Metabolomic study of the effects of perfluorinated compounds on the fatty acid metabolism during the development of Gallus gallus domesticus

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Metabolomic study of the effects of

perfluorinated compounds on the fatty

acid metabolism during the

development of Gallus gallus

domesticus

Viktoria Wigh

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Abstract

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are two commonly found contaminants associated with various manufacturing products, such as firefighting foam, non-stick coatings, electronics and water repellants. These compounds are persistent, bioaccumulative, and toxic and may therefore pose a serious health risk to living organisms. Earlier studies have shown that PFOS and PFOA affected the fatty acid β-oxidation, i.e. the energy metabolism in liver. This study evaluates the effects of PFOS and PFOA on fatty acid metabolism in domestic chicken liver cells. Liver tissues were obtained from chicken embryos treated in ovo with PFOS or with PFOA at low (0.1 µg/g) and high (1.0 and 1.6 µg/g) concentration levels. The fatty acids were extracted and derivatized into fatty acid methyl esters (FAMEs). The analysis was conducted by gas chromatography coupled with mass spectrometry. Results showed that a lower concentration of PFOS and a lower percentage of DMSO significantly affected the concentrations of fatty acids in livers of chicken embryos. PFOA-treated samples also showed some significant elevated fatty acid concentrations. Almost all fatty acid concentrations of treated liver samples exceeded the concentrations of the untreated samples.

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1. Introduction

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are both perfluorinated compounds (PFCs) which means that the carbon backbone with a carboxylic acid or sulfonic acid is only bound to fluorine atoms. Both PFOS and PFOA are commonly found contaminants and give rise for concern regarding living organisms’ health [1]. The effects of PFOA and PFOS on living

organisms are relevant topics considering both compounds may pose a serious health risk and at the same time are persistent [2]. It has been shown in previous studies that both PFOA and PFOS affected the fatty acid metabolism in liver, the mechanisms behind this, however, are not completely

understood. The oxidation of triglycerides takes particularly place in the peroxisome and mitochondria in the liver cells. The liver cells perform β-oxidation on large quantities of fatty acids and export it to other tissues in the organism [3]. A disrupted fatty acid metabolism may give rise to numerous health issues. This study focuses therefore on the effects of these PFCs on β-oxidation of fatty acids and thus, the energy metabolism which are of great interest to further understand the effects these compounds have on living organisms.

This study uses domestic chicken as an avian model. Chickens are a common model object within these kinds of studies, particularly when parallels to avian wildlife are to be drawn [4]. Birds often accumulate contaminants from the environment and are therefore of interest to study when it comes to the effects that the contamination may give to wild life [5]. The bird embryo is more sensitive to certain contaminants and is of interest when observing the developmental effects [6].

1.1 Objective

The aim of this project was to further understand how certain perfluorinated compounds (PFCs), such as PFOA and PFOS, affect the energy metabolism in the liver cells of living organisms, in this case the domestic chicken. More precisely, the overall goal was to study the effects of these compounds on β-oxidation of fatty acids.

1.2 Demarcations

This study investigated the effects of PFOS and PFOA on the fatty acids from C12 to C22 in liver cells derived from chicken embryos dissected on the 19th_{day of incubation. In biological tissues}

however, specific fatty acids are present in very low concentrations and thus they are difficult to detect. Therefore the following fatty acids C13:0, C15:0, C18:3, C18:4, C19:0, C20:1, C20:3, C20:5, C21:0, C22:1 and C22:4 were excluded from the study.

2. Ethical considerations

This study is based on liver tissues and therefore it is appropriate to mention the ethical aspects. All liver tissues used in this project work were derived from chicken embryos. Domestic chickens are a common model object within toxicology studies, especially when drawing parallels to avian wildlife [4].

The samples of the present investigation were archived from a previous study [7] which the Swedish Animal Welfare Agency had approved.

Working with animal tissues can give rise to ethical dilemmas, however, in order perform this project and get viable results it was necessary.

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3. Background

Perfluorinated compounds (PFCs) are a subgroup of organic contaminants where the carbon backbone is only bonded to fluorine atoms and obtains either a carboxylic acid or sulfonic acid for example as a functional group. PFCs have become a common contaminant as a consequence for the widespread use within the manufacturing of food packages, non-stick cookware, firefighting foams, stain and water repellants etc. Two compounds that are a cause for concern and belong to this group of contaminants are perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). PFOA is most

commonly derived from the production of chemicals. Ammonium perfluorooctane has been used within the manufacturing of fluoropolymers as a processing aid. PFOA consists of seven

perfluorinated carbons and a carboxylic acid and its chemical formula is C7F15COOH. The compound

is persistent, reproduction disruptive and is also suspected to be a carcinogenic compound. PFOS was used as a surfactant and is made up of eight perfluorinated carbons and a sulfonic acid at the end of the carbon chain, its chemical formula is C8F17SO3H. PFOS is also persistent, bioaccumulative and toxic

to mammalian species. Within EU the use of PFOS is forbidden since 2008 but relatively high levels can still be found in the environment, and through food intake and other sources mammals get a continuous exposure to PFOS and PFOA [1, 2, 8, 9].

Birds are a top consumer that often accumulates contaminants which can give certain unwanted effects [5]. Bird embryos are more sensitive to certain contaminants, and these contaminates have been found to be transferred from the mother to the embryo [6]. PFOS and PFOA have been found in avian wildlife, both in fully grown birds but also in embryos. PFOS has been found in great cormorant for example in levels up to 3800 ng/g in egg yolk, and with an average concentration of 690 ng/g in the whole egg. PFOA on the other hand has been found in great cormorant as well but at lower

concentrations with an average of 4 ng/g (whole egg). These samples were collected at Lake Vänern, Sweden [10].

The β-oxidation for fatty acids is a process that produces energy by breaking down the fatty acids during multiple steps (Figure 1). Fatty acid β-oxidation occurs in both mitochondria and peroxisomes. These processes are similar but they, however, have different enzymes performing different steps in the fatty acid break down. It is believed that peroxisomes are involved in the initial breakdown of fatty acids containing a long carbon chain and some fatty acids with methyl branches (α-oxidation). The commencing enzyme in peroxisome β-oxidation is acyl-CoA oxidase which is probably also the most significant difference between the two different oxidations sites. Acyl-CoA oxidase produces

hydrogen peroxide instead of FADH2 which is a product from the enzyme Acyl-CoA dehydrogenase

present in mitochondrial β-oxidase. However, in both mitochondrial and peroxisome β-oxidation the fatty acyl-CoA intermediates formed are the same.

It is necessary that prior entering the cell the fatty acid converts to fatty acyl-CoA, otherwise the molecule cannot enter the mitochondria. Fatty acyl-CoA synthase (FACS) is the enzyme responsible for this transformation [3].

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Figure 1. Overview of mitochondrial fatty acid β-oxidation.

Nuclear receptor proteins may interact with some fatty acids or their derivatives. These nuclear receptor proteins can be connected to particular DNA regulatory regions and by association with fatty acids alter the transcription. Earlier studies on gene expression have led to the belief that the

peroxisome proliferator activated receptors (PPARs) are affected by both PFOS and PFOA as

agonists. PPAR is connected to the lipid metabolism, where fatty acids pose as a weak activator of this receptor. It is assumed that if for some unknown reason the oxidation of fatty acids is blocked they stimulate PPAR with greater efficiency, and the energy metabolism is thereby regulated. This is caused by elevated levels of PGC-1α when more free fatty acids are present [3, 11].

An earlier investigation studied the gene expression in chicken [12] where genes relating to the metabolism of fatty acids where affected by both PFOA and PFOS. When exposed to PFOS or PFOA the results showed that down-regulation of metabolic tendencies were more prominent [12].

Another study observed the effects PFOS had on fatty acid β-oxidation in chicken embryo liver [10]. In this study the effects observed started to occur at concentrations of 0.1 µg/g, and the conclusion was that the β-oxidation rate of C16:0 (palmitic acid) was induced. However, at concentrations of PFOS that exceeds 0.3 µg/g the effect was reduced but still resulted in an up-regulated fatty acid β-oxidation [10].

Regulations of β-oxidation of fatty acids are at multiple stages; however, the most recurring levels include PPAR. This is a receptor that acts in the nucleus and increases the transcription of several genes including genes for fatty acid applications. Nevertheless PGC-1α is a transcriptional factor well known for its involvement in fatty acid β-oxidation. The activity of PPAR increases when PGC-1α connects to it. PPAR regulates several proteins involved in the oxidation of fatty acids, for instance both medium and long-chain acyl-CoA dehydrogenase (MCAD and LCAD). PPARα is particularly expressed in tissues as heart, liver and skeletal muscles, i.e. tissues with high metabolic characteristics [3].

This study focuses on the fatty acids C12 to C22 in triglycerides and phospholipids. A fatty acid is a carboxyl acid with a long aliphatic chain which either can be unsaturated or saturated. The most common ones are linear and have an even number of carbons. Among these common fatty acids C16 and C18 stand out because of its widespread presence in animals, plants and microorganisms [11]. Phospholipids and triglycerides are usually the source for free fatty acids. Most phospholipids are made up by one unsaturated fatty acid located at C2 and one saturated fatty acid on the C1-position

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6 attached to the phosphate head group. Fatty acids are stored as triglycerides; this is a structure made up of three fatty acids connected by glycerol through esterification. C16:0 and C18:0 combined are the most common fatty acids in triglycerides [13, 14].

