GC-MS screening and PCB analysis of sediment from central Kattegat

Transcription

1 GC-MS screening and PCB analysis of sediment from central Kattegat Emma Eriksson Örebro University

2 Abstract Five sediment samples were collected in Bua on the Swedish west coast, near two industries, a paper mill, and a nuclear power plant. The two industries use water in their processes and have long been associated with releases of different substances, such as PCBs, and other chlorinated compounds. The environmental impact by the two industries is believed to be significant. The aim of the project was to examine the sediments close to both the water intake and water output to determine if these industrial activities have in any way changed the composition of the sediments. The sediments were extracted by Soxhlet extraction, followed by a deactivated silica and an acidic silica clean-up and then analysed by using a gas chromatograph coupled to a mass spectrometer, (GC-MS) with electron ionization, EI+, mode used in full scan mode. Each mass spectra were analysed by comparing them to the NIST database from The results were inconclusive since the peaks were not properly resolved, causing a poor correlation to the NIST database. One batch was specifically analysed for polychlorinated biphenyls (PCB) by using an atmospheric pressure gas chromatograph (APGC) coupled to a mass spectrometer (MS). The PCB analysis provided accurate results, except for the Ringhals intake where the MS became saturated due to the high levels. The river Viskan also showed high levels of PCB. The congener pattern from PCBs found near Ringhals intake resembled an Aroclor pattern from Aroclor Since the Aroclor pattern is only seen in Ringhals intake, the source is most likely from the small harbour and not from either of the industries. Key words: Paper mill, nuclear power plant, screening, GC-MS, PCB, APGC i

3 Table of Content Abstract... i List of Figures... iii List of Tables... iv 1. Introduction Aim and objective Background and theory Material and Method Sampling Soxhlet extraction Metallic copper clean-up Silica clean-up Results and Discussion Ringhals Indicative ions for chlorine and bromine Södra Cell Värö Polychlorinated biphenyls Conclusion References Appendix A- Batch 1, without minisilica Appendix B: Batch 1 with minisilica Appendix C: Batch Appendix D NIST Database matches and analyses Appendix E APGC results ii

4 List of Figures Figure 1: Sampling locations, marked as circles, with the industries, marked as squares, and actual intake and discharge, marked as diamonds (Google Maps 215) Figure 2: Silica columns... 7 Figure 3: Total ion chromatogram, TIC, of Ringhals input, batch Figure 4: TIC of Ringhals output, batch Figure 5: TIC of Ringhals intake, batch Figure 6: TIC for Ringhals output, batch Figure 11: Indicative ions for chlorine, m/z 35 and Figure 12: Indicative ions for bromine, m/z 79 and Figure 7: TIC of Södra Cell Värö intake, batch Figure 8: TIC of Södra Cell Värö output, batch Figure 9: TIC of Södra Cell Värö intake, batch Figure 1: TIC of Södra Cell Värö output, batch Figure 13: PCB levels in sediment samples from the different sites. The blue stars indicates peaks with too high concentration of PCBs which saturated the MS. The red stars indicate peaks with bad peak shape Figure 14: PCB levels in sediment samples from the different sites, the blue stars indicate saturation of the MS, and the red stars indicate peaks with bad peak shape Figure 15: Indicative ions for PCB with one chlorine substitution Figure 16: Indicative ions for PCB with two chlorine substitution... 2 Figure 17: Indicative ions for PCB with three chlorine substitution... 2 Figure 18: Indicative ions for PCB with four chlorine substitutions Figure 19: Indicative ions for PCBs with five chlorine substitutions Figure 2: Indicative ions for PCBs with six chlorine substitutions Figure 21: Indicative ions for PCBs with seven chlorine substitutions Figure 22: Indicative ions for PCBs with eight chlorine substitutions Figure 23: Indicative ions for PCBs with nine chlorine substitutions Figure 24: Indicative ions for PCBs with ten chlorine substitutions Figure 25: TIC from Toluene Figure 26: TIC from Soxhlet blank Figure 27: TIC from Ringhals input Figure 28: TIC from Ringhals output Figure 29: TIC from Södra Cell Värös input Figure 3: TIC from Södra Cell Värö output Figure 31: TIC of sample from the bay Figure 32: TIC of toluene blank Figure 33: TIC of Soxhlet blank, after use of minisilica Figure 34: TIC of sample from the bay, after the use of minisilica Figure 35: TIC of toluene Figure 36: TIC of Soxhlet blank from batch Figure 37: TIC of sample taken in the bay from batch Figure 38: NIST database match for Ringhals batch 1, min Figure 39: NIST database match for Ringhals batch 1, min... 4 Figure 4: NIST database match for Ringhals batch 1, min Figure 41: NIST database match for Ringhals batch 1, min iii

5 Figure 42: NIST database match for Ringhals batch 2, min Figure 43: NIST database match for Ringhals batch 2, min Figure 44: NIST database match for Ringhals batch 2, 2.48 min Figure 45: NIST database match for Ringhals batch 2, min Figure 46: NIST database match for Södra Cell Värö batch 1, min The molecule suggested by the NIST database for the peak at min is 2,2 -diethyl-1,1 - biphenyl (Fig. 46). The NIST reference spectra show several similarities, mainly the peaks at m/z 181 and 165. There are two peaks from the reference spectra just above m/z 181, which seems to be missing or have a very low intensity in the sample spectra. The ions at m/z ratio of 181 and 165 seem to be very common for many polyaromatic hydrocarbons, since the first four hits from the NIST database all display the same ions, m/z 181 and 165, at similar intensities. The delta plot show some differences between the sample spectra and the NIST reference spectra. The compound with retention time minutes might be a biphenyl or another polyaromatic hydrocarbon, but the functional groups have not been determined Figure 47: NIST database match for Södra Cell Värö batch 1, min Figure 48: NIST database match for Södra Cell Värö batch 1, min Figure 49: NIST database match for Södra Cell Värö batch 1, min... 5 Figure 5: NIST database match for Södra Cell Värö batch 2, min Figure 51: NIST database match for Södra Cell Värö batch 2, 2.4 min Figure 52: NIST database match for Södra Cell Värö batch 2, min Figure 53: NIST database match for Södra Cell Värö batch 2, min List of Tables Table 1: Sampling information... 4 Table 2: Examples of Soxhlet extraction methods used in literature... 5 Table 3: Internal standard added to the samples Table 4: GC-MS full scan conditions... 8 Table 5: APGC conditions... 9 Table 6: Swedish EPA classifications of PCB contaminated sediments, µg/g dry weight of sediment (Naturvårdsverket 1999) Table 7: Comparison of levels with the Swedish EPA * indicates peaks too large to integrate ** indicate bad peaks Table 8: PCB levels from APGC analysis iv

