parameters:

– Intensity tolerance: 10.0%

– m/z tolerance: 0.015 m/z or 15.0 ppm – Retention time tolerance: 0.17

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9 • Adduct search: Peak list methods → Identification → Adduct search

Parameters:

– RT tolerance: 0.05 absolute (min) – Adducts:

[M+Na–H] 21.9825 m/z [M+K–H] 37.9559 m/z [M+NH3] 17.0265 m/z

– m/z tolerance: 0.001 m/z or 5.0 ppm – Max relative adduct peak height: 50.0%

• Export data: Peak list methods → Export/Import → Export to CSV file Parameters:

– Export common elements: Export row ID, Export row m/z, Export row retention time, Export row identity (main ID), Export row number of detected peaks.

2.4.1. Quantitation, quality control and statistical analysis

Once the peak list (original data) was acquired, the data was normalized with the internal standards, and calibration curves were made for quantification. The calibration curves consisted of following lipids: CE(18:1), CE(18:2), LPC(18:0), LPC(18:1), LysoPE(18:1), PC(16:0/16:0), PE(16:0/18:1), TG(16:0/16:0/16:0) and TG(18:0/18:0/18:0) (see Appendix 1), normalized by the internal standards. For the quantification, all of the concentrations for the identified lipids in each sample were calculated using the following calibration curves:

• CE compounds: CE(18:1)

• Cer, PC, PI and SM compounds: PC(16:0/16:0)

• LPC compounds: LPC(18:0)

• LysoPE compounds: LysoPE(18:1)

• PE compounds: PE(16:0/18:1)

• TG compounds: TG(18:0/18:0/18:0)

When the concentrations were calculated, the average concentration for each lipid in each concentration of PFOS and HBCD (as well as Control and DMSO) was calculated, as well as the standard deviation and RSD(%).

Blank samples and standards were analyzed together with the samples. The quality of the method was evaluated by calculating the relative standard deviations of the internal standards in the samples and calculating the RSDs for standard samples.

For statistical analyses, a two-tailed t-test was then performed comparing Control with the different concentrations of HBCD and PFOS, as well as comparing DMSO with them, acquiring p values. The p values below 0.05 meant a significant change in the lipid, and 2D staple graphs were made with the 7 lowest values for PFOS and HBCD.

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3. Results and discussion

A total of 1340 lipids were found with MZmine-2.32 and of those, 153 were identified. The main lipid species identified were various glycerophospholipids (PC, LPC etc.), sphingolipids (Cer and SM), sterol lipids (CE), and the vast majority were triacylglycerols (TG).

The quality of the analyses is shown in Table 1. The combined biological and analytical variation was studied by calculating the RSDs in each exposure level, the data is presented in Appendix 2. The relative standard deviation was calculated for internal standards added to each of the samples (n= 80) in order to give an estimation of the robustness of the method. In addition, the relative standard deviation was calculated for one of the standards (c= 1 ug/ml, n=5), which included both the internal standards and the standard compounds used for the calibration curves.

Table 1. Relative standard deviation (RSD, %) of the internal standards in all samples (n = 80) and

standard compounds in standard samples (c = 1 µg/ml, n = 5).

Lipid RSD (samples) RSD(standards)

02, SM(d18:1/17:0) 14,59 28,63 03,Cer(d18:1/17:0) 17,59 31,33 04,PC(17:0/17:0) 13,89 28,06 05,LPC(17:0) 10,20 26,78 09b,PC(16:0/d30/18:1) 14,28 28,53 10, TG(17:0/17:0/17:0) 16,31 30,29 CE(18:1) 9,67 CE(18:2) 12,54 LPC(18:0) 10,22 LPC(18:1) 11,94 LysoPE(18:1) 13,92 PC(16:0/16:0) 11,36 PE(16:0/18:1) 11,47 TG(16:0/16:0/16:0) 13,02 TG(18:0/18:0/18:0) 35,42

The impact of the chemical exposure on the fish embryos was studied using a t-test, searching for differences between the control embryos (nontreated and only solvent treated). The lowest p-values (below 0.05) from the t-test performed in Excel were the lipids that were the most significantly changed. As observed in Table 2 on page 11, the most significantly changed lipids were the triacylglycerols (TG) with 11 out of 14 lowest p-values for both PFOS and HBCD. They are derived from glycerol and three fatty acids and mainly constitute in animal fat tissue. Four results showed clear patterns with a decreasing concentration of lipid the higher concentration of contaminant (Figure 9, 10, 11, 15).

The DMSO as solvent also caused some changes in the lipid profiles, however, it is difficult to evaluate what are the combinatory effects of the solvent and the pollutants, thus the values are shown for the two types of controls (Tables 3 and 4).

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Table 2. The seven significantly changed lipids of each compound (HBCD and PFOS), their p-value,

average concentration and standard deviation.

