Microsoft Word - Reviderat exjobb Malin Karlsson.docxAnalysis  of  Polycyclic  Aromatic   Hydrocarbons  in  Freshwater  Snails  of   Family  Lymnaeidae  from   Patholmsviken                  
Project  in  Chemistry:  15  HP  
Malin Karlsson 2015-05-29
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Abstract Polycyclic aromatic compounds (PAHs) are a group of organic compounds that are very stable and therefore persistent. They can be pyrogenic or petrogenic and PAHs from petrogenic sources are often enriched with alkylated PAHs while pyrogenic sources often contain more of the parent PAH. In Patholmsviken, a bay located near an abandoned wood- impregnating facility, freshwater snails were collected and analysed for PAH16, alkylated PAHs, oxy-PAHs and azaarenes using GC/MS. The concentrations of PAH16 were compared with previous analyses and the results showed that the levels had declined since 2008 and 2013. The ratio between alkylated PAHs and native PAH coincide with what could be expected from a creosote source which consist of more native PAHs. Only one oxy-PAH could be detected and the levels of alkylated PAHs were low. Freshwater snails seem to be a good bioindicator since they meet many of the desired criteria for a suitable biomonitoring organism.
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Sammanfattning Polycykliska aromatiska föreningar (PAH) är en grupp av organiska föreningar som är mycket stabila och därmed långlivade. De kan vara pyrogena eller petrogena, de petrogena källorna är ofta berikad med alkylerade PAHer medan de pyrogena källorna oftare innehåller mer av icke-substituerade PAHer. I Patholmsviken, en vik som ligger bredvid en nedlagd trä- impregneringsanläggning, har snäckor samlats in och analyserats för PAH16, alkylerade PAHer, oxy-PAHer och azaarener med hjälp av GC/MS. Koncentrationerna av PAH16 jämfördes med värden från två tidigare analyser och resultaten visade att nivåerna hade minskat sedan 2008 och 2013. Endast en oxy-PAH kunde detekteras och nivåerna av alkylerade PAHer var låga. De låga nivåerna av alkylerade PAH överensstämmer med vad som kan förväntas hitta från en kreosotkälla som avger pyrogena PAHer. Snäckor verkar vara lämpliga att använda som bioindikatorer eftersom de uppfyller många av de kriterier som finns för dessa.
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Introduction Objective
The aim of the study is to collect freshwater snails from Patholmsviken, Holmsund, Sweden and then analyse them for different polycyclic aromatic hydrocarbons (PAH); The 16 so called priority pollutants (PAH16) according to the US Environmental Protection Agency (US EPA), alkylated PAHs and oxy-PAHs and azaarenes. The PAH16 content will be compare with values from two previous analyses to see if it has changed (see Appendix A6 and A7). Can the source be determined to be pyrogenic or petrogenic? A literature search will be done to find more information about the snails that can be found at Patholmsviken. Also a literature study will be done to search for answers to the following questions:
• What are the advantages with using an aquatic organism as an indication of pollution? • What makes an organism suitable as a bioindicator? • Is freshwater snail a good indicator of pollutants?
Bioindicators and Biomonitoring In the beginning of the 20th century Ortmann (1909) observed the animal life in polluted freshwater bodies. He studied both bivalves and gastropods among others. He saw that the bivalves that live in the bottom of the streams where they breathe using water were quite sensitive and died when the pollution increased. Of the gastropods he studied both water breathing and air breathing species and found that the air breathing species, among them Lymnaea, were more resistant to the pollutants. According to Phillips (1980), three advantages with monitoring the pollutant levels in aquatic animals are
§ Many pollutants will bioaccumulate and therefore be found in higher concentrations in the animals then in the surrounding water.