6 1. Introduction Paper mills have long been associated with the release of different environmental contaminants, such as polychlorinated biphenyls (PCB) and polychlorinated dioxin and furans (PCDD/F), especially when the chlorine bleaching method was used (Ratia & Oikari 214). Urban activities have been a source of contamination for the aquatic environment, and paper mills have been considered to be the sixth largest source of contamination (Ali & Sreekrishnan 21). Chlorinated effluent from the pulp industry is an extensive toxic hazard for the aquatic environment (Wulff et al. 1993). The pulp produced only contain around 4-45 of the original raw material. The rest of the raw material is then released with the effluent, mainly as organic matter. Some suggested by-products released with the effluent are chlorinated products, fibres, fatty acids, lignin, sulphur containing compounds, chlorinated lignin, resin acids, phenols, dioxins, and furans (Ali & Sreekrishnan 21). Since PCBs and PCDD/Fs are included in the Stockholm Convention on Persistent Organic Pollutants (POP), they have been well monitored globally for the past 2 years (United Nations 1995). A POP is a substance that remains in the environment for a long period of time. It accumulates in adipose tissue, and are toxic to humans and all living organisms. A majority of the known POPs have a low solubility in water, but they dissolve easily in adipose tissue and therefore accumulate in any substance that display lipophilic properties (Secretariat of the Stockholm Convention n.d.). Some POPs display different characteristics, such as poly fluorinated compounds, that have a lower degree of hydrophobicity (Lau et al. 27). Since the beginning of the 199s, the pulp industry have changed from chlorine bleaching to chlorine-free bleaching methods, or elemental chlorine free bleaching. Decreasing levels of PCBs and PCDD/Fs have been demonstrated, although they are still present as contaminants in sediments surrounding paper mills (Ratia & Oikari 214). In another study fish from the unheated and heated area near a nuclear power plant was compared, and found that perch showed significantly higher levels of PCBs. However, there were no significant difference for another fish species (Edgren et al. 1981) The cooling water from the nuclear power plant Ringhals, does normally only affect the surface water. Surface water is considered water down to 7 meters depth. Under extreme conditions during the winter, the sediments can be exposed to the heated water. The heat increase will affect the uptake of PCBs and DDT. The first studies on the subject showed that the uptake were twice as big in fish when the temperature increased from 5 C to 15 C. The initial study was performed in a laboratory, and the effect was much less prominent in the environment (Elforsk 29). 1.1 Aim and objective The aim of this project was to compare the sediments from the inlet and outlet of the selected industries. This approach was chosen to determine if the changed characteristics of the water (such as heat increase and organic components) have affected the composition of the sediments. This will be examined through a GC-MS screening where the chromatographic pattern will be examined and through a specific PCB analysis of the sediments. The sediment near the two industries was also compared to a sample from the nearby bay, to determine if any potential contamination can be linked to either of the two industries. The PCB analysis is 1

7 also to be compared to known congener profiles of Aroclor to possibly link the PCBs to a specific Aroclor. 2. Background and theory Bua is a village situated 2 km north of Varberg, on Värö peninsula. In the surrounding area there are two larger industries, Södra Cell Värö, a paper mill, and Ringhals, a nuclear power plant. Both industries use the sea and river water in their processes. Ringhals uses the water from the sea as cooling water for the reactors. The water is only used for heat exchange and is not a part of the nuclear fission itself. Södra Cell Värö uses water from Viskan, a larger river that ends in Kattegatt, in its process. The water is after usage released into the sea from a 2 km long pipeline. The pipeline is situated at near 2 meters depth and the release takes place through 18 holes along the pipeline (Eriksson 215). Figure 1: Sampling locations, marked as circles, with the industries, marked as squares, and actual intake and discharge, marked as diamonds (Google Maps 215). Södra Cell Värö was built in From the beginning until 199, the bleaching used chlorine, when they started the transition to chlorine free bleaching. In 1993, the bleaching process at Södra Cell Värö was totally free of chlorine, being one of the first paper mills in the wold to apply a chlorine free bleaching method. The chlorine bleaching was replaced with oxygen bleaching. The oxygen bleaching is then followed by further bleaching with hydrogen peroxide, oxygen and peracetic acid, if further beaching is requested. In 22, Södra Cell Värö installed a facility for biological cleaning of the waste water (Södra Cell Värö 214). In 21, Södra Cell Värö was the first paper mill in the world to be independent of fossil fuels in their normal production (Södra Cell Värö 215). Södra Cell Värö produces around 425 2

8 metric tonnes of paper pulp each year of witch about 9 is exported, mainly to Europe. Södra Cell Värö mainly produce paper pulp, but in recent years they have also started producing green electricity, biofuel, and long distance heating to the surrounding area (Södra Cell Värö 214). Previous investigations have been conducted by Södra Cell Värö in , where they analysed 2,3,7,8-tetrachlorodibenzodioxin (TCDD), 3,4,3,4 -tetrachlordibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), and extractable organic chlorine (EOCl). The samples were taken from an affected point near the industry and included crabs and common whelk, Buccinum undatum (Stibe 215; Nationalencyklopedin n.d.). The samples were compared to a reference site in Fladen. The study ended soon after Södra Cell Värö changed bleaching process from the chlorine bleaching to oxygen bleaching since the levels from the affected station approached the levels in the reference samples (Stibe 215; Södra Cell Värö 214). Ringhals is situated on the northern side of Värö peninsula, and produces 2 of the electricity consumed in Sweden today. The construction of the power plant started in 1969, and in May 1975 the first reactor was put into operation. There are currently four reactors in operation, Ringhals 1 through 4. In 22, Ringhals became certified according to Environmental Product Declaration (EPD), which means that they for each kwh produced can calculate the environmental effects. The sea water is used as cooling water for the steam produced in the reactors. When operating at full power, around 17 cubic meters of seawater is used per second for the cooling of the reactors. (Vattenfall 212a). The cooling water leaves the reactor around 1 C warmer than the initial temperature. Ringhals have constantly tried to minimize the different emissions to the environment, such as regular and dangerous waste, chemicals, cooling water discharge water treatment and radioactive emission. Ringhals has since 199 been registered with Eco Management and Audit Scheme (Emas) and since 1998 it is also certified according to ISO 141 (Vattenfall 212b). Their ammonia emission to water is less than one tenth of the natural amount found in the sea. The boric acid release is near one hundredth of the natural content found in the sea. Furthermore, chlorine is released at around.1 ppm per year (Vattenfall 212b). PCB are considered to be persistent and toxic in the environment. The PCBs have different degrees of chlorination, from one to ten, where each chlorine is attached to a carbon in a biphenyl backbone. PCB is a mixture of 29 different congeners. When producing PCBs, they were sold under the trade name Aroclor. Commensally, there were nine different formulations sold, all containing different weight percentage of chlorine. Aroclor were used in a vast number of purposes, such as dielectric fluid, inks, and pesticide extenders (Murphy & Morrison 27). 3