Lipid Contaminant

and conc. (µg/l) Sample Solvent P-value Average conc. (µg/ml) Standard deviation TG (56:6) HBCD, 0.014 Control 0,0023 0,4129 0,0614 TG (54:4) HBCD, 0.014 Control 0,0110 0,4498 0,0514 TG (18:1/18:2/18:2) HBCD, 1.4 Control 0,0110 2,6908 0,7617 TG (50:0) HBCD, 14 Control 0,0002 0,0379 0,0057 TG (16:0/16:0/16:0) HBCD, 14 Control 0,0002 0,2013 0,0390 TG (52:0) HBCD, 14 Control 0,0173 0,0258 0,0026 TG (54:1) HBCD, 0.014 DMSO 0,0041 0,1193 0,0284 LPC (20:4) PFOS, 58 DMSO 0,0163 0,0200 0,0078 TG (54:6) PFOS, 58 DMSO 0,0217 1,4425 0,3154

PC(O-38:5) PFOS, 0.58 Control 0,0365 0,0373 0,0089

LysoPE (18:1) PFOS, 5.8 DMSO 0,0380 0,0931 0,0136

TG (18:1/18:2/18:2) PFOS, 5.8 Control 0,0398 2,3961 0,7094 TG (16:0/18:0/18:1) PFOS, 58 DMSO 0,0463 0,6529 0,1229

TG (50:1) PFOS, 58 DMSO 0,0468 1,2301 0,1776

The exposure to HBCD caused changes in TGs levels, and no significant changes were observed in other identified lipid classes. The exposure caused increased levels of TGs where a clear trend could be seen in TG(50:0), TG (16:0/16:0/16:0) and TG(52:0) with decreasing TG concentration in higher exposure concentration.

Table 3. The lipids affected by HBCD (µg/l) compared with control and DMSO (t-test).

Control versus DMSO versus

Lipid HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 TG(56:6) 0,002 0,051 0,421 0,224 0,003 0,047 0,366 0,195 TG(54:4) 0,011 0,027 0,155 0,029 0,264 0,230 0,483 0,206 PE(34:2) 0,018 0,098 0,097 0,936 0,486 0,667 0,890 0,145 TG(55:5) 0,020 0,105 0,424 0,315 0,016 0,076 0,313 0,225 TG(56:5) 0,024 0,047 0,227 0,162 0,164 0,189 0,471 0,353 TG(60:7) 0,037 0,295 0,157 0,838 0,127 0,210 0,359 0,630 TG(16:0/22:5/18:1) or TG(20:4/ 0,040 0,376 0,279 0,344 0,014 0,134 0,161 0,173 TG(18:1/18:2/18:2) 0,192 0,071 0,011 0,015 0,418 0,247 0,070 0,076 Cer(d18:1/24:1) 0,218 0,232 0,027 0,923 0,116 0,134 0,014 0,788 LysoPE(18:1) 0,285 0,162 0,028 0,284 0,161 0,095 0,018 0,158 PC(O-38:5) 0,073 0,128 0,041 0,788 0,487 0,539 0,300 0,596 PC(35:1) 0,135 0,416 0,045 0,998 0,667 0,873 0,255 0,395 TG(50:0) 0,260 0,904 0,330 0,000 0,038 0,206 0,313 0,046 TG(16:0/16:0/16:0) 0,221 0,683 0,148 0,000 0,046 0,188 0,522 0,085 TG(52:0) 0,223 0,516 0,825 0,017 0,045 0,121 0,328 0,329 TG(54:1) 0,158 0,715 0,856 0,030 0,004 0,127 0,076 0,729 TG(54:5) 0,372 0,075 0,606 0,033 0,424 0,105 0,570 0,063 Fragment: CE signature ion 0,913 0,369 0,544 0,034 0,507 0,070 0,673 0,000 TG(52:4) 0,057 0,055 0,165 0,038 0,257 0,144 0,351 0,120 TG(52:5) 0,270 0,079 0,171 0,046 0,434 0,189 0,345 0,131 Fragment: CE signature ion 0,081 0,106 0,310 0,047 0,239 0,294 0,058 0,145

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Table 4. The lipids affected by PFOS (µg/l) compared to control and DMSO (t-test). Control vs DMSO vs

Lipid PFOS 0,58 PFOS 5,8 PFOS 58 PFOS 0,58 PFOS 5,8 PFOS 58

PC(O-38:5) 0,036518 0,06937 0,537448 0,213524 0,456464 0,693321 TG(18:1/18:2/18:2) 0,466054 0,039835 0,158613 0,763906 0,170522 0,389429 LPC(20:4) 0,660075 0,689923 0,5257 0,047979 0,128774 0,016308 LysoPE(18:1) 0,734249 0,066959 0,300362 0,402697 0,037981 0,16596 TG(54:6) 0,854106 0,118769 0,933823 0,065148 0,54321 0,021748 TG(16:0/18:0/18:1) 0,201361 0,855505 0,877021 0,586771 0,121041 0,046304 TG(50:1) 0,434409 0,661376 0,603593 0,444777 0,125836 0,046677