§ Only the bioavailable part of the pollutant will be measured. § If the uptake and excretion rates are known it is possible to make a time-averaged
index of the pollutions. In the literature there are more studies performed on heavy metals and trace elements than there are on organic pollutants (Menta & Parisi, 2001; Coughtrey & Martin, 1977; Mahmoud & Abu Taleb, 2013; Laskowski & Hopkin, 1996). In a study from 1977, Coughtrey & Martin examined the metal uptake in the pulmonate mollusc Helix Aspera. They found a relationship between the size of the snail and the uptake of heavy metals and therefore they drew the conclusion that it is desirable to use snails of the same size for biomonitoring. In Germany a long-term monitoring program is running where three types of terrestrial snails are used. Each year 5 to 10 adult snails of similar size are collected from different monitoring points and analysed for both organic and inorganic pollutants (Oehlmann & Schulte-Oehlmann, 2003). In 2003 Salánki et al. examined how the locomotion of Lymnaea Stagnalis (L. Stagnalis) was affected when exposed to four heavy metals (Hg, Cu, Pb and Sn) both acute and chronically. They observed that depending on the metal the locomotion could be either depressed or stimulated by them. For Pb they first saw a stimulation that later turned into a depression. Their conclusion where that L. Stagnalis can work as an indicator for different heavy metals and could also be applied for other pollutants.
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According to Oehlmann & Schulte-Oehlmann (2003) molluscs have a number of characteristics that make them suitable as bioindicators: Both gastropods and bivalves can be found all around the world both in marine and freshwater, some of the gastropods can also be found in terrestrial environments. Some of the species can even be found on different continents so this facilitates comparison between different countries. Since molluscs lack an exoskeleton they will be in direct contact with the ambient surrounding and they will therefore have two pathways for the uptake of pollutants, both from the diet and via absorption through their bodies. This means that they can accumulate pollutants more quickly than species that only take up pollutants via their diet and this can also make them more vulnerable to pollutants. Also many molluscs are important for a functioning ecosystem, so large pollution that can affect a mollusc population can further affect other parts of that system. Many gastropods are quite situated in their habitat so the population in a certain bay will represent the contamination in that area well. Bivalves are more widely used as bioindicators than gastropods. In 1986 USA introduced the “Mussel Watch” which is a biomonitoring program that analyses both biological and chemical contaminants in the Great Lakes and the US coastal waters (Kimbrough et al. 2008). By doing this they can see long-term changes in the environment. In 2010 Losso & Ghirardini published an overview of different ecotoxicological studies that have been performed in the Venice lagoon. Among the different bioindicators used mytilus galloprovincialis, crassostrea gigas, tapes philippinarum, scapharca inaequivalvis and cerastoderma glaucum have been used, all members of the bivalve family. Another way to biomonitor aquatic pollutants is by using different semi permeable membrane devices (SPMD). These are constructed so that they will mimic the uptake in aquatic organisms. In a study from 2001, Baussant et al. compared the uptake and excretion between a passive sampler: semipermeable membrane device (SPMD) and two aquatic species: the blue mussels Mytilus Edulis and the turbot Scophthalmus Maximus. After an eight day long exposure period of 1 mg/L the PAH profiles for SPMD and the blue mussels showed a good correlation with the seawater. After an elimination period of 10 days the PAH levels in the fish were back at the background level. For the mussels the concentration had dropped to 63% of the levels that could be measured after the exposure period and for the SPMD it had dropped to 55%.
Molluscs Molluscs are divided into seven different classes where gastropods and bivalves make up the larger part, 80% and 15% respectively (Oehlmann & Schulte-Oehlmann, 2003) The bivalves are characterized by being enclosed within a pair of shells while the gastropods have one part that is enclosed within the shell and one part that it outside the shell that is used for locomotion and feeding (Barnes et al. 1988). Most of the gastropods have an asymmetrical shell that serves as a retreat that can be used for protection (Ruppert & Barnes, 1994). Most molluscs are found in the marine environment but they have also spread to freshwater and terrestrial environments. Pulmonata is a subclass of the gastropods and it contains both land snails and freshwater snails, among them the family Lymnaeidae, which can be found all around the world. They have their organs located on the right side of their body and also their lung that is developed from the mantle cavity. The mantle cavity has become almost completely sealed to the back of the snail except for a small opening at its right side called the pneumostome. And since they have developed lungs their gills have disappeared and the roof of the mantle cavity has become much vascularised (Ruppert & Barnes, 1994). Most snails feed by crawling over a food source while stuffing food into their mouth. Lymnaea have a
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cuticle-covered jaw that can aid them when ripping apart larger particles (Dillon, 2000). The Lymnaeidaes have a number of eggs that can be laid throughout the summer. They are hermaphrodites but when mating one individual will act as a female and the other one as a male and their egg-laying period starts in April or May and last until the end of summer (Dillon, 2000).