9 3. Material and Method 3.1 Sampling Duplicate samples for each site were acquired by using an Ekman sediment grab sampler. The samples were collected from five different locations along the coastline. Each location was chosen so that it could be compared to the other locations. Table 1: Sampling information Sample Sediment type Location description Coordinates Ringhals Clay Small harbour N intake E Ringhals Sand Open water, near N output coastline E Södra Cell Clay Open water, N intake middle of river E Södra Cell Clay Open water, near N output coastline E Bay Sand Open water, near N coastline E N E Depth (m) The sample vials were filled to the best capacity in amber glass jars, 25 ml, and kept frozen until the extraction took place. The sampling point from Södra Cell Värös intake, were acquired from two different locations, from both sides of a delta at the end of the river. 3.2 Soxhlet extraction Before extraction began, the samples were thawed and dried overnight. The sediments were spread out on aluminium foil and left in room temperature in the fume hood until dryness. There are several problems associated with the drying technique, mainly the loss of analyte. Drying times of only 4 hours have been associated with loss of PCBs from sediments. This, when the temperature was 2 C and the relative humidity was 25. The main part of the total PCB losses, up to 9, occurred after the first 4 hours of drying. The heat, air flow, water content and grain size are believed to affect the amount of PCBs in sediments. The congener pattern found in the sediments may also be altered in the drying process. The change in congener pattern are most distinct in sediments with PCB source from lighter Aroclors (Chiarenzelli et al. 1996). For the first batch, dryness was achieved after 12 hours and for batch 2 after 48 hours. Different amounts of sediment were dried between the different batches and that can account for the difference in drying time. The Soxhlet extraction chambers were cleaned by starting an extraction with clean thimbels and toluene while the samples were drying. The Soxhlet blank, which is a method blank were treated in the same way as the samples. The wash continued for 24 hours before the extractions were stopped at reflux and the toluene was discarded. The dry sediments were then weighed, and 1 g of sediments were placed in each thimble. The decision to use 1 g of sediments was based on previous studies which used similar amount and method for extraction of sediments (Table 2). 4

10 Table 2: Examples of Soxhlet extraction methods used in literature Amount of Solvent (h) Reference sediment used 5 g n-hexane:dcm 1:1 v/v 16 (Parera et al. 24) 1 g Hexane, acetonitrile, (US EPA 1996) toluene/methanol, acetone:hexne 1:1, 2-propanol, cyclohexane, or acetone:hexane 1 g Methanol 18 h (Jurado-Sánchez et al. 213) 2 g Chloromethyl: Acetone 18 h (Hawthorne et al. 2) Since the sediments contained varying amounts of water, the water present in the sediments had to be estimated with the intent of having 1 g of sediment present for the analysis. For batch 1, the sample representing Södra Cell Värös intake, only 8.17 g were used since the amount of water in the sediment was underestimated. After the drying process were completed, only 8.17 g of sediment were present and used for the analysis. A PCB internal standard was added to the sediments, 25 µl was added (Table 3), and the extraction started. The extraction was then discontinued after 24 hours and the toluene used for the Soxhlet extraction was evaporated using a rotary evaporator until ~1 ml of extract remained in the round bottom flask. The rotary evaporator was washed with hexane before usage. The temperature of the water bath was 45 C, and the pressure was 1 mbar for hexane and 8 mbar for toluene. Table 3: Internal standard added to the samples. Compound Amount (pg µl -1 ) 13 C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C PCB # C OCDD Metallic copper clean-up If any of the samples at this stage had a yellow discolouration, metallic copper was added to the eluate to react with sulphur that can disturb the analysis at higher levels. The copper was added to the round bottom flask and allowed to react. The extract was then removed from the 5

11 round bottom flask and placed in a new flask. The copper was rinsed with hexane three times to ensure that most of the analyte had been transferred to the new round bottom flask. The new round bottom flask was evaporated until ~1 ml of the extract remained. Batch 1 was at this point split, around half of the solvent was removed and stored in 8 ml amber glass vials. The partition occurred in case the clean-up method were to be altered and the extract would be needed for a second clean up. 3.4 Silica clean-up The extract was then added to a 1 deactivated silica for purification. The deactivated silica was prepared by weighing 7 g of silica, and adding 7 ml of MilliQ to the silica. The silica was shaken for three hours on a shaker, and additionally shaken by hand every 15 minutes under the three hours to ensure that the silica was homogenized. This method for the silica preparation was used since there was no access to the rotary shaker at the time of the experiment. The setup can be seen in fig. 2 To prepare the deactivated silica column, a piece of glass wool was placed at the bottom of a column and rinsed with ethanol, hexane, and dichloromethane, DCM. To the column, 1 g of silica was added, followed by a layer of anhydrous sodium sulphate to remove traces of water from the organic solvents used. The columns were then rinsed with 4 ml of hexane before the addition of the sample. The eluates were collected after the samples were added to the column. After the samples were added the round bottom flasks were rinsed three times with hexane and added to the silica. The analytes were then eluted by addition of 1 ml hexane. The extract was then evaporated using the rotary evaporator until ~ 1 ml of the extract remained. For the first batch, the extracts were transferred to 8 ml amber glass vials, and the previous vials washed with hexane three times. The extracts were then evaporated until near dryness with nitrogen gas. The extracts were then transferred to GC vials and 25 µl of toluene was added. The 8 ml amber glass vials were rinsed 3 times with hexane. The GC vials were then placed under nitrogen gas. 6

12 Figure 2: Silica columns The samples were then analysed using GC-MS. When evaluating the results, it was noticed that the samples were too impure to provide useful mass spectra and chromatograms, see Appendix A. The samples were after the GC-MS analysis further purified by using a minisilica column. The minisilica column were prepared in a Pasteur pipette, with glass wool acting as a plug at the end. The pipette and the glass wool was initially rinsed with ethanol, hexane, and DCM. The last drops of DCM were pushed out, the column was then packed. Around 2 cm of KOH silica and 2 cm 4 H2SO4 silica were added to the pipette (Fig. 2). The silica was then covered with sodium sulphate. The minisilica column was then rinsed with two pipettes of hexane before the sample was added. The collection vials were placed under the pipettes as soon as the samples were added. The analytes were eluted with 2 pipette volumes of hexane. The eluate was collected in 8 ml amber glass vials and then evaporated until near dryness before being transferred to the GC vial, in the same manner as stated previously. The chromatograms were matched to the NIST library. The NIST database match comes in four parts, one part gives the name of the suggested structure, denoted hit list, and a visual representation of the first compound on the hit list. The third part is the mass spectra of the sample and the first three hits on the hit list. The fourth part is a delta plot where the sample spectra and the NIST reference spectra is compared to each other. For an ideal match, the delta plot should show no peaks. On the y-axis, the positive side indicates a peak present in the sample and negative side represents a peak present in the reference spectra. 7

13 Batch 2 was analysed in the same manner as the previous batch, the only difference was that the GC-MS analysis only took place after the minisilica clean-up, not before and after. Table 4: GC-MS full scan conditions Inlet Injection type Splitless Volume 1 µl Heater 25 C Pressure psi Total flow 24.3 ml/min Purge flow Split vent 5. 2 min Gas saver min Column Constant flow 14.4 psi Flow 1.4 ml/min Average velocity 44 cm/sek Manufacturer specifications Model number J & W Column DB-5 Capillary 3 m * 25 µm *.25 µm nominal Oven settings Oven ramp Temperature increase C/min Next C Hold Initial 9 2 min Ramp min Ramp 2 Run time min Solvent delay 4 min GC-MS Brand Hewlett & Packard Model GC 689 plus series Model MS 5973 Ionization mode Positive Electron ionization (EI+) Scanning range Batch 1: m/z 5-5 Batch 2: m/z