When analyzing fatty acids with GC-MS they need to be volatile, this is achieved by derivatizing them into FAMEs (fatty acid methyl esters). However, this method primary derivatizes the triglycerides and phospholipids but is not as efficient for free fatty acids. The free fatty acids will therefore not be present in the results from the analysis [15].

4. Methods

4.1 Exposure procedure

The liver samples were archived from an earlier study [7]. The eggs from G. gallus domesticus (white leghorn chicken) were bought fertilized and unincubated from Ova production, Vittinge, Sweden. When incubated, the eggs were kept in 60 % humidity at 37.5°C and were monitored every 1-3 days. The eggs were then injected with either PFOS, PFOA or DMSO (dimethyl sulfoxide) at the fourth day of incubation with different concentrations; however in this study the concentrations of 0.1 µg/g and 1.0 µg/g PFOS, 1.6 µg/g PFOA, 2.5 % and 5 % DMSO were chosen as these concentrations of PFOS and PFOA have previously been found in avian wildlife [10]. The water solution with 2.5 % DMSO was used for the PFOA solutions and 5 % DMSO for PFOS due to difficulties dissolving PFOS in the same concentration of DMSO as PFOA. The incubation continued after injection and dissected on the 19th_{day of incubation when the hatching process began. The chicken embryos hearts and livers were}

collected [7, 10].

4.2 Liver samples

The samples analyzed were liver tissues derived from chicken embryos. Eight samples were treated in

ovo with PFOS and additionally four more with PFOA. Furthermore, eight samples that were only

treated with the solvent used in the earlier mentioned treatments, in this case dimethyl sulfoxide (DMSO). There were also four samples that were completely untreated and untouched. All of these samples have been stored at -80°C since dissection. The concentrations of treatments and their names are shown in Table 1.

Table 1. A list of treatments and the sample names.

Sample number: Content: Label:

1-4 Untreated liver LS1, LS2, LS3, LS4

5-8 Liver treated with 5 % DMSO LS5, LS6, LS7, LS8

9-12 Liver treated with 0.1 µg/g PFOS LS9, LS10, LS11, LS12

13-16 Liver treated with 1.0 µg/g PFOS LS13, LS14, LS15, LS16

17-20 Treated with 2.5 % DMSO LS17, LS18, LS19, LS20

21-24 Treated with 1.6 µg/g PFOA LS21, LS22, LS23, LS24 .

4.3 Sample preparation

When extracting the fatty acids, the liver tissue was added to a pre-weighed homogenizer tube containing 200 µl or 50 µl PBS buffer depending on the amount of tissue sample. The samples were then weighed and the difference between the weighed liver sample and the amount PBS was fixed to

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7 the ratio of 1 mg liver tissue to 1 µl PBS buffer. The mixture was then homogenized. A fixed amount of 50 µl homogenate was transferred into a new vial; each sample was duplicated except for LS1 which was made as a triplicate for statistical reasons. 25 µl 600 ppm internal standard (methyl

undecanoate), and as a control, 25 µl 600 ppm triheptadecanoate TG C17:0 was added to each sample. 1.5 ml methanol was added and the samples vortexed. 5 ml MTBE (methyl-tert-butyl ether) was also added. The samples were then incubated in room temperature for 60 minutes placed on a shaker plate. 1.25 ml MQ-water was added to each sample. After a 10 minute long incubation at room temperature the samples were centrifuged at 1000 RCF for 10 minutes. The centrifugation creates two phases, 5 ml of the organic phase was transferred to a new vial and the solvent was evaporated under a flow of nitrogen.

To be able to analyze the fatty acids with GC-MS the analytes need to be volatile, this study uses FAMEs (fatty acid methyl esters). This derivatization step was performed by using petroleum ether, sodium methoxide and hydrochloric acid (HCl). The samples were dissolved in 700 µl petroleum ether and mixed by vortex. If the whole mixture was not dissolved, the liquid was transferred to a new vial. 250 µl of 0.5 M sodium methoxide was added to the dissolved samples and the mixture was vortexed. A couple of boiling stones were added as well and the sample boiled at 45°C for 5 minutes using a heating plate. 500 µl of 15 % HCl and 200 µl petroleum ether were added, and the samples were vortexed after additives. The mixture was transferred into a new vial and centrifuged at 1000 RCF for 10 minutes, creating two phases and 400µl of the petroleum ether layer was transferred to a GC-vial. After the transmethylation there should be a solvent swap to hexane since it is a better choice of solvent for GC-analysis than the former. The solvent swap was performed by evaporation of

petroleum ether under a flow of nitrogen. Hexane was then added giving the mixture a total volume of 500 µl in each sample and after being thoroughly mixed the samples were ready for analysis.

4.4 Standards

The use of standards is crucial for quantitative analysis. In this study an internal standard, recovery standard and external calibration curve were used for calculating the concentrations and recovery for the fatty acids. External calibration was done by using a standard mixture, the components are

presented in Table 2. However, not all of these compounds were detected in the liver samples; only the fatty acids C12:0 to C22:0 were quantified. The calibration curve of C18:3 was used to calculate concentrations for C20:4 and C22:6 since the compounds were detected with the same quantification ion. The calibration curve was made of six points with the concentrations of 1 ppm, 3ppm, 10 ppm, 30 ppm, 100 ppm and 150 ppm of each fatty acid. The internal standard was added to all samples post homogenization as a fixed amount 600 ppm methyl undecanoate, with the final concentration of 30 ppm in the sample. Also 600 ppm triheptadecanoate TG C17:0 was used as a recovery standard, with the final concentration of 30 ppm, which was added at the same time as the internal standard.

Table 2. Compounds included in the external standard mixture.

Components in the external calibration: Internal standard (Methyl undecanoate) Dimethyl Suberate (C8) Dimethyl Azelate (C9) Dimethyl Sebacate (C10) Caprylic acid (C8:0) Capric acid (C10:0) Lauric acid (C12:0)

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4.5 Analysis

The analysis was performed by gas chromatography coupled with mass spectrometry (Agilent Technologies). The GC settings are shown in Table 3. The samples were analyzed in randomized order.

Table 3. Settings for the gas chromatograph.

Injection type Split

Injection 1:10 µl

Oven program 40 ºC for 1 min, 5ºC/min to 225ºC, 40ºC/min to 300ºC with a 15 min hold

Column Non-polar, DB-5MS, 122-5532 (30 m x 0.250 mm x 0.25 µm), -60°-325°C

Flow rate 1.5 ml/min

Total run time 54,88 min

The mass spectrometer used in this project is a quadropol. When carrying out the analysis the selective ion monitoring (SIM) was used to get a better selectivity and higher sensitivity. The quantification ions used for the liver samples were m/z 74, 79 and 81.

5. Results

The results for this study are presented in Tables 4-9 and in Figure 2. In Table 4, the fold changes of the different fatty acids are presented. The fold change is the ratio between the treated samples and the untreated samples, using median concentrations. Table 5 shows the significance of the concentration values in the form of a t-test. Table 6 presents the difference in concentrations between the treated samples towards the untreated samples. The standard deviations of the triplicates are shown in Table 7. The recovery values are presented in Table 9 and Table 8 represents the equations for the calibration curves. Lastly, Figure 2 shows an overview of all the compounds concentrations and their standard deviation.

The liver weights, peak areas, retention times and concentrations for each compound in every sample can be found in appendix I.

Myristic acid (C14:0) Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0)

Vaccenic, Oleic, Elaidic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Arachidic acid (C20:0) Behenic acid (C22:0) Erucic acid (C22:1) Lignoceric acid (C24:0)

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Table 4. Fold change for the different compounds and treatments when compared to the untreated samples (median

concentration of treated samples/median concentration of untreated samples).

Table 5. T-test: Comparison of the different treatments to the untreated liver samples. The PFOA-treated liver samples have

also been compared to the 2.5 % DMSO-treatment. A= Untreated liver samples, B= 5 % DMSO-treated liver samples, C= 0.1 µg/g PFOS-treated liver samples, D= 1.0 µg/g PFOS-treated liver samples, E= 2.5 % DMSO-treated liver samples, F= 1.6 µg/g PFOA-treated liver samples.