Lymnaea Stagnalis
L. Stagnalis is one of seven species that can be found in Sweden where it inhabits ponds, lakes and rivers (Kemenes & Benjamin, 2009). Their life expectancy is 2-5 years according to Ted von Proschwitz. Individuals that live in a moderate climate will feed during May to October and their diet consists mainly of algae but they can also feed on macro vegetation but also dead organic materials. During the summer Lymnaea Stagnalis can use its lung to breathe atmospheric air (Meshcheryakov, 1990). If it needs to it can also fill its pulmonary cavity with water and breathe by using the oxygen dissolved in the water. During the winter it will use skin respiration instead and by doing that it can dig itself down into the ground (Meshcheryakov, 1990). According to von Proschwitz it is hard to find these snails in shallower water when the temperature is low since they don’t go up to the surface to fill their pulmonary cavity until later in the spring. They are ready to reproduce when they are around one year old and they can lay their eggs several times during a summer.
Stagnicola Sp
Stagnicola Sp consists of three related species of Lymnaeidae snails. To tell them apart an anatomically examination has to be observed. They do not live as long as L. Stagnalis, 1-2 years is most common according to von Proschwitz. They are ready to reproduce when just before they reach one year. Their diet consists mostly of plants and dead organic materials but also algae.
Polycyclic aromatic hydrocarbons Origin and Chemical Properties of PAHs
Polycyclic aromatic hydrocarbons, PAHs, are a group of organic compounds that are very stable when bound to particles and therefore persistent in the environment. They can also bioaccumulate in some living organism e.g. molluscs (Wenning & Martello, 2014). But for organisms higher up in the food chain this will not happen, both humans and other predators like fishes have the ability to break down PAHs (Jakoby, 1982; Baumard, 1998). The PAHs does not fulfil all the criteria to be in the Stockholm conventions list of persistent organic pollutants (POPs) since they do not bioaccumulate in all organisms (Kemikalieinspektionen, 2006). However, they are listed in the protocol to the 1979 convention on longe-range transboundary air pollution on persistent organic pollutants (LRTAP POP) how action can be taken to lower the PAH produced during different processes for example coke production (United Nations, 1998). They are made of two or more aromatics that have hydrogen or alkyl groups attached to it. Heavier PAHs are considered to be immobile because of their large size, their low solubility in water and low volatility. The US EPA has determined 16 different PAHs that are so called priority pollutants and among them seven are considered to be carcinogen to mammals, see figure 1 (ATSDR, 1995).
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Figure 1. Structure of US EPAs 16 priority PAHs.
PAHs are a result of incomplete combustion of organic compounds, this means that they can be both naturally and anthropogenic. Natural sources are diagenesis at low temperature, formation of petroleum and coal, incomplete combustion at moderate to high temperature, e.g. forest fires, or biosynthesis. Among the anthropogenic sources are heating by different fuels, e.g. petroleum, wood, coal or natural gas. Each combustion source gives rise to a specific fingerprint depending on how the distribution of the different PAHs looks like (Wenning & Martello, 2014). Apart from being naturally or anthropogenic they can also be classified as pyrogenic or petrogenic and the main difference between these two groups are the temperature during the formation (Murphy & Morrison, 2006). It is common to find PAHs with one or more alkyl groups attached to it and these PAHs are referred to as alkylated PAHs. To distinguish the different levels of alkylation they are often classified in groups depending on how many alkyl carbons they contain, e.g. methylnaphthalene is called C1-naphthalene while ethylpyrene will be named C2-pyrene, see figure 2 (Murphy & Morrison, 2006). Petrogenic sources are often enriched with alkylated PAHs while pyrogenic sources often contain more of the parent PAH (Murphy & Morrison, 2006).