14 Batch 2 was also analysed for PCBs using atmospheric pressure gas chromatography, APGC. Table 5: APGC conditions Inlet Injection type Splitless Pulse time 1 min Heater 28 C Pulse pressure 45 kpa Column Constant pressure 14.4 psi Flow 1.4 ml/min Average velocity 44 cm/sek Manufacturer specifications Model number Cat #: Serial #: 9419 Column Rtx -5MS Capillary 3 m *.25 mmid *.25 µm no Oven settings Oven ramp Temperature Next C increase C/min Initial 18 Ramp min Hold time Ramp min Run time 31.1 min Solvent delay 3 min Ion source Transferline Temperature 28 C Make up gas Nitrogen Make up gas flow 37 ml/min APGC GC Agilent 789 MS Waters XEVO TQS 9

15 4. Results and Discussion The results from the full scan experiment are presented as chromatograms that can be seen in Appendix A, B and C. The NIST spectral database from 1998 was used to determine which compounds were present in the sediments from the full scan experiment. Each match was of varying quality since many of the mass spectra obtained did not match well with the suggested spectra. The chromatograms from the intake and output from the same industry and batch are compared to each other to determine which peaks are present in the output but not in the input. This, in order to determine which compounds might have been released from the two industries, the chromatograms were examined, and attempts were made to identify peaks that appears in the output but not in the input. The chromatographic pattern were examined and each pair of chromatograms provided a varying number of peaks that can only be found in the output.. This approach resulted in several peaks that were examined, and the peaks with the highest probability of obtaining a good match were chosen from each sample. The chromatograms are examined for peaks only present in the output and NIST matches are generated for each peak. The NIST comparisons with the highest probability of providing and accurate match have been presented in the thesis. The selection process is based on the delta plot and if the main peaks, such as molecular ions or indicative ions, are present in the spectra. The chromatograms seen in Appendix A, batch 1 before the minisilica clean-up, have not been matched to the NIST library, due to the poor peak resolution. Appendix B show the total ion chromatogram, TIC, from batch 1 for toluene, the Soxhlet blank and the sample from the bay. Appendix C show the TIC for toluene, the Soxhlet blank and the sample from the bay for batch 2. 1

16 4.1 Ringhals When comparing the samples from Ringhals batch 1 (Fig. 3,4) some peaks appeared in the output, but not in the input. Some of these peaks were suggested to be 2,3-dihydro-1,1,3- trimethyl-1h-indene, chlorocycloheptane, 2,3,4,6-tetramethyl-bibenzyl and, erucylamide. R1 LRH_15528_ TIC 3.26e Figure 3: Total ion chromatogram, TIC, of Ringhals input, batch 1 R2 LRH_15528_ TIC 3.95e Figure 4: TIC of Ringhals output, batch 1 The samples from Ringhals batch 2 (Fig. 5,6) were n-methyl-n-phenyl benzamine, 3-(1,1 biphenyl-4-yl)butanenitrile, 1H-indene, 2,3-dihydro-1,1,3-trimethyl-3-phenyl and, 1(2- bromoethyl)-4-methoxy-benzene. 11

17 R1 LRH_15613_ TIC 5.29e Figure 5: TIC of Ringhals intake, batch 2 R2 LRH_15613_ TIC 5.67e Figure 6: TIC for Ringhals output, batch 2 The NIST database output can be seen in Appendix D together with an analysis of the spectra (Fig ). These substances are highly possible matches for some of the peaks found in the output of Ringhals. Each match is of varying quality, and the peaks with the highest probability of providing an accurate match have been included in the thesis. Erucylamide is used as a slip and antiblocking agent for polyolefins (Fine Organics 212). Polyolefins is a polymer made from any simple alkene and is for example used in detergent to strengthen the package (Fine Organics 212; Vasile 2). Erucylamide is also found in leaf essential oil from Citrus medica. Since C. medica has its origin in Bangladesh (Bhuiyan et al. 29), it is unlikely to be found in sediment in Kattegat from the C. medica plant. 12

18 For the other compounds, no information on their uses or origin have been found. Since no information have been obtained about the other substances, it is difficult to determine their origin Indicative ions for chlorine and bromine Indicative ions for chlorine and bromine have been examined from the sample from Ringhals intake (Fig. 11). There are some peaks present in both chromatograms for chlorine, m/z 35, and 37. This supports the presence of chlorine at several retention times, among them 5.92, and minutes. The noise levels are much higher in the chromatogram displaying m/z 35. The signal for m/z 37 is nearly ten times higher than for m/z 35 for the main peak, this suggests that the peak with retention time 16.7 and is from an impurity. The other peaks present, 5.92, 7.54, 24.25, 25.88, 26.68, and 27.92, seems to conform to the theoretical distribution of chlorine. In conclusion, chlorine seems to be present in the sample, since several peaks appear with similar retention times. R1 LRH_15613_ e LRH_15613_ e Figure 7: Indicative ions for chlorine, m/z 35 and 37 The indicative ions for bromine (Fig. 1), show some peaks with retention time between and minutes, since peaks are present in both chromatogram, it supports the theory that bromine is present in the sample from Ringhals. The isotope ratios for bromine are acceptable for some peaks, such as 15.27, where the isotope ratio are nearly ideal. For the main peak, min, the isotope ratio does not resemble bromine, the abundance for the lighter isotope is This is far from the ideal value, suggesting that the peak at min does not contain bromine. In conclusion, there are evidence of bromine being present in the sample from Ringhals. 13

19 R1 LRH_15613_ e LRH_15613_ e Figure 8: Indicative ions for bromine, m/z 79 and 81 14

20 4.2 Södra Cell Värö When comparing the samples from Södra Cell Värö batch 1 (Fig. 7,8) some peaks appeared in the output, but not in the input. Some of these peaks were suggested to be 2,2 -diethyl-1,1 - biphenyl, (2-methylphenyl)-methyl-carbamic acid, 2,6,1,14-tetramethyl-nonadecane, and 2,6,1,14,18-pentamethyl-eicosane. VB1 LRH_15528_ TIC 4.37e Figure 9: TIC of Södra Cell Värö intake, batch 1 VB2 LRH_15528_ TIC 3.6e Figure 1: TIC of Södra Cell Värö output, batch 1 The samples from Södra Cell Värö batch 2 (Fig. 9,1) were suggested to be 1-chloro-3- methyl-benzene, (E)-stilbene, 3,4,5,6-tetramethyl-phenanthrene and, erucylamide. 15

21 VB1 LRH_15613_ TIC 5.49e Figure 11: TIC of Södra Cell Värö intake, batch 2 VB2 LRH_15613_ TIC 5.52e Figure 12: TIC of Södra Cell Värö output, batch 2 The NIST database output can be seen in Appendix D together with an analysis of the spectra (Fig ). These substances are highly possible matches for some of the peaks found in the output of Södra Cell Värö. Each match is of varying quality, and the peaks with the highest probability of providing an accurate match have been included in the thesis. The substances may have its origin from the paper mill, since the long carbon chains may be a component from the raw material used in the process. Since erucylamide is found near both Ringhals and Södra Cell Värö, either its source is of a natural origin, or the chemical is released from either of the industries and is transported with the currents in the sea. For the other compounds, no information on their uses or origin have been found. Since no information have been obtained about the other substances, it is difficult to determine their origin. 16