5 % DMSO 0.1 µg/g PFOS 1.0 µg/g PFOS 2.5 % DMSO 1.6 µg/g PFOA Compound Fold change Fold change Fold change Fold change Fold change

C12:0 1.01 1.02 1.00 1.03 1.02 C14:0 1.00 1.00 1.00 1.00 1.00 C16:0 1.26 1.97 1.08 3.81 5.23 C16:1 1.07 1.12 0.95 1.11 1.41 C18:0 1.25 1.78 0.98 3.58 4.80 C18:1 1.10 1.41 0.95 1.68 2.70 C18:2 1.09 1.82 0.90 3.44 4.24 C20:0 1.00 1.01 1.00 1.03 1.04 C20:4 1.06 1.69 0.96 3.92 4.66 C22:0 1.00 1.00 1.00 1.01 1.01 C22:6 1.06 1.74 0.96 4.02 4.86 T-test A vs C A vs D A vs B A vs E A vs F E vs F Compound P P P P P P C12:0 0.0511 0.8591 0.3496 0.162 0.1059 0.5604 C14:0 0.6551 0.3821 0.7549 0.7419 0.7175 0.9176 C16:0 0.0009 0.7051 0.4446 0.0327 3E-05 0.2802 C16:1 0.0354 0.1929 0.5189 0.2337 0.0023 0.0422 C18:0 0.0091 0.7923 0.3532 0.0178 0.0006 0.3237 C18:1 0.0085 0.2133 0.4949 0.109 0.0004 0.0558 C18:2 0.0066 0.6476 0.5568 0.0107 0.0007 0.2215 C20:0 0.0733 0.503 0.8555 0.0663 0.0007 0.5251 C20:4 0.0212 0.7999 0.5658 0.0053 0.0007 0.3354 C22:0 0.0696 0.8147 0.9534 0.067 0.0348 0.838 C22:6 0.031 0.9274 0.7184 0.0211 0.0006 0.5234

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Table 6. The values represent the difference in concentrations (ppm), the median and mean concentrations for the untreated

samples are subtracted from the median and mean concentrations from the treated liver samples. Positive values show that those compounds exist in higher concentrations in the treated liver than in the untreated liver samples. The PFOA-treated liver samples have also been compared to the 2.5 % DMSO-treatment.

5 % DMSO 0.1 µg/g PFOS 1.0 µg/g PFOS

Compound Median(T)-Median(UT) Mean(T)-Mean(UT) Median(T)-Median(UT) Mean(T)-Mean(UT) Median(T)-Median(UT) Mean(T)-Mean(UT) C12:0 0.0035 0.0051 0.0130 0.0133 0.0003 -0.0006 C14:0 0.0002 -0.0011 0.0006 -0.0014 -0.0004 -0.0028 C16:0 6.9398 5.1714 26.4526 24.7977 2.0687 -1.5681 C16:1 0.4762 0.2380 0.8495 0.8008 -0.3664 -0.3799 C18:0 14.0842 10.1691 44.6897 48.4878 -1.1168 -1.5709 C18:1 1.4989 1.2298 5.8249 5.3453 -0.7601 -1.4968 C18:2 7.5200 9.4557 72.3515 87.3860 -9.2227 -6.5831 C20:0 0.0100 0.0029 0.0498 0.0547 -0.0114 -0.0068 C20:4 2.8627 5.7776 34.9151 46.4048 -2.1752 2.1905 C22:0 0.0039 0.0006 0.0181 0.0219 0.0025 -0.0024 C22:6 1.5364 1.5214 18.5649 23.6218 -0.9333 0.4341

2.5 % DMSO 1.6 µg/g PFOA PFOA-2.5 % DMSO

Compound Median(T)-Median(UT) Mean(T)-Mean(UT) Median(T)-Median(UT) Mean(T)-Mean(UT) Median Mean C12:0 0.0203 0.0326 0.0125 0.0189 -0.0079 -0.0138 C14:0 0.0007 0.0018 0.0040 0.0013 0.0033 -0.0005 C16:0 76.2766 77.5712 115.0606 112.4421 38.7840 34.8709 C16:1 0.8239 0.9808 3.0103 3.4133 2.1864 2.4325 C18:0 147.2730 154.5781 217.2280 216.1840 69.9549 61.6058 C18:1 9.7078 9.4732 24.3223 23.4900 14.6145 14.0167 C18:2 214.8947 203.6123 285.6869 299.5488 70.7922 95.9365 C20:0 0.1868 0.2536 0.3099 0.3377 0.1231 0.0842 C20:4 147.2009 145.8865 184.5070 192.6049 37.3060 46.7184 C22:0 0.0734 0.1006 0.0901 0.1136 0.0166 0.0130 C22:6 75.3906 75.6906 96.3532 94.5376 20.9626 18.8470

Table 7. Standard deviation for each compound of the technical triplicates (LS1).

Compound Standard deviation C12:0 Lauric acid 0.014381 C14:0 Myristic acid 0.014665 C16:0 Palmitic acid 7.568798 C16:1 Palmitoleic acid 0.521003 C18:0 Stearic acid 15.5417

C18:1 Vaccenic, Oleic and Eladic acid 3.18951 C18:2 Linoleic acid 13.71386

C20:0 Arachidic acid 0.073889

C20:4 Arachidonic acid 6.97325

C22:0 Behenic acid 0.005955

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Table 8. Equations for calibration curves. Observe that compound C18:3 was used as calibration equation for C20:4 and

C22:6 and that C16:0 calibration equation was used for C17:0, the recovery standard.

Table 9. Recovery values for all samples using triheptadecanoate.

C17:0 TG standard. Triheptadecanoate Sample Text Recovery (%) C17:0 LS1 rep.1 75.02 LS1 rep.2 123.7 LS1 rep.3 74.3 LS2 rep.1 80.9 LS2 rep.2 104.2 LS3 rep.1 167.1 LS3 rep.2 140.3 LS4 rep.1 86.0 LS4 rep.2 105.4 LS5 rep.1 114.8 LS5 rep.2 108.3 LS6 rep.1 107.7 LS6 rep.2 174.9 LS7 rep.1 116.4 LS7 rep.2 96.4 LS8 rep.1 88.9 LS8 rep.2 90.1 LS9 rep.1 95.8 LS9 rep.2 135.4 LS10 rep.1 141.1 LS10 rep.2 159.6 LS11 rep.1 186.5 LS11 rep.2 149.7 LS12 rep.1 112.1 LS12 rep.2 135.0 LS13 rep.1 90.0 LS13 rep.2 105.3 LS14 rep.1 131.7 LS14 rep.2 104.4 LS15 rep.1 105.0 Compounds Equations C12:0 y = 0.7309x - 0.5391 C14:0 y = 0.9441x - 1.9122 C16:0 y = 0.9527x - 4.1943 C16:1 y = 0.0601x - 0.3638 C18:0 y = 0.6165x - 3.3517 C18:1 y = 0.0875x - 0.4942 C18:2 y = 0.0638x - 0.4446 C18:3 y = 0.1311x - 0.9052 C20:0 y = 0.4367x - 3.0265 C22:0 y = 0.4301x - 2.9911

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12 LS15 rep.2 107.5 LS16 rep.1 77.6 LS16 rep.2 101.5 LS17 rep.1 508.9 LS17 rep.2 122.1 LS18 rep 1 215.3 LS18 rep.2 135.6 LS19 rep.1 168.0 LS19 rep.2 90.4 LS20 rep.1 244.2 LS20 rep.2 319.3 LS21 rep.1 216.8 LS21 rep.2 1557.5 LS22 rep.1 370.5 LS22 rep.2 188.8 LS23 rep.1 279.8 LS23 rep.2 455.2 LS24 rep.1 208.3 LS24 rep.2 229.1 Mean value: 185.8

Figure 2. Median concentrations and their standard deviations. The asterix display that the concentration is of significance

when compared to the concentrations of the untreated liver samples.

6. Discussion

Fold change is a way to show the response treated liver samples have against untreated samples. The interval values for fold change ranged from 0.90 to 5.23. All data for fold change are presented in Table 4. The closer the fold change value is to 1 the less difference there is between treatments. Most of the treated liver samples obtain values very close to the value of 1; however, some samples and compounds stand out. Several of the PFOA and 2.5 % DMSO-treated samples resulted in a much

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13 higher fold change value. Particularly, the compounds C16:0, C16:1, C18:0, C18:1, C18:2, C20:4 and C22:6 show more distinct differences than the other fatty acids, with the highest values within their sample treated with 0.1µg/g PFOS, 2.5 % DMSO and 1.6 µg/g PFOA. This would mean that these are most distinguished in comparison to the untreated liver samples, and are most likely to be of

significance. However, when observing the values from the t-test, the previous statement appears to be somewhat misleading especially regarding the compounds found in the PFOA-treated samples.