Figure 2. C1-naphthalene and C2-pyrene. (Murphy & Morrison, 2006).
Since the PAHs have a wide range in molecular weight their properties will also differ within the group. In general the water solubility will decrease for heavier PAHs while the boiling point, melting point and the octanol/water partitioning coefficient (log Kow) will increase. Low molecular weights PAHs are more volatile than the heavier ones (Wenning & Martello, 2014). Naphthalene is the most volatile PAH and it will be more present in the air than in the
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water or soil (Naturvårdsverket, 2007). Because of PAHs low water solubility they will tend to adsorb to particles or sediment in aquatic environments. PAHs can enter the water column via sewage water or with precipitation among other things. Also it is possible that PAHs from contaminated soils can reach the ground water and end up in the surface water. Once in the water system they will tend to adsorb to the sediment due to its hydrophobicity, but also in suspended particles in the water column and in aquatic organisms. The half-life time for PAHs in sediment are between 0.2-5 years (Wenning & Martello, 2014). Two other types of polycyclic aromatics are oxy-PAHs and azaarenes. Oxy-PAHs will be created from incomplete combustion when there is oxygen present and azaarenes when nitrogen is present, and since both of these elements are found in the atmosphere they will always be created when incomplete combustion occurs in the atmosphere. Another way for oxy-PAHs to be created is by oxidation of PAHs, either chemical or biological processes that can occur both in soil and in water (Lundstedt et al., 2007). Oxy-PAHs are PAHs that have been substituted with a ketone group while azaarenes have a nitrogen atom incorporated in their aromatic structure, see figure 3.
Figure 3. The oxy-PAH 1-indanone to the left and the azaarene acridine to the right.
Since these types of compounds contain electronegative elements they can have a small dislocation of charge within the molecule. This will lead to higher water solubility then what is seen for PAHs. Another thing that is special with the azaarenes is that depending on how the nitrogen atom is bound within the molecule they can show either acidic or basic properties (Herod, 1998). This means that they can interact with the surrounding in ionic form.
Metabolism of PAHs
When PAHs enter the body the main metabolic pathway is by an enzyme known as cytochrome P450, also known as mixed function oxidase (MFO). The main function for this system is to make compounds that are poorly water-soluble more soluble so that they can be excreted more easily. It will oxidise NADPH to NADP+ so that the following reaction occurs (Jakoby, 1982): RH + O2 → ROH + H2O where R is a chemical like, PAHs. Thus, when PAHs are metabolised by P450 a functional group such as –OH, -NH2, and - COOH will be added to them (Sette et al., 2013). Both molluscs and crustaceans will tend to bioaccumulate PAHs and other lipophilic compound in their hepatopencreas or digestive gland (Walker & Livingstone. 1992). In molluscs the P450 system is mainly located in the digestive gland, while it for different fish species can be found in the organs that are directly exposed to the surrounding environment like the gills and intestines (Eisler, 1987). The presence and activity of P450 are generally lower in molluscs than in fishes, which is why PAH can bioaccumulate in molluscs while it’s rapidly broken down in fish (Oehlmann & Schulte-Oehlmann, 2003; Stegeman & Lech, 1991, Livingstone, 1998). This means that PAHs will not biomagnify in the same extent higher up in the food chain when it comes to
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aquatic organisms. Baumard et al. (1998) analysed PAHs in different marine organisms and found that high molecular weight PAHs were more present in mussels than in fishes. The fishes had mostly low molecular weight PAHs in their tissue. This could be due to the fact that they can metabolise high molecular weight PAHs better than the molluscs (Baumard et al., 1998).