22 pg/g d.w. sediment 4.3 Polychlorinated biphenyls The results from the APGC can be seen in Table 8, appendix E. The results from the PCB analysis (Fig. 13), are shown below. The peaks representing Ringhals intake saturated the detector, and only the base of the peaks were present. Therefore the peaks were not integrated. There were no attempts made to integrate the peaks since the levels were believed to be completely misguided if prefrormed. The peaks from all samples are also shown in graphical form below in Fig. 14, which is a cropped axis of Fig. 13. In table 5 in appendix E, it can be seen that the blank contain high levels of the analysed PCBs. This might be due to contaminations that occurred in the sample preparation. The samples may contain additional analytes than the PCB that may interfere with the PCB analysis, this is seen in the large shift in retention time which could be up to.5 minutes. This problem should also affect the samples, and should be kept in mind when examining the results. Due to the high levels found in the blank, the levels are only indicative PCB 28 PCB 52 PCB 11 PCB 118 PCB 153 PCB138 PCB 18 Ringhals intake Ringhals output Södra Cell Värö intake Södra Cell Värö output Bay Figure 13: PCB levels in sediment samples from the different sites. The blue stars indicates peaks with too high concentration of PCBs which saturated the MS. The red stars indicate peaks with bad peak shape. 17

23 pg/g d.w. sediment PCB 28 PCB 52 PCB 11 PCB 118 PCB 153 PCB138 PCB 18 Ringhals intake Ringhals output Södra Cell Värö intake Södra Cell Värö output Bay Figure 14: PCB levels in sediment samples from the different sites, the blue stars indicate saturation of the MS, and the red stars indicate peaks with bad peak shape. The sample from Ringhals intake, PCB #11, #118, #153 and #138 saturated the detector, since only the base of the peaks were seen in the chromatograms. PCB #18 from Ringhals intake is several times larger than the other sites, supporting the saturation theory. The intake of both industries seems to contain higher levels of PCBs. Although, this may be a biased statement, both intake points come from places believed to contain higher levels of contaminants. Ringhals intake comes from a small harbour near the intake where the extra activity associated with the harbour might increase the PCB levels. This is due to a sampling error, since the sediments were too well packed to obtain samples closer to the actual input. The only sediment that were obtained from Ringhals intake were from the harbour, initially the effect of the harbour was unknown. The intake for Södra Cell Värö was taken from the river Viskan, which passes through Borås, a town associated with the textile industry. Kattegat can therefore receive contaminants from Viskan, as it absorbs pollutants from the nearby land and industries as it passes through Sweden, and deposits them in the sea (Forsman & Edlund n.d.). Since the intake is situated in the river Viskan, no other sampling point was available for the intake from Södra Cell Värö. PCB #118 showed signs of co-elution in Ringhals output and Södra Cell Värös output, and they were therefore not integrated in those samples. In general, the peaks had a poor peak shape and the co-elution is most likely from several PCBs, so obtaining any useful information is hard. In another report from Swedish EPA, the levels of several congeners of PCBs (PCB #28, #69, #73, #52, #84, #89, #9, #11, #16, #118, #153, #138, #164, #163, and #18) show a maximum level of 25 for the PCBs previously mentioned in pg g -1 d.w in the Baltic Proper region in 199 (Wiberg et al. 213). The PCB 18 levels from Ringhals intake for alone account for around 4 of the concentration in the highest level found in the study. 18

24 A study showed that the mean concentration along the Baltic Sea were for PCB #11 21 pg g -1 d.w., PCB #118 was 13pg g -1 d.w., PCB #153 was 4 pg g -1 d.w., PCB #138 was 11 pg g -1 d.w and for PCB #18 it was 22 pg g -1 d.w. in 1985 (Sobek et al. 215). For PCB #11 the levels are all lower than the mean. For PCB #118 all sampling sites were lower than the levels from the Baltic Sea coast. For PCB #153, all but Södra Cell Värös intake were lower than the mean. The intake from Södra Cell Värö had around 7 pg g -1 d.w, the highest level at a sampling site in the Baltic Sea was 13 pg g -1 d.w. For PCB #18 the levels were lower than the mean from the Baltic Sea, except for Södra Cell Värös intake and Ringhals intake. The levels near Södra Cell Värö are close to the mean value from the study. The levels near Ringhals intake are much higher than the mean level for PCB #18, but the highest level measured in the study was 17 pg g -1 d.w. from one of the sampling sites. Ringhals intake are much lower than the highest level measured. The PCB levels from Ringhals intake are believed to be the sampling point with the highest concentration of PCBs. The levels from Ringhals intake saturates the detector causing the top of the peak to be removed in some cases the base were nearly invisible. No attempt at integrating the levels of those PCBs have been made. Since Ringhals intake showed high levels, 1171 pg/g sediment of PCB #18, the remaining PCBs were believed to be seen in the chromatograms from the GC-MS screening. The indicator ions for PCBs with one chlorine substitution can be seen fig. 15. The two ions displayed are m/z 188 and 19, these are the two ions with the highest and second highest intensity. Since the peaks displayed in either chromatogram are not present in the other chromatogram, the single chlorine substituted PCBs are most likely found in levels below the GC-MS limit of detection, (LOD). R1 LRH_15613_ e LRH_15613_ e Figure 15: Indicative ions for PCB with one chlorine substitution In fig. 16 the indicative ions for PCBs with two chlorine substitutions. Some peaks are visible in both chromatograms, such as There is a large peak disturbing the chromatogram displaying m/z 224, suggesting that the chromatogram suffers from impurities. Since this peak is visible in the GC-MS full scan experiment, the levels are believed to be quiet high. 19

25 R1 LRH_15613_ e LRH_15613_ Figure 16: Indicative ions for PCB with two chlorine substitution e In fig. 17 the indicative ions for PCBs with three chlorine substitution can be seen. Several peaks can be seen in both chromatograms, especially peaks around retention time and minutes. Since the peaks can be seen with the GC-MS full scan, it indicates high levels, supporting the saturation theory from the PCB analysis with the APGC. Since the standards for the APGC analysis are not visible in the samples when using the GC-MS approach, the peaks cannot be matched to a specific PCB. R1 LRH_15613_ e LRH_15613_ e Figure 17: Indicative ions for PCB with three chlorine substitution Fig. 18 show the two largest ions for PCBs with four chlorine substitutions. They show a similar pattern in both chromatogram, suggesting that PCBs with three chlorine atoms are present in high levels. Peaks with retention time of 22.53, 24.27, 27.25, 27.92, 29.18, minutes are possible PCBs. 2