When presenting a great deal of comparative data a t-test can be used for evaluating the significance of the results. The results show which samples and compounds that have a significant value, the results are shown in Table 5. If the probability value (P) is < 0.05 the null hypothesis (i.e. there is no difference between the test values) is rejected which means that there is a difference between the samples, and the value is significant. If the compound in a sample however has P > 0.05 the null hypothesis is to be followed since the value is not significant. The results of t-tests conclude that C16:0, C16:1, C18:0, C18:1, C18:2, C20:4, C22:6 when treated with 0.1 µg/g PFOS show significant difference in comparison with the untreated liver samples. The solvent (5 % DMSO) which was used for the PFOS treatment did not show any significant difference, thus it did not have an impact on the findings. There are also some values of significance from the liver samples treated with 2.5 % DMSO, the compounds that obtain relevant values are C16:0, C18:0, C18:2, C20:4 and C22:6. Fatty acids C16:0, C16:1, C18:0, C18:1, C18:2, C20:0, C20:4, C22:0 and C22:6 are all of statistical significance regarding the PFOA-treated samples. Although, when the PFOA-treated liver samples are compared to 2.5 % DMSO-treated samples only PFOA-treated C16:1 seems to be of significance. These results do not only confirm that lower concentrations of both PFOS and DMSO indeed affect the fatty acid metabolism but also give rise for suspicion that PFOA-treated samples also influences the metabolism of fatty acids. It is also important to mention that although 0.1 µg/g PFOS-treated C12:0 and PFOA-treated C18:1 (when compared to 2.5 % DMSO) exceeds the P-value of 0.05 these may still be of statistical significance. When observing Table 6 it is obvious that most samples treated have distinctively higher concentrations of fatty acids than the untreated samples. This leads to the belief that the β-oxidation of most fatty acids in the liver are down-regulated when exposed lower

concentrations of PFOS (0.1 µg/g) and a lower percentage of DMSO (2.5 %). The concentrations of fatty acids in 1.0 µg/g PFOS-treated samples, however, seem overall to be lower than the untreated samples which could mean that the opposite effect was given when compared to the samples treated with a lower concentration of PFOS. Nonetheless, since the t-test shows no significant values regarding 1.0 µg/g PFOS-treated samples; further study is needed to confirm these results. These results regarding fatty acid metabolism correlate with the earlier mentioned study on gene expression [12]. However, when comparing the results to the study on avian developmental toxicity [10] the results given seem to be contradictive. A possible reason for this could be that there are several more factors that influenced the results than the fatty acid β-oxidation alone.

There is often an uncertainty when conducting an experiment. The contamination risk in this project for instance, is not very high but since fatty acids are present on our skin there is still a possibility for contamination. To check for possible contaminants blanks were also made and analyzed, these showed no indications of being contaminated. Nevertheless, these blanks were prepared in relation to the final step when preparing the liver samples for analysis and do therefore not account for earlier steps in the sample preparation. Systematic and random errors within the processing of the data are also a potential risk for non-viable results. There is a possibility that a random error has occurred regarding LS21 rep.2 since all fatty acid concentrations were much higher than that of the other samples within the same treatment group (appendix I) and its recovery was calculated to 1557.5 % (Table 9). Because of this possibility, LS21 rep.2 was excluded from the calculations of t-test, fold change and the difference in

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14 concentrations between samples. This sample was also excluded from the concentrations and standard deviations presented in Figure 2.

The median concentrations of the compounds and their sample treatments are shown in Figure 2. The most distinguished difference between compounds in the same treatment groups are concerning C16:0, C18:0, C18:1, C18:2, C20:4 and C22:6. These are also some of the compounds with a higher

abundancy within the tissues. However, these compounds also obtain an uncertainty higher than the other fatty acids when looking at the standard deviation also presented in the figure.

The recovery of the samples was determined through the use of triheptadecanoate C17:0, a triglyceride standard. The recovery is presented in Table 9 and shows a mean value of 185.8 % which is too high to be tolerable. However, if the recovery of LS21 rep.2 is excluded this number will drop to 157.2 % which is more acceptable. When observing the intensity of the internal standard (appendix I) it is noticeable that variations between samples are present, these should however be more or less the same. A possible reason that might give this result for both the recovery and internal standard is if these do not dissolve in the solvent used in this method. If these are not completely dissolved in the organic phase when transferring the phase to a new vial after centrifugation there will be a difference in the concentration between samples.

The standard deviations of the different compounds are shown in Table 7. These are from the

untreated liver samples 1 (LS1) as those samples were made triplicates. The standard deviation varies between the different fatty acids but is higher for C16:0, C18:0, C18:1, C18:2, C20:4 and C22:6. An explanation to this can be that the presence of these fatty acids is more common within the liver cells. When evaluating results regarding the PFOA-treated samples, not much can be determined. This uncertainty caused by huge variations in concentrations needs further investigation to figure out what generated these results. However, natural variation is always present but in this case should not stand for all of the uncertainty. Nonetheless, one theory that can contribute to the level of uncertainty is that the solvent used (2.5 % DMSO) perhaps was not the best choice for dissolving PFOA. The liver tissues treated with 2.5 % DMSO and PFOA show a higher deviation and thereby a greater uncertainty. Meanwhile liver tissues treated with 5 % DMSO and PFOS obtain a noticeable lower deviation. For proving that the solvent (2.5 % DMSO) is the reason for this, however, a study obtaining new samples where both PFOS and PFOA are dissolved in 5 % DMSO needs to be conducted.

To be able to confirm these results there is a need for more extensive studies including more samples and multiple concentrations for the treated liver samples. This study however shows that at least PFOS indeed affect the fatty acid metabolism in the liver.

7. Conclusions

This study concludes that the chicken embryo liver when exposed to a lower concentration of PFOS (0.1 µg/g) or a lower percentage of DMSO (2.5 %) is affected, which implies that the β-oxidation of fatty acids is down-regulated. This causes the concentrations of fatty acids to be elevated. The energy metabolism is therefore down-regulated and not as effective. PFOA-treated samples also show significance regarding some of the fatty acid concentrations and may indicate a regulated fatty acid metabolism.

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15

8. References

[1] Minghong Wua, Rui Suna, Mingnan Wangb, Huanhuan Lianga, Sihan Maa, Tao Hana, Xiaoyu Xiaa, Jing Maa, Liang Tanga, Yanfeng Suna, Gang Xua, Analysis of perfluorinated compounds in human serum from the general population in Shanghai by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Volume 168, February 2017, Pages 100–105, Chemosphere, doi.org/10.1016/j.chemosphere.2016.09.161 (2016)

[2] Högfluorerande ämnen - PFAAs. Kemikalieinspektionen (2015)

http://www.kemi.se/om-kemikalieinspektionen/verksamhet/handlingsplan-for-en-giftfri-vardag/hogfluorerade-amnen-pfas

[3] N. Fillmore, O.A. Alrob, G.D. Lopaschuk, Fatty acid beta-oxidation. AOCS Lipid library, doi: 10.21748/lipidlibrary.39187 (2016)

[4] J.C. DeWitt, E.B. Meyer, D.S. Henshel, Environmental toxicity studies using chickens as surrogates for wildlife: effects of injection day. Archives of Environmental Contamination and Toxicology, doi:10.1007/s00244-004-2006-8 (2005)

[5] S. Godshe, B. Bharucha, G. Padate, Quantification and assessment of various environmental toxicants from feather of Black kite (Milvus migrans Govinda): A preliminary study. International Journal of Pharma and Bio Sciences, Volume 3, Issue 4, October 2012, Pages 465-474 (2012) [6] A. Bertoleroa, J. Vicenteb, J. Meyerb, S. Lacorteb, Accumulation and maternal transfer of perfluorooctane sulphonic acid in yellow-legged (Larus michahellis) and Audouin's gull (Larus audouinii) from the Ebro Delta Natural Park. Elsevier, Environmental Research, Volume 137, February 2015, Pages 208–214 (2015)

[7] M. Nordén, U. Berger, M. Engwall, Developmental toxicity of PFOS and PFOA in great

cormorant (Phalacrocorax carbo sinensis), herring gull (Larus argentatus) and chicken (Gallus gallus domesticus). Environmental Science and Pollution Research, doi: 10.1007/s11356-016-6285-1 (2016) [8] R.C. Buck, J. Franklin, U. Berger, J.M. Conder, I.T. Cousins, P. de Voogt, A.Astrup Jensen, K.Kannan, S.A. Mabury, S.PJ. van Leeuwen, Perfluoroalkyl and Polyfluoroalkyl Substances in the Environment: Terminology, Classification, and Origins. Integrated Environmental Assessment and Management, doi: 10.1002/ieam.258 (2011)

[9] K. Inoue, F. Okada, R. Ito, S. Kato, S. Sasaki, S. Nakajima, A. Uno, Y. Saijo, F. Sata, Y. Yoshimura, R. Kishi, H. Nakazawa, Perfluorooctane sulfonate (PFOS) and related perfluorinated compounds in human maternal and cord blood samples: assessment of PFOS exposure in a susceptible population during pregnancy. Environmental Health Perspectives, doi: 10.1289/ehp.6864 (2004) [10] M. Nordén, Comparative avian developmental toxicity of PFAAs. Örebro Universitet, ISBN 978-91-7668-959-2 (2013)

[11] A.C. Rustan, C.A. Drevon, Fatty acids: structures and properties. Encyclopedia of life sciences, doi: 10.1038/npg.els.0003894 (2005)

[12] L.W.Y. Yeung, K.S. Guruge, N. Yamanaka, S. Miyazaki, P.K.S. Lam, Differential expression of chicken hepatic genes responsive to PFOA and PFOS. Elsevier, Toxicology, Volume 237, Issues 1–3, 31 July 2007, Pages 111–125 (2007)

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16 [13] M.W. King, Synthesis of triglycerides. themedicalbiochemistrypage.org (1996–2016, Last

modified: April 29, 2016)

[14] M.W. King, Phospholipid synthesis. themedicalbiochemistrypage.org (1996–2016, Last modified: April 29, 2016)

[15] E. Stauffer, A review of the analysis of vegetable oil residues from fire debris samples: Analytical scheme, interpretation of the results and future needs. Journal of Forensic Sciences, Vol.51 No.5 (2006)

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17

Appendix I

Table 10. Weight of each liver sample used in this study.

Table 11. C12:0 areas, concentrations and retention times for all samples.