There are very limited number of studies on the impact of HBCD exposure on the lipid metabolism, so it is not possible to compare and directly correlate the results with the literature. There is one study that did a gene expression profile analysis by using liver tissues from Wistar rats and found that several pathways were affected by HBCD exposure, one of them being triacylglycerol metabolism (Cantón et al., 2008). The results from the study showed that the female rats were more sensitive to HBCD than males due to having a higher number of regulated genes. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females and phase I and II metabolism were up-regulated in males. The study further discusses that these specific differences in the gene expression profiles could underlie sex-specific differences in toxicity, e.g. decreased thyroid hormone or increased serum cholesterol levels.

The exposure to PFOS caused significant levels in TGs as well as three glycerophospholipids; LPC, PC and LysoPE. One clear trend is shown in PC(O-38:5) with decreasing PC concentration in higher exposure concentration. The results agree with the literature studies which have indicated that PFOS exposure disturbs lipid metabolism, particularly glycerophospholipids (Ortiz-Villanueva et al., 2018). The changes in lipids from this study are also in alignment with another study not yet published analyzing liver lipid metabolism on chicken embryos where PFOS exposure showed main changes in glycerophospholipids, cholesterol esters and TGs (Geng et al., 2018).

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13 Hexabromocyclododecane (HBCD)

Figure 6. The concentration of lipid TG(56:6) in control fish, fish treated with DMSO only and fish treated with different concentrations of HBCD (µg/l).

Exposure to HBCD caused significant increase of the TG(56:6) concentration in the lowest HBCD concentration level, showing a slightly decreasing correlation with the exposure concentration, except in the highest exposure concentration.

TG(54:4) is another lipid that was significantly changed by the exposure to HBCD, but no clear correlation with the exposure concentration can be observed except that every other lipid concentration is lower/higher, and this can be seen in the majority of the lipids as well.

0 0,1 0,2 0,3 0,4 0,5 0,6 CTRL DMSO HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 Co nce nt ra tio n ( µg/m l)

TG (56:6)

0 0,1 0,2 0,3 0,4 0,5 0,6 CTRL DMSO HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 Co nce nt ra tio n ( µg/m l)

TG (54:4)

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14 Figure 8. The concentration of lipid TG(18:1/18:2/18:2) in control fish, fish treated with DMSO only and fish treated with different concentrations of HBCD (µg/l).

Exposure to HBCD caused significant increase of the TG(50:0) concentration in the lowest HBCD concentration level in the control sample, showing a decreasing correlation with the exposure concentration. Similar trends could be seen for other lipids as well (Figures 9, 10, 11 and 15), showing that the changes in lipid profiles were associated with the exposure concentration, and the strongest changes were not necessarily observed at the highest concentration. 0 0,5 1 1,5 2 2,5 3 3,5 4 CTRL DMSO HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 Co nce nt ra tio n ( µg/m l)

TG (18:1/18:2/18:2)

0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 CTRL DMSO HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 Co nce nt ra tio n ( µg/m l)

TG (50:0)

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15 Figure 10. The concentration of lipid TG(16:0/16:0/16:0) in control fish, fish treated with DMSO only and fish treated with different concentrations of HBCD (µg/l).

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5 CTRL DMSO HBCD 0,014 HBCD 0,14 HBCD 1,4 HBCD 14 Co nce nt ra tio n ( µg/m l)

TG (16:0/16:0/16:0)

TG (52:0)

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16 Figure 12. The concentration of lipid TG(54:1) in control fish, fish treated with DMSO only and fish treated with different concentrations of HBCD (µg/l).

TG (54:1)

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17 Perfluorooctane sulfonate (PFOS)

Figure 13. The concentration of lipid LPC(20:4) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

As in the majority of the lipids, every other lipid concentration is higher/lower.

Figure 14. The concentration of lipid TG(54:6) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

0 0,005 0,01 0,015 0,02 0,025 0,03 0,035

CTRL DMSO PFOS 0,58 PFOS 5,8 PFOS 58

Co nce nt ra tio n ( µg/m l)

LPC (20:4)

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

TG (54:6)

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18 Figure 15. The concentration of lipid PC(O-38:5) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

Exposure to PFOS caused significant increase of PC(O-38:5) concentration in the lowest PFOS concentration level in the control sample, also showing a decreasing correlation with the exposure concentration as in Figure 9, 10 and 11.