26 R1 LRH_15613_ e LRH_15613_ e Figure 18: Indicative ions for PCB with four chlorine substitutions Fig. 19 show the two largest indicative ions for PCBs with five chlorine substitutions. Many peaks are present in both chromatograms, such as the peaks with retention time 24.27, 24.96, 25.88, 26.68, and minutes. Those peaks are suggested to be PCBs with four chlorine substitutions. R1 LRH_15613_ e LRH_15613_ e Figure 19: Indicative ions for PCBs with five chlorine substitutions Fig 2 show the two largest indicative ions for PCBs with six chlorine substitutions. Several peaks are present in both chromatograms, among them the peaks with retention time 25.73, 26.5, 27.25, 27.92, and minutes. 21

27 R1 LRH_15613_ e LRH_15613_ e Figure 2: Indicative ions for PCBs with six chlorine substitutions Fig. 21 show the two main indicative ions for PCBs with seven chlorine substitutions. Peaks with retention time 27.47, 28.29, 28.87, 29.69, and 3.37 minutes, among other are suggested to be PCB with six chlorine substitutions. R1 LRH_15613_ e LRH_15613_ e Figure 21: Indicative ions for PCBs with seven chlorine substitutions Fig. 22, 23 and 24 show indicative ions for PCBs with eight, nine and ten chlorine substitutions. All six chromatogram show noise at the end of the chromatogram, and the levels of PCBs are believed to be below LOD for the GC-MS screening. 22

28 R1 LRH_15613_ e LRH_15613_ e Figure 22: Indicative ions for PCBs with eight chlorine substitutions R1 LRH_15613_ e LRH_15613_ e Figure 23: Indicative ions for PCBs with nine chlorine substitutions 23

29 R1 LRH_15613_ e LRH_15613_ e Figure 24: Indicative ions for PCBs with ten chlorine substitutions Swedish EPA have proposed five levels of PCB concentrations, which is no levels, low levels, average levels, high levels and very high levels. Ringhals intake is 5 times higher than the level characterised as very high levels for PCB #18. The saturated peaks are most likely of higher concentrations than PCB #18 and are therefore most likely placed in the very high levels category. Table 6: Swedish EPA classifications of PCB contaminated sediments, µg/g dry weight of sediment (Naturvårdsverket 1999) PCB congener No levels Low levels Average levels High levels Very High levels # >2. # >2. # >3.5 # >4.1 # >1.9 Table 7: Comparison of levels with the Swedish EPA * indicates peaks too large to integrate ** indicate bad peaks PCB Ringhals Ringhals Södra Cell Södra Cell Bay congener intake output Värö intake Värö output #11 * Low Low Low Low #118 * ** Low ** Low #153 * Average High Average Average #138 * Low Average Low Low #18 Very high Low Low Average Low Comparing the results obtained to the data from Swedish EPA, the values are shown as levels instead of numerical values (Table 7). The result indicates that there are PCBs present in the sediments, in high levels at some of the sampling sites, Södra Cell Värö intake, and at very high levels in some samples, Ringhals intake. The industries seem to have no effect on the 24

30 PCB concentration in sediments surrounding the two industries since the levels are lower in the output than the input. The levels from Ringhals intake indicates very high levels of PCBs, since peaks are visible in the GC-MS experiment. PCB #18 is believed to have the lowest concentration of all the PCBs measured. PCB #18 is according to the Swedish EPA very high suggesting that the levels for the other PCBs indicative very high levels. The levels indicated with an asterisk, probably falls in the category of very high levels, since they have a higher concentration than PCB #18 in the GC-MS screening, and PCB #18 falls under the category of very high levels. The levels proposed by the Swedish EPA have in recent years been out dated, but it is still applied routinely. The pattern of the congeners may by lined to a specific Aroclor. The conclusion that PCB #18 which was the only peak small enough to integrate, is supported since PCB #18 is a heptachlor, shown in fig. 21, and only noise is visible, suggesting that the levels of the other PCBs with two, three, four, five, and six chlorine substitutions are higher than the PCB #18 levels from the APGC. For the patter determination, the chromatograms from the GC-MS indicative ions were used. The chromatogram with the highest intensity belonged to the heptachlorobiphenyls. The second highest intensity was hexachlorobiphenyls, followed by trichlorobiphenyls and tetra chlorobiphenyls. The highest intensity is plotted, and the other peaks are plotted by their relative intensity, providing a plot used to match against the congener patterns from EPA. When examining the PCB congeners, the highest peaks belong to heptachlorobiphenyls, PCB #4 PCB #81. The Aroclor patterns from EPA show higher levels of PCBs with other degrees of chlorination, such as Aroclor 116 and 1242, where the maximum concentration is between PCB #15 and PCB #3. Aroclor 1254a and 1254g have a maximum between PCB #11 and #12. Aroclor 126 have its maximum around PCB #18. Neither of those match the pattern from the sample collected from Ringhals intake. The Aroclors that match these high levels of heptaklorobifenyls, is Aroclor 1248a and 1248g from EPA. Aroclor 1248a and 1248g display a very similar pattern, and since the specific PCB congener cannot be determined from the GC-MS, the Aroclor cannot be differentiated between Aroclor 1248a and 1248g (US EPA n.d.) 5. Conclusion The aim of this study was to determine if there were any differences between the intake and output of the two industries and if potential contaminants linked to either industry can be found in the bay. This was done by applying a full scan approach. With this approach, numerous errors were encountered. Many of the peaks were not sufficiently resolved to provide reliable results, and most of the results cannot give any conclusive information regarding the presence of contaminants in the samples. Often, the type of compound group present can be matched with the reference library, but the specific compound cannot be determined. The samples from Ringhals generally contained longer carbon chains, whereas Ringhals generally contained more PAHs. The high levels found in the Soxhlet blank may be due to the fact that the sample was analysed late in a sequence, causing a memory effect. This may explain the high levels found 25

31 in the Soxhlet blank. The levels in the Soxhlet blank are higher than the levels in some samples, so any levels below that of the Soxhlet blank may be used as indicative levels of the PCB concentration. Since the study is performed by using a screening method, identification of analytes may not be feasible due to the co-elution of different analytes with similar retention time. Some compounds will co-elute and overlapping chromatogram and mass spectra will most likely be obtained. Overlapping mass spectra will then be much harder to match to databases since the mixed mass spectra will generate a different spectra and the possible matches will not fit the mass spectra suggested by the database. Not only will the purity of mass spectra be of issue, many compounds have several congeners, often with similar mass spectra and they cannot be differentiated by only using the mass spectra. For this kind of study, it is impossible to acquire standards for all compounds analysed, and therefore each match to the database is a tentative identification. There are other indications that can be used for the confirmation of the compound than the mass spectra and retention time. If possible, a MS/MS spectra, and 13 C standards for important ions can be used to further strengthen the conclusion. With the MS/MS approach, the limits of detection, LOD, are lowered, but all other analytes are lost in the process. The MS/MS approach does lower the LOD, but the risk of overcharging the detector increases with higher levels. The spectra from the full scan, all follow the criteria from the mass spectra, and the retention time in many cases seems plausible. The screening method provided some good matches for each chromatogram. The chromatogram do indicate differences in the sediments from the five locations. Each chromatogram show different peaks, suggesting that the industries have affected the composition of the sediments. Since the peaks have not been identified, to maintain within the scope of the thesis, only differences in the pattern have been examined. The main part of the thesis was to determine what substances have been released by the industries, and that was proven to be difficult. The industries could not be linked to any compounds found in the sediments. But the general difference in chromatographic pattern suggests that there is a difference in the sediments. The differences in the sediments might be worth additional studies with another approach. There appear to be less extractable material in the outtake of the two industries, suggesting that they have not affected the superficial environment surrounding Värö peninsula. Since the samples acquired for the intakes are from sites believed to show additional contamination, such as a harbour for Ringhals intake, and the fabric industry for Södra Cell Värö intake. Again, the intake for Ringhals should have been acquired closer to the actual intake and not in the harbour, but due to sampling errors, this was unavailable. The sediments were packed too hard for the Ekman sediment grabber to acquire a sample. The results show a general decrease in the intensity in the chromatogram from the Ringhals comparison, but the decrease might be from the additional elements added by the harbour. The same problem may arise with the water from Södra Cell Värö, the contamination from the previous releases in Viskan. Several of compounds suggested by the NIST library, any information on the origin of the compound were unattainable. In many instances medical safety data sheets (MSDS) have been found, and they do not provide information on the sources of the compounds. To maintain within the scope of the thesis, the sampling was limited to five locations. Preferably a reference point should have been obtained, further out from the coast. There is a 26