C12:0 Lauric acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 1.584 174.894 0.009056914 0.749975256 24.79 LS1 rep.2 2.131 78.011 0.02731666 0.774957806 24.78 LS1 rep.3 1.339 542.958 0.002466121 0.740957889 24.8 LS2 rep.1 2.698 807.357 0.003341768 0.742155929 24.81 LS2 rep.2 1.145 273.994 0.004178924 0.743301305 24.8 LS3 rep.1 1.221 113.668 0.010741809 0.752280489 24.8 LS3 rep.2 1.219 137.651 0.008855729 0.749699999 24.79 LS4 rep.1 2.339 508.077 0.004603633 0.743882382 24.8 LS4 rep.2 1.304 350.016 0.003725544 0.742681001 24.79 LS5 rep.1 1.015 123.389 0.008226017 0.748838442 24.82 LS5 rep.2 2.123 171.791 0.01235804 0.754491777 24.79 LS6 rep.1 1.825 174.816 0.010439548 0.751866942 24.79 LS6 rep.2 3.015 102.568 0.029395133 0.777801523 24.8 LS7 rep.1 2.064 264.802 0.007794503 0.748248055 24.79 LS7 rep.2 2.205 398.569 0.005532292 0.745152951 24.79 LS8 rep.1 1.739 314.083 0.005536753 0.745159055 24.8 LS8 rep.2 4.686 391.333 0.011974457 0.753966967 24.8 LS9 rep.1 6.508 193.468 0.033638638 0.783607385 24.8 LS9 rep.2 5.517 314.268 0.01755508 0.761602244 24.8 LS10 rep.1 1.445 122.279 0.011817238 0.753751864 24.8 LS10 rep.2 2.72 182.043 0.014941525 0.75802644 24.79 LS11 rep.1 3.575 140.383 0.025466046 0.77242584 24.8 LS11 rep.2 2.866 208.664 0.013735 0.756375701 24.8 LS12 rep.1 4.064 381.533 0.010651765 0.752157293 24.8 LS12 rep.2 2.642 238.421 0.011081239 0.752744888 24.78 LS13 rep.1 2.739 449.509 0.006093315 0.74592053 24.81 LS13 rep.2 2.202 199.048 0.011062658 0.752719467 24.8 LS14 rep.1 1.047 161.819 0.006470192 0.746436164 24.8

Name Content Weight

(g)

Name Content Weight

(g) LS1 Uninjected liver 0.4505 LS13 1.0 µg/g PFOS-treated liver 0.2078

LS2 Uninjected liver 0.4004 LS14 1.0 µg/g PFOS-treated liver 0.4196

LS5 5 % DMSO-treated liver 0.4129 LS17 2.5 % DMSO-treated liver 0.2149

LS9 0.1 µg/g PFOS-treated liver 0.4440 LS21 1.6 µg/g PFOA-treated liver 0.0516

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18 LS14 rep.2 2.747 652.486 0.004210052 0.743343894 24.79 LS15 rep.1 1.769 187.657 0.009426773 0.750481288 24.8 LS15 rep.2 2.521 289.803 0.008699013 0.749485583 24.8 LS16 rep.1 5.969 1143.926 0.005217995 0.744722937 24.79 LS16 rep.2 2.232 339.766 0.006569227 0.746571661 24.8 LS17 rep.1 2.95 24.733 0.119273845 0.900771439 24.79 LS17 rep.2 4.055 152.462 0.026596791 0.773972898 24.8 LS18 rep 1 0.874 117.524 0.007436779 0.747758625 24.8 LS18 rep.2 4.107 285.277 0.014396534 0.757280796 24.82 LS19 rep.1 3.673 267.334 0.013739367 0.756381676 24.8 LS19 rep.2 2.816 540.117 0.005213685 0.744717041 24.8 LS20 rep.1 3.936 113.874 0.034564519 0.784874153 24.79 LS20 rep.2 3.215 103.827 0.030964971 0.779949337 24.8 LS21 rep.1 2.409 134.902 0.017857408 0.762015881 24.82 LS21 rep.2 2.149 10.276 0.209128065 1.023707847 24.82 LS22 rep.1 1.596 61.434 0.0259791 0.773127787 24.79 LS22 rep.2 0.42 155.096 0.002708 0.741288822 24.8 LS23 rep.1 1.316 79.143 0.016628129 0.760334011 24.82 LS23 rep.2 0.344 49.026 0.007016685 0.747183862 24.81 LS24 rep.1 8.03 183.72 0.043707816 0.797383796 24.8 LS24 rep.2 7.767 194.39 0.039955759 0.79225032 24.79

C14:0 Myristic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 1.009 174.894 0.005769209 2.031531839 27.64 LS1 rep.2 2.518 78.011 0.032277499 2.05960968 27.68 LS1 rep.3 0.383 542.958 0.000705395 2.026168198 27.65 LS2 rep.1 1.481 807.357 0.001834381 2.02736403 27.69 LS2 rep.2 1.197 273.994 0.004368709 2.030048415 27.68 LS3 rep.1 0.34 113.668 0.002991167 2.02858931 27.63 LS3 rep.2 0.243 137.651 0.001765334 2.027290895 27.66 LS4 rep.1 0.696 508.077 0.001369871 2.026872017 27.62 LS4 rep.2 0.264 350.016 0.000754251 2.026219946 27.63 LS5 rep.1 1.225 123.389 0.009927951 2.03593682 27.62 LS5 rep.2 1.159 171.791 0.00674657 2.032567069 27.61 LS6 rep.1 0.453 174.816 0.002591296 2.028165762 27.66 LS6 rep.2 0.45 102.568 0.004387333 2.030068142 27.68 LS7 rep.1 0.458 264.802 0.001729594 2.027253039 27.66 LS7 rep.2 1.146 398.569 0.002875286 2.028466567 27.64 LS8 rep.1 0.463 314.083 0.001474133 2.026982452 27.63 LS8 rep.2 0.385 391.333 0.000983817 2.026463104 27.63 LS9 rep.1 1.436 193.468 0.007422416 2.033282932 27.62 LS9 rep.2 1.093 314.268 0.003477923 2.029104886 27.65 LS10 rep.1 0.188 122.279 0.001537468 2.027049537 27.68 LS10 rep.2 0.721 182.043 0.003960603 2.029616145 27.67 LS11 rep.1 0.891 140.383 0.006346922 2.032143758 27.64 LS11 rep.2 0.258 208.664 0.001236438 2.026730683 27.66 LS12 rep.1 0.999 381.533 0.002618384 2.028194454 27.63

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19 LS12 rep.2 0.419 238.421 0.001757396 2.027282487 27.64 LS13 rep.1 0.845 449.509 0.001879829 2.027412169 27.67 LS13 rep.2 0.923 199.048 0.004637072 2.030332669 27.7 LS14 rep.1 0.249 161.819 0.001538756 2.027050902 27.67 LS14 rep.2 0.271 652.486 0.000415335 2.025860962 27.68 LS15 rep.1 0.77 187.657 0.004103231 2.029767218 27.64 LS15 rep.2 0.65 289.803 0.002242903 2.027796741 27.63 LS16 rep.1 0.271 1143.926 0.000236903 2.025671966 27.62 LS16 rep.2 0.991 339.766 0.002916713 2.028510447 27.63 LS17 rep.1 0.853 24.733 0.034488335 2.06195142 27.64 LS17 rep.2 0.525 152.462 0.003443481 2.029068405 27.65 LS18 rep 1 1.013 117.524 0.008619516 2.034550912 27.7 LS18 rep.2 0.199 285.277 0.000697568 2.026159906 27.67 LS19 rep.1 0.203 267.334 0.00075935 2.026225347 27.64 LS19 rep.2 0.213 540.117 0.000394359 2.025838745 27.62 LS20 rep.1 0.107 113.874 0.000939635 2.026416307 27.63 LS20 rep.2 0.353 103.827 0.003399886 2.029022229 27.63 LS21 rep.1 1.246 134.902 0.009236335 2.035204252 27.64 LS21 rep.2 0.529 10.276 0.051479175 2.079948284 27.63 LS22 rep.1 0.419 61.434 0.006820328 2.032645194 27.67 LS22 rep.2 0.213 155.096 0.001373343 2.026875694 27.68 LS23 rep.1 0.421 79.143 0.005319485 2.031055487 27.62 LS23 rep.2 0.621 49.026 0.012666748 2.03883778 27.62 LS24 rep.1 0.45 183.72 0.002449379 2.028015443 27.63 LS24 rep.2 0.329 194.39 0.001692474 2.027213721 27.61