Figure 16. The concentration of lipid LysoPE (18:1) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04 0,045 0,05

PC(O-38:5)

0 0,02 0,04 0,06 0,08 0,1 0,12

LysoPE (18:1)

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19 Figure 17. The concentration of lipid TG(18:1/18:2/18:2) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

Figure 18. The concentration of lipid TG(16:0/18:0/18:1) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

0 0,5 1 1,5 2 2,5 3 3,5

TG (18:1/18:2/18:2)

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

TG (16:0/18:0/18:1)

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20 Figure 19. The concentration of lipid TG(50:1) in control fish, fish treated with DMSO only and fish treated with different concentrations of PFOS (µg/l).

Conclusions

The exposure to HBCD and PFOS caused changes in the lipid profiles of particularly triacylglycerols, and the exposure to PFOS also caused changes in three glycerophospholipids. This indicates that these chemicals have a disturbance effect on the lipid metabolism of especially triacylglycerols and may affect e.g. the storage of metabolic energy in organisms as they are the most important lipids regards to that (Bruss, 2008). This might in turn predispose the organism to diseases associated with changes in triacylglycerols such as hypertriglyceridemia, but further research would have to be implemented on what the effects are of disturbances in lipid metabolism due to environmental pollutants such as PFOS and HBCD. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8

TG (50:1)

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21

Acknowledgements

Thanks to Tuulia Hyötyläinen, Ph.D., professor in chemistry at Örebro university, Sweden, who was my supervisor for this project.

Special thanks to Dawei Geng, Ph.D., postdoctoral researcher at Örebro university, Sweden, for helping me throughout the project with the practical work and always being available. To Cecilia Carlsson and Daniel Duberg for also being available in the laboratory to help and assist me in certain parts of practical work.

And also a special thanks to Heli Ratia, Ph.D., ecotoxicologist from University of Jyväskylä, Finland, that assisted me in a part of a tedious moment of the practical work.

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22

References

Baker, J. and Hites, R. (2006). Persistent organic pollutants in the Great Lakes. 1st ed. Springer, p.404.

Bossi, R., Riget, F., Dietz, R., Sonne, C., Fauser, P., Dam, M. and Vorkamp, K. (2005). Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands. Environmental Pollution, 136(2), pp.323-329. Bruss, M. (2008). Lipids and Ketones. Clinical Biochemistry of Domestic Animals, pp.81-115. Cantón, R., Peijnenburg, A., Hoogenboom, R., Piersma, A., van der Ven, L., van den Berg, M. and Heneweer, M. (2008). Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats. Toxicology and Applied Pharmacology, 231(2), pp.267-272. Du, G., Hu, J., Huang, H., Qin, Y., Han, X., Wu, D., Song, L., Xia, Y. and Wang, X. (2012).

Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine-related genes in vitro and in vivo. Environmental Toxicology and Chemistry, 32(2), pp.353-360.

Geng, D., Au Musse, A., Wigh, V., Carlsson, C., Engwall, M., Orešič, M., Scherbak, N. and

Hyötyläinen, T. (2018). Effect of perfluorooctanesulfonic acid (PFOS) on the liver lipid metabolism of the developing chicken embryo.

Kannan, K., Corsolini, S., Falandysz, J., Oehme, G., Focardi, S. and Giesy, J. (2002).

Perfluorooctanesulfonate and Related Fluorinated Hydrocarbons in Marine Mammals, Fishes, and Birds from Coasts of the Baltic and the Mediterranean Seas. Environmental Science & Technology, 36(15), pp.3210-3216.

Kemi.se. (2017). Perfluorooctane sulphonate (PFOS). [online] Available at:

https://www.kemi.se/en/prio-start/chemicals-in-practical-use/substance-groups/perfluorooctane-sulphonate-pfos [Accessed 4 Jun. 2018].

Khan, H. and Ali, J. (2015). UHPLC/Q-TOF-MS Technique: Introduction and Applications. Letters in

Organic Chemistry, 12(6), pp.371-378.

Labcompare.com. (2018). Quadrupole Time of Flight Mass Spectrometer (QTOF MS) |

Labcompare.com. [online] Available at:

https://www.labcompare.com/Mass-Spectrometry/130-

Quadrupole-Time-of-Flight-Mass-Spectrometer-QTOF-MS/Compare/?compare=9077305,11129986&catid=130 [Accessed 4 Jun. 2018].

Lipidmaps.org. (2018). LIPID MAPS Lipidomics Gateway : Lipidomics Resources. [online] Available at: http://www.lipidmaps.org/resources/tutorials/lipid_tutorial.html#L [Accessed 25 May 2018]. Ortiz-Villanueva, E., Jaumot, J., Martínez, R., Navarro-Martín, L., Piña, B. and Tauler, R. (2018). Assessment of endocrine disruptors effects on zebrafish ( Danio rerio ) embryos by untargeted LC-HRMS metabolomic analysis. Science of The Total Environment, 635, pp.156-166.