32 small river that flows into the bay in the middle of the peninsula, which should have been investigated as well. When running the APGC, there are some limitations with the standards, as the standard added only contains a few different congeners of PCBs, and the analysis is thereby limited to those PCBs alone. The large time shift that appeared in retention time due to the high content in the samples made the identification via retention time nearly impossible. The samples were analysed in the end of a long sequence, causing several of the peaks to tail. The samples were also highly contaminated, since no further clean-up took place between the GC-MS screening and the APGC analysis, causing the retention time to shift. This shift in retention time makes the identification of PCB congeners hard. Since the retention time from the PCB standard does not match the retention times in the samples. The industries seems to not affect the levels of PCB in the sediments, since they should be elevated at the output sites, and that is not the case. The PCB levels are much lower in the output locations suggesting that the industries do not affect the levels of PCBs in the sediments. Ringhals output, Södra Cell Värös output and the bay seem to contain similar levels of PCBs, suggesting that the sites are exposed to similar sources. Since an Aroclor pattern can be determined from the GC-MS analysis, the levels of PCB #11, PCB #118, PCB #153, and PCB #138, are most likely higher than the levels found for PCB # 18. The Aroclor found near Ringhals intake probably have its origin in the activity associated with the harbour, not the neighbouring industries. This since the elevated levels are only found near Ringhals intake, and all other points display lower levels. The initial intention was to compare the samples taken in the bay to the findings in the other samples; this was proven to be difficult, it has been excluded as a part of the experiment. Sop for further investigations, the bay should be more extensively compared to the two industries to determine if any releases takes place and affects the industries. Another possibility is to widen the search and examine more than the indicator PCBs. This may be done by using principal component analysis to determine the source of the PCB congeners. It would also be helpful in the Aroclor pattern recognition, since the levels in this thesis is only based on the peak height from the GC-MS screening. To continue the studies, several additional sample should be examined for PCBs, which seems to be present at quite high levels in some of the sample locations. Preferably, a clean-up adapted for the analysis of PCBs should be used. Another possibility is to examine the exposure to seals, fish, crabs, or clams to determine the impact on wildlife. Preferably crabs and clams, since they live in the sediments. Water samples is another possibility to continue the study. The results are inconclusive, so further studies are needed to achieve reliable results. The sampling should have taken place at another location than the harbour for the Ringhals intake. Since this location most likely do not represent what can be found closer to the intake, further from the harbour. The harbour near Ringhals intake is in a small bay, probably with limited water exchange, causing the levels released by the boats to increase. If sediment samples are to be acquired again, the Ringhals intake should not be taken from the small harbour but closer to the actual intake, on open water. The congener profile found in the harbour is probably due to the activity in the harbour. This since a similar profile is present in all sites 27

33 except for Ringhals intake, suggesting that the release of Aroclor is due to the activity in the harbour. 28

34 6. References Ali, M. & Sreekrishnan, T.R., 21. Aquatic toxicity from pulp and paper mill effluents: A review. Advances in Environmental Research, 5(2), pp Bhuiyan, M.N.I. et al., 29. Constituents of Peel and Leaf Essential Oils of Citrus Medica L. Journal of Scientific Research, 1(2), pp Chiarenzelli, J. et al., Volatilization of polychlorinated biphenyls from sediement drying at ambient conditions. Chemosphere, 33(5), pp Edgren, M., Olsson, M. & Reutergårdh, L., A one year study of the seasonal variations of sddt and PCB levels in fish from heated and unheated areas near a nuclear power plant. Chemosphere, 1(5), pp Elforsk, 29. Miljöeffecter av stora kylvattenutsläpp - Erfarenheter från de svenska kärnkraftverken, Eriksson, S.G., 215. Personal communication. Fine Organics, 212. Material safety data sheet - Erucamide., pp.1 8. Forsman, A. & Edlund, L.-E., Viskan. Available at: [Accessed July 1, 215]. Google Maps, 215. Google maps. Hawthorne, S.B. et al., 2. Comparisons of Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction for environmental solids: Recovery, selectivity and effects on sample matrix. Journal of Chromatography A, 892(1-2), pp Jurado-Sánchez, B., Ballesteros, E. & Gallego, M., 213. Comparison of microwave assisted, ultrasonic assisted and Soxhlet extractions of N-nitrosamines and aromatic amines in sewage sludge, soils and sediments. Science of the Total Environment, , pp Available at: Lau, C. et al., 27. Perfluoroalkyl acids: A review of monitoring and toxicological findings. Toxicological Sciences, 99(2), pp Murphy, B.L. & Morrison, R.D., 27. Introduction to environmental forensics 2nd ed., Elsevir Inc. Nationalencyklopedin, Valthornssnäcka. Nationalencyklopedin. Available at: [Accessed June 21, 215]. Naturvårdsverket, Rapport 4919: Bedömningsgrunder för miljökvalitet Kust och hav, 29

35 Parera, J., Santos, F.J. & Galceran, M.T., 24. Microwave-assisted extraction versus Soxhlet extraction for the analysis of short-chain chlorinated alkanes in sediments. Journal of Chromatography A, 146(1-2), pp Ratia, H. & Oikari, a., 214. Vertical distribution of AhR-activating compounds in sediments contaminated by modernized pulp and paper industry. Water Research, 5, pp Available at: Secretariat of the Stockholm Convention, What are POPs? Sobek, A. et al., 215. Baltic Sea sediment records: Unlikely near-future declines in PCBs and HCB. Science of The Total Environment, , pp Available at: Stibe, L., , personal communication. Södra Cell Värö, 215. Massafabrik i ständig utveckling. Available at: [Accessed June 2, 215]. Södra Cell Värö, 214. Välkommen till Södra Cell Värö - En av världens största och mest moderna massaindustrier, United Nations, Stockholm convention. US EPA, Aroclor plot EPA. Available at: [Accessed July 3, 215]. US EPA, EPA method 354c., pp.1 8. Vasile, C., 2. Handbook of Polyolefins (2nd Edition) 2nd ed., New York: CRC Press. Vattenfall, 212a. Ringhals The largest Nordic power plant, Vattenfall, 212b. Technical information on Ringhals, Wiberg, K. et al., 213. Managing the Dioxin Problem in the Baltic Region With Focus on Sources To Air and Edible Fish, Wulff, F. et al., A Mass-Balance Model of Chlorinated Organic Matter for the Baltic Sea: A Challenge for Ecotoxicology. AMBIO, 22(1), pp Available at: 3