C16:0 Palmitic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 2243.704 174.894 12.82893638 17.86841229 33.61 LS1 rep.2 2221.539 78.011 28.47725321 34.2936425 33.61 LS1 rep.3 7361.912 542.958 13.55889774 18.63461503 33.61 LS2 rep.1 11237.9 807.357 13.91936281 19.0129766 33.61 LS2 rep.2 7751.186 273.994 28.28961948 34.09669306 33.61 LS3 rep.1 4141.625 113.668 36.43615617 42.64769201 33.6 LS3 rep.2 3996.323 137.651 29.03228455 34.87623024 33.6 LS4 rep.1 8055.842 508.077 15.85555339 21.04529589 33.6 LS4 rep.2 10074.59 350.016 28.78323562 34.61481644 33.6 LS5 rep.1 5272.485 123.389 42.73059187 49.25463616 33.61 LS5 rep.2 6815.718 171.791 39.67447654 46.0467897 33.61 LS6 rep.1 4560.675 174.816 26.08843012 31.78621824 33.61 LS6 rep.2 3477.94 102.568 33.90862647 39.99467458 33.61 LS7 rep.1 8054.253 264.802 30.41613356 36.3287851 33.6 LS7 rep.2 9119.639 398.569 22.88095411 28.41949629 33.61 LS8 rep.1 5677.984 314.083 18.07797302 23.37805502 33.6 LS8 rep.2 5686.043 391.333 14.52993486 19.65386256 33.6 LS9 rep.1 6528.422 193.468 33.74419542 39.8220798 33.61 LS9 rep.2 16651.4 314.268 52.98469777 60.01784169 33.61 LS10 rep.1 5606.386 122.279 45.8491319 52.52800662 33.61

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20 LS10 rep.2 9985.548 182.043 54.85268865 61.97857526 33.61 LS11 rep.1 8193.336 140.383 58.3641609 65.66438638 33.6 LS11 rep.2 9419.997 208.664 45.14433252 51.78821509 33.61 LS12 rep.1 15475.74 381.533 40.56200381 46.97838124 33.6 LS12 rep.2 11060.24 238.421 46.38952525 53.09522961 33.6 LS13 rep.1 6359.121 449.509 14.14681575 19.25172221 33.61 LS13 rep.2 3594.147 199.048 18.05668482 23.3557099 33.61 LS14 rep.1 4058.505 161.819 25.08052206 30.7282692 33.61 LS14 rep.2 16263.84 652.486 24.9259555 30.56602865 33.61 LS15 rep.1 4516.793 187.657 24.06940855 29.66695555 33.6 LS15 rep.2 7149.667 289.803 24.67078326 30.29818753 33.61 LS16 rep.1 14675.14 1143.926 12.82875116 17.86821787 33.6 LS16 rep.2 11267.39 339.766 33.16220575 39.21119529 33.6 LS17 rep.1 7049.85 24.733 285.0382081 303.59243 33.61 LS17 rep.2 5580.51 152.462 36.60262885 42.82242978 33.61 LS18 rep 1 11332.83 117.524 96.42995473 105.6200847 33.6 LS18 rep.2 12517.8 285.277 43.87946102 50.46054478 33.6 LS19 rep.1 16822.12 267.334 62.92548273 70.45217039 33.61 LS19 rep.2 9733.344 540.117 18.0208066 23.31805039 33.61 LS20 rep.1 11626.62 113.874 102.1007341 111.5724091 33.6 LS20 rep.2 14028.32 103.827 135.1124178 146.2230689 33.6 LS21 rep.1 14682.99 134.902 108.8418852 118.6482473 33.61 LS21 rep.2 12806.43 10.276 1246.246594 1312.523243 33.61 LS22 rep.1 11174.59 61.434 181.8958069 195.329177 33.6 LS22 rep.2 13897.3 155.096 89.60449657 98.45575372 33.61 LS23 rep.1 8406.603 79.143 106.2204238 115.8966346 33.6 LS23 rep.2 9641.297 49.026 196.6568148 210.8230448 33.61 LS24 rep.1 23270.96 183.72 126.6653549 137.3566232 33.6 LS24 rep.2 24717.65 194.39 127.154936 137.8705111 33.6

C16:1 Palmitoleic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 8.843 174.894 0.050562055 6.89454334 33.16 LS1 rep.2 8.51 78.011 0.10908718 7.868339102 33.15 LS1 rep.3 20.031 542.958 0.036892356 6.667094115 33.17 LS2 rep.1 29.887 807.357 0.03701832 6.669190021 33.17 LS2 rep.2 19.562 273.994 0.071395724 7.241193411 33.15 LS3 rep.1 14.352 113.668 0.126262449 8.15411728 33.16 LS3 rep.2 15.184 137.651 0.110307953 7.88865146 33.17 LS4 rep.1 32.388 508.077 0.063746243 7.113914196 33.15 LS4 rep.2 36.071 350.016 0.103055289 7.767974857 33.16 LS5 rep.1 16.686 123.389 0.135230855 8.303342018 33.17 LS5 rep.2 18.639 171.791 0.108498117 7.858537719 33.17 LS6 rep.1 14.071 174.816 0.080490344 7.392518205 33.17 LS6 rep.2 13.996 102.568 0.136455815 8.323724038 33.16 LS7 rep.1 29.238 264.802 0.110414574 7.890425523 33.15 LS7 rep.2 33.856 398.569 0.084943887 7.466620412 33.17 LS8 rep.1 17.372 314.083 0.055310221 6.973547763 33.15

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21 LS8 rep.2 17.966 391.333 0.045909749 6.817133935 33.16 LS9 rep.1 17.188 193.468 0.088841566 7.531473636 33.17 LS9 rep.2 39.002 314.268 0.124104268 8.118207449 33.16 LS10 rep.1 15.106 122.279 0.123537157 8.108771328 33.17 LS10 rep.2 26.553 182.043 0.145861143 8.48021868 33.17 LS11 rep.1 20.446 140.383 0.145644416 8.476612573 33.15 LS11 rep.2 18.156 208.664 0.087010697 7.501009927 33.16 LS12 rep.1 49.449 381.533 0.129606089 8.209751899 33.16 LS12 rep.2 43.683 238.421 0.183217921 9.101795695 33.16 LS13 rep.1 15.179 449.509 0.033767956 6.615107416 33.17 LS13 rep.2 12.211 199.048 0.061347012 7.07399354 33.15 LS14 rep.1 13.44 161.819 0.08305576 7.435203991 33.15 LS14 rep.2 47.134 652.486 0.072237565 7.255200753 33.17 LS15 rep.1 9.459 187.657 0.050405794 6.89194332 33.17 LS15 rep.2 15.378 289.803 0.053063633 6.936166936 33.16 LS16 rep.1 38.145 1143.926 0.033345688 6.608081339 33.15 LS16 rep.2 24.776 339.766 0.072920775 7.266568634 33.16 LS17 rep.1 10.892 24.733 0.440383294 13.38075364 33.17 LS17 rep.2 9.429 152.462 0.061844919 7.082278182 33.15 LS18 rep 1 14.224 117.524 0.121030598 8.067064859 33.16 LS18 rep.2 18.737 285.277 0.065680023 7.146090238 33.17 LS19 rep.1 23.679 267.334 0.088574592 7.527031482 33.17 LS19 rep.2 14.956 540.117 0.027690297 6.513981643 33.17 LS20 rep.1 13.021 113.874 0.11434568 7.955834947 33.15 LS20 rep.2 20.23 103.827 0.194843345 9.295230369 33.16 LS21 rep.1 33.243 134.902 0.24642333 10.15346638 33.17 LS21 rep.2 27.085 10.276 2.635753211 49.90937124 33.17 LS22 rep.1 18.009 61.434 0.293143862 10.93084628 33.16 LS22 rep.2 26.919 155.096 0.17356347 8.941155913 33.16 LS23 rep.1 13.91 79.143 0.175757805 8.977667313 33.17 LS23 rep.2 17.301 49.026 0.352894383 11.92503132 33.16 LS24 rep.1 71.741 183.72 0.390490965 12.55059841 33.15 LS24 rep.2 78.775 194.39 0.405242039 12.79604059 33.15

C18:0 Stearic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 2970.354 174.894 16.98373872 32.98530206 37.44 LS1 rep.2 3017.339 78.011 38.67837869 68.17531012 37.44 LS1 rep.3 10900.93 542.958 20.07693229 38.00264767 37.44 LS2 rep.1 18874.78 807.357 23.37848436 43.35796328 37.43 LS2 rep.2 12979.47 273.994 47.37134755 82.27582733 37.43 LS3 rep.1 5005.255 113.668 44.03398494 76.86242488 37.43 LS3 rep.2 4826.374 137.651 35.06239693 62.30997069 37.43 LS4 rep.1 10114.79 508.077 19.90798245 37.72860089 37.43 LS4 rep.2 12853.39 350.016 36.72229841 65.00243051 37.42 LS5 rep.1 6099.598 123.389 49.43388795 85.62139164 37.43 LS5 rep.2 8136.929 171.791 47.36528107 82.26598713 37.43 LS6 rep.1 6746.351 174.816 38.59115298 68.03382479 37.43