Shi, Y., Pan, Y., Yang, R., Wang, Y. and Cai, Y. (2010). Occurrence of perfluorinated compounds in fish from Qinghai-Tibetan Plateau. Environment International, 36(1), pp.46-50.

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23 US EPA. (2017). Risk Management for Hexabromocyclododecane (HBCD) | US EPA. [online]

Available at: https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/risk-management-hexabromocyclododecane-hbcd [Accessed 4 Jun. 2018].

van der Ven, L., Verhoef, A., van de Kuil, T., Slob, W., Leonards, P., Visser, T., Hamers, T., Herlin, M., Håkansson, H., Olausson, H., Piersma, A. and Vos, J. (2006). A 28-Day Oral Dose Toxicity Study Enhanced to Detect Endocrine Effects of Hexabromocyclododecane in Wistar Rats. Toxicological

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24

Appendix 1

Calibration curves

Figure 20. Calibration curve for CE(18:1). The normalized lipid area is lipid area/ISTD area.

Figure 21. y = 0,3483x R² = 0,9709 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

CE (18:1)

y = 0,378x R² = 0,8446 0 0,5 1 1,5 2 2,5 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

CE (18:2)

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25 Figure 22. Figure 23. Figure 24. y = 1,1232x R² = 0,9974 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

LPC (18:0)

y = 1,0686x R² = 0,9984 0 1 2 3 4 5 6 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

LPC (18:1)

y = 0,2416x R² = 0,9922 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

LysoPE (18:1)

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26 Figure 25. Figure 26. Figure 27. y = 1,5561x R² = 0,9854 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

PC (16:0/16:0)

y = 0,5477x R² = 0,9196 0 0,5 1 1,5 2 2,5 3 3,5 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

PE (16:0/18:1)

y = 0,093x R² = 0,9697 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

TG (16:0/16:0/16:0)

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27 Figure 28. y = 0,4441x R² = 0,9836 0 0,5 1 1,5 2 2,5 0 1 2 3 4 5 6 N or m al ize d lip id a re a Concentration (µg/ml)

TG (18:0/18:0/18:0)

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28

Appendix 2

Table 3. Standard deviation on all lipids in all samples.

Lipid CTRL DMSO HBCD

0.014 HBCD 0.14 HBCD 1.4 HBCD 14 PFOS 0.58 PFOS 5.8 PFOS 58

TG(56:6) 0,06 0,07 0,06 0,11 0,11 0,09 0,09 0,10 0,07 TG(54:4) 0,04 0,12 0,05 0,08 0,10 0,09 0,06 0,07 0,04 PE(34:2) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 TG(55:5) 0,03 0,04 0,02 0,04 0,04 0,03 0,04 0,02 0,03 TG(56:5) 0,07 0,14 0,09 0,11 0,12 0,13 0,20 0,11 0,06 TG(60:7) 0,07 0,09 0,07 0,13 0,08 0,13 0,12 0,07 0,06 TG(16:0/22:5/18:1) or TG(20:4/18:1/18:1) 0,78 0,63 0,98 0,51 1,31 0,96 1,26 0,88 0,80 TG(18:1/18:2/18:2) 0,65 1,08 0,81 0,70 0,76 0,84 0,64 0,71 0,74 Cer(d18:1/24:1) 0,03 0,03 0,04 0,04 0,04 0,04 0,05 0,03 0,06 LysoPE(18:1) 0,01 0,02 0,02 0,03 0,02 0,02 0,01 0,01 0,02 PC(O-38:5) 0,00 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 PC(35:1) 0,01 0,01 0,01 0,02 0,01 0,01 0,02 0,01 0,01 TG(50:0) 0,01 0,01 0,02 0,02 0,01 0,01 0,01 0,01 0,01 TG(16:0/16:0/16:0) 0,02 0,04 0,11 0,10 0,04 0,04 0,07 0,06 0,09 TG(52:0) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 TG(54:1) 0,02 0,02 0,03 0,02 0,03 0,02 0,02 0,03 0,02 TG(54:5) 0,04 0,07 0,03 0,07 0,05 0,06 0,05 0,04 0,04 Fragment: CE signature ion 0,02 0,01 0,04 0,03 0,02 0,02 0,03 0,02 0,03 TG(52:4) 0,03 0,05 0,02 0,05 0,04 0,05 0,04 0,03 0,02 TG(52:5) 0,04 0,07 0,03 0,04 0,03 0,04 0,05 0,03 0,03 Fragment: CE signature ion 0,01 0,01 0,01 0,01 0,01 0,01 0,02 0,02 0,02 CE(18:2) 0,03 0,02 0,05 0,05 0,02 0,05 0,06 0,02 0,05 TG(47:2) 0,01 0,01 0,01 0,01 0,00 0,00 0,00 0,01 0,00 TG(18:2/18:1/18:1) 0,74 1,42 0,68 0,98 1,16 1,17 0,65 0,83 0,55 TG(52:3) 0,04 0,09 0,05 0,08 0,06 0,09 0,05 0,05 0,03 TG(53:5) 0,03 0,05 0,02 0,04 0,04 0,04 0,04 0,03 0,02 TG(18:1/18:2/18:2) 0,53 1,04 0,40 0,71 0,66 0,73 0,62 0,48 0,48 TG(16:0/18:2/18:2) 0,53 0,83 0,37 0,67 0,65 0,68 0,41 0,55 0,33 TG(16:0/18:2/18:3) 0,60 0,88 0,44 0,56 0,53 0,50 0,54 0,50 0,38 TG(54:6) 0,10 0,13 0,11 0,08 0,10 0,12 0,11 0,10 0,10 TG(54:3) 0,07 0,15 0,09 0,12 0,12 0,13 0,07 0,08 0,05 PC(O-38:6) 0,02 0,02 0,02 0,03 0,02 0,08 0,03 0,02 0,14 TG(50:0) 0,00 0,01 0,00 0,00 0,00 0,00 0,00 0,00 0,00 TG(14:0/18:2/18:2) 0,26 0,37 0,17 0,25 0,23 0,25 0,20 0,24 0,16 TG(50:1) 0,00 0,01 0,00 0,00 0,01 0,00 0,00 0,00 0,00 TG(54:5) 0,04 0,03 0,05 0,07 0,07 0,06 0,04 0,06 0,06 TG(18:1/18:1/18:1) 0,90 1,73 0,99 1,32 1,50 1,46 0,74 1,03 0,67 TG(50:3) 0,02 0,03 0,02 0,03 0,02 0,03 0,02 0,02 0,01 10, TG(17:0/17:0/17:0) 380410,12 946326,77 700366,52 952468,89 502825,60 842030,61 916960,70 619291,40 513824,78