History about Patholmsviken Patholmsviken is a bay located next to the road E12 in Holmsund, Umeå municipality, see figure 4. The area north of Patholmsviken has been used for wood impregnation since 1944 (Umeå Kommun, 2014). In the beginning they used a method called Bolidenmetoden, which used arsenic salt but later the facility expanded and impregnation with creosote came in use in 1953 (Karlsson & Sjöström, 2008). From 1953 until 1976 they shifted between these two impregnation techniques. After 1976 and until the closedown in 1981 only arsenic salt were used. The ground were examined and partly sanitised in 1983 and in 2012 a major remediation were performed (Umeå Kommun, 2014). Nowadays a marina is located in the bay.
Figure 4. Map showing Patholmsviken, Holmsund.
Creosote
Creosote is a dark coloured oily liquid that can be made from either coal tar or wood tar. The wood tar derived creosote has mainly pharmaceutical uses while the coal tar creosote can be used to impregnate wood, use for example as railway ties. This type of creosote can contain around 85% PAH, for example acenaphthene, anthracene, fluorene, phenanthrene and pyrene (Murphy & Brown, 2005). Creosote will contribute with pyrogenic PAHs (Murphy & Brown, 2005).
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Method Material
The composition of the different standards is listed under each analysis together with their trace in table A1 and in table A.2 they are also listed together with purity grade and supplier.
Field work Equipped with waders and rubber gloves, Malin Karlsson and Cecilia Hagberg collected the snails between the 22th and 24th April 2015 from one site called Patholmsviken in Holmsund. The area can be seen as red lines in figure 2.1 and the coordinates can be found in table 2.2. The coordinate system used to present them is WGS84 decimal (lat, lon). The snails were collected from rocks and other debris in the water see figure 5. Most of the snails were found on the backside of stones when turning them.
Table 1. The coordinates for where the fieldwork was carried out.
Coordinates   Patholmsviken West: 63.699569, 20.358036 East: 63.699384, 20.359261
To avoid contamination the snails were collected in a floating metallic sieve and later stored in glass containers. Two types of snail were collected, Stagnicola Sp and Lymnaea Stagnalis. Stagnicola Sp is a group name, which consists of three related species. The only way to tell them apart is by doing an anatomical examination. Ideally all the collected snails would have been in the same size but since there were hard to gather enough snails there was not possible to do any size exclusion. The collection work were carried out in the shallowly water approximately 0 - 0.6 m depth. After the snails had been collected they were left in the glass container to empty their stomachs, called depuration. This step should have been 12-24 h long but due to lack of time and travel back to Orebro the time window were extended to 72-120 h. When back at Örebro University the snails were stored in a freezer.
Figure 5. The red lines represent the site from where the snails were collected in Patholmsviken.