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22 LS6 rep.2 5082.203 102.568 49.54959637 85.80907764 37.43 LS7 rep.1 11408.4 264.802 43.08276373 75.31948698 37.43 LS7 rep.2 12311.12 398.569 30.88829538 55.53932746 37.43 LS8 rep.1 8536.917 314.083 27.18044912 49.52497829 37.42 LS8 rep.2 8230.408 391.333 21.0317249 39.55137859 37.43 LS9 rep.1 8261.549 193.468 42.70240557 74.70252322 37.44 LS9 rep.2 21452.35 314.268 68.26133746 116.1606447 37.44 LS10 rep.1 7118.202 122.279 58.21279206 99.86130099 37.43 LS10 rep.2 12453.25 182.043 68.40830463 116.3990343 37.43 LS11 rep.1 12952.04 140.383 92.26219699 155.0914793 37.43 LS11 rep.2 14715.12 208.664 70.52065042 119.8253859 37.43 LS12 rep.1 17430.85 381.533 45.68634954 79.54265944 37.42 LS12 rep.2 11934.61 238.421 50.05688677 86.63193312 37.43 LS13 rep.1 10995.05 449.509 24.46012872 45.11245534 37.43 LS13 rep.2 6277.769 199.048 31.5389705 56.59476156 37.43 LS14 rep.1 5554.976 161.819 34.3283298 61.11926975 37.43 LS14 rep.2 22359.38 652.486 34.26798583 61.0213882 37.43 LS15 rep.1 5948.387 187.657 31.69818872 56.85302306 37.43 LS15 rep.2 9318.434 289.803 32.15437383 57.5929827 37.42 LS16 rep.1 18436.78 1143.926 16.11710635 31.57957235 37.43 LS16 rep.2 15172.7 339.766 44.65631052 77.87187432 37.43 LS17 rep.1 8805.078 24.733 356.0052561 582.8985501 37.43 LS17 rep.2 7121.188 152.462 46.70795346 81.1997623 37.43 LS18 rep 1 16819.24 117.524 143.1132194 237.5748896 37.43 LS18 rep.2 19118.93 285.277 67.0188203 114.1452073 37.43 LS19 rep.1 26265.35 267.334 98.24917893 164.8027233 37.43 LS19 rep.2 15031.82 540.117 27.83067002 50.57967562 37.43 LS20 rep.1 13877.28 113.874 121.8652282 203.1093726 37.43 LS20 rep.2 16462.62 103.827 158.5581496 262.6274933 37.43 LS21 rep.1 20339.83 134.902 150.7748588 250.0025284 37.43 LS21 rep.2 18832.34 10.276 1832.652783 2978.109462 37.43 LS22 rep.1 15035.92 61.434 244.7491617 402.4344878 37.43 LS22 rep.2 18109.85 155.096 116.7654356 194.8372029 37.42 LS23 rep.1 11705.91 79.143 147.9082799 245.3527655 37.43 LS23 rep.2 13452.79 49.026 274.4010729 450.5316673 37.42 LS24 rep.1 21759.22 183.72 118.4368659 197.5483632 37.42 LS24 rep.2 23205.89 194.39 119.3779876 199.074919 37.43

C18:1 Vaccenic, Oleic and Eladic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 1129.136 174.894 6.456116276 12.10411628 36.94 LS1 rep.2 941.797 78.011 12.07261796 17.72061796 36.93 LS1 rep.3 2476.553 542.958 4.561223888 10.20922389 36.93 LS2 rep.1 4337.609 807.357 5.372603446 11.02060345 36.93 LS2 rep.2 3249.638 273.994 11.86025241 17.50825241 36.93 LS3 rep.1 1508.577 113.668 13.27178274 18.91978274 36.93 LS3 rep.2 1453.723 137.651 10.56093308 16.20893308 36.93 LS4 rep.1 3384.208 508.077 6.66081716 12.30881716 36.93

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23 LS4 rep.2 3787.91 350.016 10.82210528 16.47010528 36.93 LS5 rep.1 1833.818 123.389 14.86208657 20.51008657 36.93 LS5 rep.2 2316.16 171.791 13.48242923 19.13042923 36.93 LS6 rep.1 1563.177 174.816 8.941841708 14.58984171 36.93 LS6 rep.2 1051.987 102.568 10.2564835 15.9044835 36.93 LS7 rep.1 3213.224 264.802 12.13444007 17.78244007 36.93 LS7 rep.2 3738.021 398.569 9.378604457 15.02660446 36.93 LS8 rep.1 2448.824 314.083 7.796741626 13.44474163 36.93 LS8 rep.2 2711.025 391.333 6.927667741 12.57566774 36.92 LS9 rep.1 2077.532 193.468 10.73837534 16.38637534 36.93 LS9 rep.2 5182.959 314.268 16.49216274 22.14016274 36.93 LS10 rep.1 1729.349 122.279 14.14264919 19.79064919 36.93 LS10 rep.2 3029.623 182.043 16.64234824 22.29034824 36.93 LS11 rep.1 2768.595 140.383 19.72172557 25.36972557 36.93 LS11 rep.2 3003.276 208.664 14.39288042 20.04088042 36.93 LS12 rep.1 4228.833 381.533 11.08379354 16.73179354 36.93 LS12 rep.2 3216.406 238.421 13.49044757 19.13844757 36.92 LS13 rep.1 2338.774 449.509 5.202952555 10.85095255 36.93 LS13 rep.2 1449.264 199.048 7.280977453 12.92897745 36.93 LS14 rep.1 1579.93 161.819 9.763562993 15.41156299 36.93 LS14 rep.2 5248.832 652.486 8.044359572 13.69235957 36.93 LS15 rep.1 1528.64 187.657 8.145925811 13.79392581 36.93 LS15 rep.2 2293.51 289.803 7.914031256 13.56203126 36.93 LS16 rep.1 5783.496 1143.926 5.055830534 10.70383053 36.93 LS16 rep.2 3587.831 339.766 10.55971168 16.20771168 36.92 LS17 rep.1 1331.248 24.733 53.82476853 59.47276853 36.93 LS17 rep.2 1027.255 152.462 6.737777282 12.38577728 36.93 LS18 rep 1 2254.905 117.524 19.18676185 24.83476185 36.93 LS18 rep.2 2426.985 285.277 8.50746818 14.15546818 36.93 LS19 rep.1 3259 267.334 12.19074267 17.83874267 36.93 LS19 rep.2 1851.339 540.117 3.427662895 9.075662895 36.93 LS20 rep.1 2248.112 113.874 19.74210092 25.39010092 36.92 LS20 rep.2 2710.976 103.827 26.11051075 31.75851075 36.92 LS21 rep.1 3383.18 134.902 25.07879794 30.72679794 36.94 LS21 rep.2 2992.664 10.276 291.2284936 296.8764936 36.93 LS22 rep.1 2980.736 61.434 48.51932155 54.16732155 36.93 LS22 rep.2 3626.015 155.096 23.37916516 29.02716516 36.93 LS23 rep.1 2184.028 79.143 27.59597185 33.24397185 36.93 LS23 rep.2 2554.412 49.026 52.10321054 57.75121054 36.93 LS24 rep.1 5551.935 183.72 30.21954605 35.86754605 36.92 LS24 rep.2 5809.712 194.39 29.88688719 35.53488719 36.92

C18:2 Linoleic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 276.709 174.894 1.582152618 31.76728242 36.8

LS1 rep.2 286.975 78.011 3.678647883 64.62770976 36.8

LS1 rep.3 1219.032 542.958 2.245168135 42.15937515 36.8

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24 LS2 rep.2 1908.801 273.994 6.96657956 116.162689 36.8 LS3 rep.1 809.821 113.668 7.124441356 118.6370118 36.8 LS3 rep.2 776.694 137.651 5.64248716 95.40888965 36.8 LS4 rep.1 1858.853 508.077 3.658604897 64.31355637 36.79 LS4 rep.2 2287.261 350.016 6.534732698 109.3939294 36.8 LS5 rep.1 634.741 123.389 5.144226795 87.59916606 36.8 LS5 rep.2 937.519 171.791 5.457323143 92.50663233 36.8 LS6 rep.1 935.308 174.816 5.350242541 90.82825299 36.8 LS6 rep.2 685.208 102.568 6.68052414 111.6790618 36.8 LS7 rep.1 1951.757 264.802 7.370627865 122.4957346 36.8 LS7 rep.2 2110.215 398.569 5.294478497 89.95420841 36.8 LS8 rep.1 1413.66 314.083 4.500912179 77.51586488 36.8 LS8 rep.2 1374.355 391.333 3.5119834 62.0154138 36.79 LS9 rep.1 1202.334 193.468 6.214640147 104.3768048 36.8 LS9 rep.2 3570.732 314.268 11.36206041 185.0573732 36.8 LS10 rep.1 1177.957 122.279 9.633354869 157.9616751 36.8 LS10 rep.2 2178.554 182.043 11.9672495 194.5430955 36.8 LS11 rep.1 2118.717 140.383 15.09240435 243.526714 36.8 LS11 rep.2 2409.805 208.664 11.54873385 187.9832892 36.79 LS12 rep.1 3157.312 381.533 8.275331361 136.6760401 36.79 LS12 rep.2 2143.923 238.421 8.992173508 147.9118105 36.79 LS13 rep.1 1431.449 449.509 3.184472391 56.8820124 36.8 LS13 rep.2 769.266 199.048 3.864726096 67.54429618 36.8 LS14 rep.1 729.349 161.819 4.507190132 77.61426539 36.8 LS14 rep.2 3176.149 652.486 4.867765745 83.26592077 36.8 LS15 rep.1 817.487 187.657 4.356283006 75.24894993 36.8 LS15 rep.2 1341.308 289.803 4.628344082 79.51323013 36.8 LS16 rep.1 3135.621 1143.926 2.741104757 49.93267644 36.79 LS16 rep.2 2369.595 339.766 6.974196947 116.2820838 36.79 LS17 rep.1 1047.719 24.733 42.36117737 670.9369494 36.8 LS17 rep.2 891.172 152.462 5.84520733 98.58632179 36.8 LS18 rep 1 2364.074 117.524 20.11566999 322.2612851 36.8 LS18 rep.2 2827.199 285.277 9.910364313 162.3035159 36.8 LS19 rep.1 3856.919 267.334 14.42734183 233.1025365 36.8 LS19 rep.2 2279.161 540.117 4.219754238 73.1090006 36.8 LS20 rep.1 2268.234 113.874 19.91880499 319.1756269 36.79 LS20 rep.2 2658.957 103.827 25.60949464 408.3713893 36.79 LS21 rep.1 2426.553 134.902 17.98752428 288.9047692 36.8 LS21 rep.2 2589.643 10.276 252.0088556 3956.950715 36.8 LS22 rep.1 2147.64 61.434 34.95849204 554.9073987 36.8 LS22 rep.2 2640.267 155.096 17.0234371 273.7936849 36.8 LS23 rep.1 1670.832 79.143 21.11155756 337.8708082 36.79 LS23 rep.2 1993.726 49.026 40.66670746 644.3778599 36.8 LS24 rep.1 3738.521 183.72 20.34901481 325.9187274 36.79 LS24 rep.2 4138.477 194.39 21.28955708 340.6607692 36.79