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29 CE(18:1) 0,09 0,10 0,13 0,18 0,09 0,16 0,18 0,08 0,16 SM(d38:2) 0,00 0,00 0,00 0,00 0,01 0,00 0,00 0,00 0,00 TG(18:2/18:1/16:0) 0,74 1,24 0,65 1,01 0,96 1,09 0,56 0,83 0,45 04,PC(17:0/17:0) 3579 73,36 599147,24 709220,26 1029173,18 20,32 3117 778910,45 826641,63 524695,23 515979,71 02, SM(d18:1/17:0) 1572 45,64 217776,85 286527,52 373254,83 18,64 1213 312668,56 321891,16 235936,45 243084,49 TG(18:2/18:2/18:2) or TG(18:3/18:2/18:1) 0,87 1,37 0,84 0,68 1,25 1,12 0,80 0,88 0,74 TG(51:4) 0,03 0,04 0,02 0,03 0,03 0,03 0,03 0,02 0,02 TG(52:6) 0,30 0,77 0,49 0,43 0,44 0,70 0,59 0,60 0,31 09b,PC(16:0/d30/18:1 ) 67002,37 108434,91 124339,05 181555,03 54931,92 141955,24 151246,96 88591,10 98569,37 TG(18:0/18:0/18:0) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 TG(50:2) 0,04 0,07 0,04 0,06 0,04 0,07 0,04 0,05 0,02 TG(50:5) 0,16 0,23 0,12 0,18 0,12 0,17 0,15 0,13 0,09 TG(50:3) 0,38 0,54 0,26 0,40 0,34 0,42 0,28 0,37 0,19 TG(50:5) 0,04 0,04 0,04 0,04 0,05 0,04 0,06 0,10 0,06 TG(48:3) 0,10 0,12 0,07 0,09 0,07 0,09 0,07 0,09 0,05 TG(53:4) 0,05 0,07 0,03 0,07 0,06 0,06 0,04 0,04 0,03 TG(54:6) 0,26 0,62 1,23 1,08 0,36 0,68 1,48 0,32 0,22 SM(d36:1) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 TG(56:5) 0,22 0,31 0,18 0,26 0,31 0,29 0,19 0,20 0,16 TG(16:0/18:0/18:1) 0,11 0,11 0,17 0,19 0,16 0,16 0,13 0,20 0,12 TG(51:3) 0,06 0,09 0,04 0,08 0,06 0,08 0,04 0,06 0,04 SM(d18:0/14:0) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 PC(O-38:6) 0,09 0,09 0,09 0,10 0,08 0,09 0,09 0,06 0,09 05,LPC(17:0) 3065 4,35 71800,99 59041,48 59233,14 6,54 6398 45698,32 60339,90 62352,53 59709,96 TG(14:0/18:1/18:1) 0,56 0,79 0,47 0,67 0,56 0,72 0,41 0,62 0,31 TG(49:3) 0,02 0,02 0,01 0,02 0,01 0,01 0,01 0,01 0,01 TG(51:2) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,00 03,Cer(d18:1/17:0) 1342 20,98 174043,27 234610,16 272577,67 24,73 1001 234639,35 243224,29 205169,03 198516,02 PC(30:0) 0,05 0,04 0,03 0,07 0,04 0,04 0,05 0,04 0,04 TG(52:2) 0,06 0,09 0,07 0,09 0,08 0,11 0,05 0,07 0,04 TG(53:3) 0,09 0,12 0,08 0,12 0,11 0,12 0,06 0,09 0,06 TG(58:6) 0,06 0,10 0,13 0,10 0,09 0,10 0,15 0,06 0,06 TG(18:1/18:1/16:0) 0,70 1,04 0,73 1,03 0,94 1,08 0,56 0,86 0,49 TG(49:2) 0,03 0,04 0,03 0,04 0,03 0,04 0,03 0,03 0,02 PC(O-34:2) 0,00 0,00 0,00 0,00 0,00 0,00 0,01 0,00 0,00 TG(56:4) 0,32 0,43 0,27 0,35 0,44 0,37 0,26 0,27 0,21 TG(18:1/12:0/18:1) or TG(18:2/16:0/14:0) 0,21 0,25 0,16 0,20 0,15 0,21 0,15 0,20 0,09 TG(51:2) 0,08 0,11 0,06 0,11 0,08 0,11 0,06 0,09 0,06 TG(47:1) 0,01 0,01 0,01 0,01 0,00 0,01 0,01 0,01 0,00 TG(52:6) 0,17 0,22 0,16 0,24 0,11 0,21 0,13 0,23 0,15 LPC(20:4) 0,01 0,00 0,01 0,01 0,01 0,01 0,01 0,01 0,01 PC(18:0p/18:1(9Z)) 0,00 0,00 0,00 0,00 0,00 0,00 0,01 0,00 0,00