36 Appendix A- Batch 1, without minisilica Toluen LRH_15527_ TIC 6.53e Figure 25: TIC from Toluene Blank LRH_15527_ TIC 5.11e Figure 26: TIC from Soxhlet blank 31

37 R1 LRH_15527_ TIC 2.71e Figure 27: TIC from Ringhals input R2 LRH_15527_ TIC 2.8e Figure 28: TIC from Ringhals output 32

38 VB1 LRH_15527_ TIC 3.35e Figure 29: TIC from Södra Cell Värös input VB2 LRH_15527_ TIC 3.19e Figure 3: TIC from Södra Cell Värö output 33

39 F LRH_15527_ TIC 3.54e Figure 31: TIC of sample from the bay 34

40 Appendix B: Batch 1 with minisilica Toluen LRH_15528_ TIC e Figure 32: TIC of toluene blank Blank LRH_15528_ TIC 3.65e Figure 33: TIC of Soxhlet blank, after use of minisilica 35

41 F LRH_15528_ TIC 3.63e Figure 34: TIC of sample from the bay, after the use of minisilica 36

42 Appendix C: Batch 2 Toluene LRH_15613_ TIC 2.31e Figure 35: TIC of toluene Blank LRH_15613_ TIC 4.93e Figure 36: TIC of Soxhlet blank from batch 2 37

43 F LRH_15613_ TIC 6.7e Figure 37: TIC of sample taken in the bay from batch 2 38

44 Appendix D NIST Database matches and analyses Figure 38: NIST database match for Ringhals batch 1, min The NIST database suggested 2,3-dihydro-1,1,3-trimethyl-1H-indene for the mass spectra generated at 6.26 min (Fig. 38). The spectra from the NIST library resembled the spectra obtained, but there were small variations between the reference and the sample spectra. The other hits from the NIST library were also resembling the mass spectra from the sample, therefore an identification was not possible. The conclusion that can be drawn was that the structure most likely have an indene, or an aromatic structure. Many of the suggested matches from the NIST library all contain indene, benzene, or naphthalene as the suggested backbone for the structure. The proposed analyte most likely have an aromatic structure, with a methyl substitution. 39

45 Figure 39: NIST database match for Ringhals batch 1, min The suggested molecule for the mass spectra at minutes is chlorocycloheptane (Fig. 39). Many of the peaks in the mass spectra are again not matching, there are additional peaks in both the reference spectra and in the sample spectra. The suggested match by the NIST library seemed poor and it could not be used to determine the structure of the compound. The series of m/z 55, 69, 83, 97, 111 suggest an alkene, cycloalkane, alkenyl-, cycloalkyl carbonyl, cyclic alcohol, ethers, or cyanides. The presence of chlorine seems to be unlikely since no clorine clusters are present in the samples. The peak at 83 should be more prominent if the structure suggested by NIST is true. In this spectra, the settings of the MS should have been different. If the scanning range included m/z 35 and 37, the presence of chlorine would have been apparent and the analysis of the spectra would have been simplified. The suggested analyte is most likely an alkene, possibly with a cyclo configuration. 4

46 Figure 4: NIST database match for Ringhals batch 1, min The NIST database suggestion for the peak at minutes was 2,3,4,6-tetramethyl-bibenzyl (Fig. 4). The peak at m/z 91 confirms the possible presence of a benzene structure in the molecule. The first two matches from the NIST library are of two bibenzyl and the third and fourth match consists of a benzene structure. When examining the delta plot, there are some differences indicating that the suggested molecule may not be the best possible match. The sample spectra have a possible molecular ion at m/z 238, but there is an additional peak at m/z 223. The additional peak at m/z ratio 223 may indicate the loss of a methyl group, this could be possible with either of the structures, since there are methyl groups on the first four spectra matches from the hit list. The analyte is probably bibenzyle, with unknown functional groups. The structure probably contains at least one benzene ring. 41

47 Figure 41: NIST database match for Ringhals batch 1, min The peak with retention time min is suggested to be erucylamide (Fig. 41). The delta plot show some discrepancies between the sample and the NIST reference spectra. The differences are minimal for large parts of the delta plot, but some key peaks are missing from the sample spectra, such as the peak between m/z 8 and 9, where around 6 of the peak is unaccounted for. The main peaks present in the sample are m/z 55, 83, 69, 97, and 112, this suggests an alkene, cycloalkane, alkyl, cycloalkyl carbonyl, cyclic alcohol, or an ether. The delta plot shows too large differences between the spectra. The peak with m/z 72 and 126 is not a part of the sample spectra. The erucylamide is a tentative identification, but the uncertainty of the match is too high to provide any reliable results. 42

48 Figure 42: NIST database match for Ringhals batch 2, min For the peak with retention time min, the mass spectra from min have been used for comparison to the NIST database (Fig. 42). The NIST library s best match is an n-methyln-phenyl benzamine. When comparing the sample spectra and the reference spectra to each other, some differences become apparent. The match is of poor quality with many additional peaks near the lower m/z ratios, but nearly all of the main peaks are present, and it is possible that the peak suffers from impurities can account for the additional peaks. The sample spectra obtained have many peaks that is not present in the NIST reference spectra, and therefore it is possible that the additional peaks are due to a contaminant that co-eluted with the analyte. Since the mass spectra displays characteristic ions such as m/z 91, and 182, it is highly possible that it is an aromatic compound even with the exact structure is not known. 43

49 Figure 43: NIST database match for Ringhals batch 2, min The structure proposed by the NIST database for the peak with retention time minutes is a 3-(1,1 -biphenyl-4-yl)butanenitrile (Fig. 43). The structure seems plausible, the spectra closely resembles the reference spectra with minor differences. The same conclusion can be drawn from the delta plot, where there are minor difference, the biggest differences is under 2 in the delta plot. It is worth noting that the spectra is not a perfect match. Since there are no standards to analyse with the GC-MS, the conclusion is not definite. To make a definite match, the retention time needs to be compared to the retention time of a standard. 44

50 Figure 44: NIST database match for Ringhals batch 2, 2.48 min The NIST database suggested that the structure is a 1H-indene, 2,3-dihydro-1,1,3-trimethyl-3- phenyl (Fig. 44). The first five matches are the same compound. The spectra obtained from the sample show some additional peaks compared to the NIST reference spectra. The match suggested seems plausible, but the additional peaks present may be due to impurities or the sample spectra might originate from a different compound. The sample spectra does display the characteristics of benzene like structure due to the peak present at m/z 91 and 128. Since The first five matches are the same compound, it is highly possible that the compound is the match suggested by the NIST database. 45