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25

C20:0 Arachidic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 2.535 174.894 0.014494494 6.963577957 39.58 LS1 rep.2 6.583 78.011 0.084385535 7.12362156 39.59 LS1 rep.3 9.488 542.958 0.017474648 6.970402217 39.59 LS2 rep.1 21.527 807.357 0.026663545 6.991443887 39.59 LS2 rep.2 14.757 273.994 0.053858844 7.053718442 39.59 LS3 rep.1 5.809 113.668 0.051104972 7.047412348 39.58 LS3 rep.2 5.198 137.651 0.037762167 7.016858637 39.59 LS4 rep.1 13.877 508.077 0.027312789 6.992930591 39.58 LS4 rep.2 15.035 350.016 0.042955179 7.028750124 39.59 LS5 rep.1 6.818 123.389 0.055256141 7.056918116 39.58 LS5 rep.2 5.926 171.791 0.034495404 7.009378073 39.58 LS6 rep.1 7.134 174.816 0.040808622 7.023834719 39.59 LS6 rep.2 4.619 102.568 0.045033539 7.033509363 39.58 LS7 rep.1 14.209 264.802 0.05365896 7.053260729 39.58 LS7 rep.2 20.848 398.569 0.052307129 7.050165168 39.59 LS8 rep.1 6.982 314.083 0.022229793 6.98129103 39.59 LS8 rep.2 9.229 391.333 0.023583495 6.984390876 39.58 LS9 rep.1 6.15 193.468 0.031788203 7.003178847 39.59 LS9 rep.2 16.569 314.268 0.052722517 7.051116366 39.59 LS10 rep.1 9.899 122.279 0.080954211 7.115764166 39.59 LS10 rep.2 11.897 182.043 0.065352691 7.080038222 39.59 LS11 rep.1 14.166 140.383 0.100909654 7.161460166 39.59 LS11 rep.2 16.214 208.664 0.077703868 7.108321201 39.59 LS12 rep.1 23.936 381.533 0.062736382 7.074047131 39.59 LS12 rep.2 8.586 238.421 0.036011928 7.012850764 39.58 LS13 rep.1 9.047 449.509 0.020126405 6.976474478 39.59 LS13 rep.2 8.229 199.048 0.041341787 7.025055615 39.59 LS14 rep.1 7.151 161.819 0.04419135 7.031580833 39.59 LS14 rep.2 32.323 652.486 0.049538228 7.043824657 39.59 LS15 rep.1 5.963 187.657 0.03177606 7.003151041 39.58 LS15 rep.2 9.137 289.803 0.031528314 7.002583728 39.59 LS16 rep.1 26.915 1143.926 0.02352862 6.984265216 39.58 LS16 rep.2 17.462 339.766 0.051394195 7.048074639 39.58 LS17 rep.1 13.726 24.733 0.554967048 8.201206888 39.59 LS17 rep.2 4.819 152.462 0.031607876 7.002765917 39.59 LS18 rep 1 18.982 117.524 0.161515946 7.300242605 39.59 LS18 rep.2 24.093 285.277 0.084454758 7.123780073 39.59 LS19 rep.1 28.198 267.334 0.10547854 7.171922464 39.59 LS19 rep.2 14.394 540.117 0.026649781 6.991412369 39.59 LS20 rep.1 12.046 113.874 0.105783585 7.172620988 39.58 LS20 rep.2 13.765 103.827 0.132576305 7.233973677 39.58 LS21 rep.1 20.522 134.902 0.152125246 7.278738829 39.59 LS21 rep.2 20.566 10.276 2.001362398 11.51330982 39.59 LS22 rep.1 15.935 61.434 0.259384054 7.524350938 39.59 LS22 rep.2 21.062 155.096 0.135799763 7.241355078 39.59 LS23 rep.1 10.901 79.143 0.137738019 7.245793493 39.58 LS23 rep.2 17.592 49.026 0.358830009 7.752072381 39.59

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26

LS24 rep.1 26.971 183.72 0.146804921 7.266555806 39.59

LS24 rep.2 30.01 194.39 0.154380369 7.283902838 39.58

C20:4 Arachidonic acid

Sample Text Area IS Area Area sample/Area IS Concentration (µg/ml) RT LS1 rep.1 253.914 174.894 1.451816529 17.97876834 39.09 LS1 rep.2 284.397 78.011 3.645601261 34.71244288 39.09 LS1 rep.3 1595.074 542.958 2.937748408 29.31310761 39.09 LS2 rep.1 3438.08 807.357 4.258438336 39.38702011 39.09 LS2 rep.2 2255.208 273.994 8.230866369 69.68776788 39.09 LS3 rep.1 863.093 113.668 7.59310448 64.82307002 39.09 LS3 rep.2 815.803 137.651 5.926604238 52.1113977 39.09 LS4 rep.1 1817.558 508.077 3.577327846 34.19166931 39.09 LS4 rep.2 2355.149 350.016 6.728689546 58.22951598 39.09 LS5 rep.1 700.394 123.389 5.676308261 50.20219879 39.09 LS5 rep.2 1050.254 171.791 6.113556589 53.53742631 39.09 LS6 rep.1 1232.271 174.816 7.048960049 60.67246415 39.09 LS6 rep.2 905.667 102.568 8.829917713 74.25719079 39.09 LS7 rep.1 1950.752 264.802 7.366832577 63.0971211 39.09 LS7 rep.2 2048.583 398.569 5.139845297 46.11018533 39.09 LS8 rep.1 1353.907 314.083 4.310666289 39.78540266 39.09 LS8 rep.2 1269.969 391.333 3.24523871 31.65857139 39.08 LS9 rep.1 1119.319 193.468 5.785551099 51.03547749 39.09 LS9 rep.2 3785.106 314.268 12.04419795 98.77496526 39.09 LS10 rep.1 1259.574 122.279 10.30082026 85.47688982 39.09 LS10 rep.2 2361.867 182.043 12.97422587 105.8689998 39.09 LS11 rep.1 2536.454 140.383 18.06809941 144.7238704 39.09 LS11 rep.2 2945.089 208.664 14.11402542 114.563123 39.09 LS12 rep.1 3204.859 381.533 8.399952298 70.97751562 39.09 LS12 rep.2 2063.354 238.421 8.654246061 72.9172087 39.09 LS13 rep.1 1675.991 449.509 3.728492644 35.34471887 39.09 LS13 rep.2 907.101 199.048 4.557197259 41.66588298 39.09 LS14 rep.1 818.481 161.819 5.058003078 45.48591211 39.09 LS14 rep.2 4003.458 652.486 6.135699463 53.70632695 39.09 LS15 rep.1 1269.483 187.657 6.764911514 58.50580865 39.09 LS15 rep.2 2105.116 289.803 7.26395517 62.31239641 39.09 LS16 rep.1 3144.177 1143.926 2.748584262 27.87020794 39.09 LS16 rep.2 2620.394 339.766 7.712349087 65.73263987 39.08 LS17 rep.1 1477.123 24.733 59.72275907 462.4558281 39.09 LS17 rep.2 1182.255 152.462 7.754424053 66.05357783 39.09 LS18 rep 1 3332.341 117.524 28.35455737 223.186555 39.09 LS18 rep.2 4057.183 285.277 14.22190713 115.3860193 39.09 LS19 rep.1 5731.076 267.334 21.43788669 170.4278161 39.09 LS19 rep.2 3119.181 540.117 5.775009859 50.95507139 39.09 LS20 rep.1 2818.828 113.874 24.753921 195.7217468 39.09 LS20 rep.2 3390.689 103.827 32.65710268 256.0053599 39.08 LS21 rep.1 3581.375 134.902 26.54797557 209.4063735 39.09 LS21 rep.2 3768.457 10.276 366.7241144 2804.190042 39.09

Metabolomic study of the effects of perfluorinated compounds on the fatty acid metabolism during the development of Gallus gallus domesticus