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30 TG(56:2) 0,06 0,06 0,05 0,07 0,08 0,06 0,06 0,08 0,06 TG(18:2/22:5/16:0) 0,88 1,42 0,96 1,00 1,22 0,99 0,99 1,05 0,57 TG(48:1) 0,02 0,02 0,01 0,02 0,01 0,02 0,01 0,02 0,01 LPC(18:0) 0,03 0,03 0,02 0,03 0,03 0,03 0,04 0,02 0,04 PC(40:8) 0,09 0,10 0,08 0,11 0,10 0,09 0,12 0,07 0,12 PC(32:1) 0,30 0,33 0,21 0,51 0,36 0,38 0,39 0,32 0,23 PC(31:0) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 LPC(22:6) 0,21 0,14 0,15 0,26 0,24 0,24 0,27 0,17 0,24 PI(18:0/20:4) 0,02 0,02 0,02 0,03 0,02 0,03 0,03 0,03 0,03 PE(16:0/18:1) 0,03 0,02 0,02 0,04 0,02 0,02 0,02 0,02 0,02 PC(37:2) 0,00 0,00 0,00 0,01 0,00 0,01 0,01 0,00 0,01 PC(33:1) 0,03 0,03 0,02 0,04 0,03 0,03 0,03 0,02 0,03 TG(14:0/16:0/18:1) 0,17 0,19 0,14 0,22 0,14 0,20 0,15 0,21 0,10 PC(37:1) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 PC(P-18:0/22:6) 0,05 0,03 0,03 0,05 0,04 0,06 0,05 0,04 0,04 PC(35:2) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 TG(56:4) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 TG(56:3) 0,41 0,49 0,35 0,49 0,52 0,45 0,30 0,42 0,34 TG(54:7) 1,16 2,09 1,27 1,54 1,49 1,64 1,28 1,37 1,14 PE(O-16:0/22:6) or PE(P-18:0/20:5) 0,02 0,02 0,02 0,05 0,02 0,03 0,02 0,02 0,01 PC(O-40:6) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 PC(42:5) 0,01 0,01 0,01 0,02 0,01 0,02 0,01 0,01 0,01 PC(36:4) 0,18 0,18 0,15 0,36 0,23 0,28 0,25 0,15 0,14 PE(38:6) 0,87 0,54 0,39 1,32 0,66 0,83 0,69 0,65 0,57 LPC(18:1) 0,05 0,04 0,04 0,06 0,06 0,06 0,07 0,04 0,06 PC(34:3) 0,04 0,04 0,03 0,05 0,05 0,05 0,06 0,03 0,04 PC(16:0/16:0) 0,08 0,07 0,07 0,12 0,07 0,08 0,06 0,07 0,05 PC(32:2) 0,05 0,05 0,04 0,05 0,04 0,04 0,05 0,05 0,04 PC(34:2) 0,25 0,30 0,21 0,39 0,29 0,32 0,37 0,22 0,31 LPC(18:2) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 PC(36:4) 0,12 0,15 0,08 0,27 0,14 0,21 0,17 0,15 0,12 PC(36:3) 0,13 0,09 0,07 0,11 0,09 0,09 0,14 0,06 0,11 PC(40:5) 0,12 0,10 0,08 0,11 0,11 0,11 0,13 0,10 0,14 PC(40:4) 0,01 0,01 0,00 0,01 0,00 0,00 0,01 0,00 0,01 TG(18:0/18:1/20:4) 0,07 0,06 0,08 0,10 0,08 0,09 0,08 0,07 0,05 LPC(22:5) 0,03 0,02 0,03 0,04 0,03 0,04 0,04 0,03 0,03 TG(49:1) 0,01 0,01 0,01 0,02 0,01 0,02 0,01 0,02 0,01 TG(18:1/18:1/22:6) 0,41 1,18 0,72 0,58 0,92 0,99 0,66 0,55 0,42 PC(16:0e/18:1(9Z)) 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 TG(58:9) 0,37 0,94 0,37 0,89 0,77 0,77 0,39 0,66 0,51 PE(P-18:0/22:6) 0,04 0,02 0,02 0,05 0,03 0,03 0,03 0,03 0,02 PC(36:4) 0,10 0,11 0,08 0,14 0,10 0,13 0,14 0,09 0,16 TG(53:2) 0,05 0,06 0,04 0,07 0,06 0,06 0,04 0,06 0,04 PC(36:2) 0,00 0,00 0,00 0,00 0,01 0,00 0,01 0,00 0,01 PC(34:1) 1,07 1,01 0,83 1,83 1,28 1,42 1,37 0,90 0,79 PC(42:6) 0,04 0,03 0,03 0,04 0,04 0,04 0,05 0,04 0,03

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31 TG(18:2/22:5/16:0) 0,42 0,88 0,58 1,41 0,33 0,93 0,57 0,53 0,43 PC(38:6) 1,30 1,80 0,98 2,46 1,47 1,86 2,29 1,29 1,76 PC(36:5) 0,64 0,85 0,57 1,02 0,65 0,96 0,97 0,63 1,05 SM(d32:1) 0,01 0,01 0,01 0,02 0,01 0,02 0,02 0,01 0,02 PC(40:6) 0,16 0,17 0,19 0,21 0,14 0,24 0,19 0,15 0,20 PC(37:4) 0,00 0,00 0,00 0,00 0,00 0,00 0,01 0,00 0,00 PC(40:6) 0,13 0,10 0,10 0,12 0,11 0,10 0,13 0,09 0,12 TG(58:6) 0,08 0,13 0,08 0,11 0,08 0,15 0,15 0,05 0,14 TG(50:1) 0,20 0,23 0,27 0,34 0,24 0,32 0,21 0,33 0,18 PC(36:2) 0,24 0,24 0,21 0,32 0,25 0,26 0,34 0,19 0,28 PC(40:5) 0,01 0,01 0,01 0,01 0,01 0,01 0,02 0,01 0,01 SM(d34:1) 0,01 0,01 0,01 0,01 0,01 0,01 0,00 0,01 0,01 PC(38:3) 0,02 0,01 0,01 0,02 0,02 0,02 0,02 0,03 0,02 PC(38:6) 0,26 0,27 0,21 0,28 0,25 0,27 0,33 0,20 0,38 TG(54:2) 0,42 0,48 0,40 0,56 0,59 0,56 0,35 0,51 0,35 PC(42:6) 0,05 0,04 0,05 0,06 0,05 0,05 0,06 0,05 0,06 PC(40:7) 0,69 0,75 0,51 0,81 0,65 0,52 0,87 0,51 0,83 PC(40:6) 0,83 0,80 0,58 0,86 0,82 0,64 0,96 0,69 1,03 PC(36:3) 0,11 0,09 0,08 0,08 0,09 0,10 0,07 0,06 0,09 TG(54:2) 0,04 0,05 0,04 0,06 0,06 0,06 0,04 0,06 0,04 PC(39:6) 0,08 0,08 0,05 0,08 0,07 0,06 0,09 0,05 0,10 TG(54:6) 0,39 0,26 0,20 0,38 0,16 0,34 0,37 0,26 0,32 PE(36:4) 0,02 0,02 0,01 0,03 0,02 0,02 0,02 0,02 0,02 PC(38:5) 0,43 0,38 0,30 0,49 0,42 0,43 0,55 0,35 0,51 PE(38:4) 0,02 0,02 0,02 0,04 0,02 0,03 0,03 0,02 0,02 PC(38:4) 0,03 0,04 0,03 0,06 0,04 0,05 0,06 0,05 0,05 TG(56:8) 1,14 2,12 1,20 1,49 1,63 1,70 1,20 1,36 1,12 LPC(16:1) 0,01 0,01 0,01 0,01 0,02 0,01 0,01 0,01 0,01