©Lantmäteriet/Metria
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Sample Preparation All utensils used throughout the method were washed in three steps with ethanol (grade: absolute, VWR, Radnor, USA), n-hexane (supersolv, Merck chemicals, Darmstadt, Germany) and dichloromethane (for analysis of dioxins, furans and PCBs, Fluka, Steinheim, Germany). First the frozen snails were separated from their shell using metallic tweezers, 30,08 g wet weight (w/w) were collected. The snails were then stored in a refrigerator (8°C) until the next day. Before the homogenisation the sample was divided into three parts; mPCB weighed 9.9398 g, mR1 weighed 9.9072 g (replicate 1) and mR2 weighed 6.4342 g (replicate 2). Replicate 1 and 2 were used for PAH analysis and the last portion was taken out and used for PCB analysis in another experiment. The replicates were hereafter recalled as samples. The samples were homogenised by using a mortar, for each sample 5 times the wet weight (w/w) of anhydrous sodium sulphate (Na2SO4) (ACS reagent, Sigma-Aldrich, Steinheim, Germany) were added into the mortar, this was done to get rid of the water content. Next step was the fat content determination. First a piece of glass wool (3950 Fiberglass, 8 micron, Corning, New York, USA) was put into two columns to prevent the sample from slipping through. Then the two samples were added into each column. The samples were spiked by adding 20 µl internal standard (IS) PAH16 (200 ng) onto the samples in the column. The lipids were eluted using a solution made of n-hexane and dichloromethane in a 1:1 relationship, and the volume was four times the column height. The eluate was collected in a pre weighed flask. The solvent was evaporated using a rotary evaporator (Laborota 4002, Heidolph rotavac valve control pump) with a water bath (Büchi waterbath B-480) temperature of 45°C and to prevent photo degradation the flask was covered with aluminium foil. First the vacuum was set to 1000 mbar so that the dichloromethane would disappear and then it was lowered to 340 mbar and this was done to evaporate the n-hexane. The flasks were weighed until the weight was constant and only the fat content (FC) remained in the flasks. To determine the fat content in the original samples equation 1 was used: FC (%) = 100 * fat weight / sample weight (w/w) (Eq 1) For further clean up the extracts, 10% deactivated silica columns were used. The deactivated silica was prepared by following steps; 30 g silica gel (High purity grade 7734, pore size 60 Å, 70-230 mesh, Sigma-Aldrich, Steinheim, Germany) was transferred into a flat-bottomed flask and sat in an oven at 550°C over night so the water content evaporated. The gel was then store in another oven at 105°C until use. To deactivate the silica 10 % of deionised water was added and the flask was left on a shaker for 2 h to let the water equilibrate with the silica gel. When not using the deactivated silica it was left in a flask with glass lid in a desiccator and the lid was also covered with Para film to avoid loss of water from the silica gel. The columns were prepared as followed; A piece of glass wool was put into the end of a column with Ø = 1 cm then the column was washed in the three steps previously mentioned. Ten grams of silica gel was packed in the column and on top of it about 2 cm of Na2SO4 was added and then 40 ml n-Hexane was used to pre wash it. When the solvent level just reached the top of the Na2SO4 layer the samples were added with the help of a pasteur pipette. The amber glass vial was washed three times with n-hexane, which also was transferred to the column. When the extract had reached the top of the Na2SO4 level, 100 mL of n-hexane was used to elute the samples and a 250 mL flask was used to collect it. The rotavapor was used to evaporate the samples to about 1 mL and then it was transferred to an 8 mL amber glass vial. To reach a sample volume of approximately 0,5 mL it was put under nitrogen gas to vaporise then they were left in a freezer (18°C) until analysis.  Hamilton syringes (10 µL, 25µL, 50 µL and 500 µL) was used to spike the two samples for analysis. First 10 µl (200 ng) perylene D-12 was
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added as a recovery standard (RS). Then 25 µl (50 ng) IS 3 mix for methylated PAHs was added and also 50 µl (50 ng) 3 IS for oxy-PAHs and azaarenes. For PAH16 a four-point calibration was made. The amounts used for these calibration standards (CS) are presented in table 2. Table 2. The following calibration standards (CS) were prepared for the analysis of PAH16.
PAH16  
Compound CS1 2 ng CS2 20 ng CS3 200 ng CS4 500 ng
PAH Mix 1 µl (2 ng) 10 µl (20 ng) 10 µl (200 ng)
25 µl (500 ng)
ng) 20 µl (200
ng) 20 µl (200
ng) 20 µl (200
ng) RS Perylene D12
10 µl (200 ng)
10 µl (200 ng)
10 µl (200 ng)
10 µl (200 ng)
Toluene 369 µl 360 µl 370 µl 345 µl Total volume 400 µl 400 µl 400 µl 400 µl Final concentration PAHs
0.005 ng/µl 0.05 ng/µl 0.5 ng/µl 1.25 ng/µl
In table 3 the amount for the CS for alkylated PAHs is presented. It was a five-point calibration were their concentrations varied from 10 ng to 1000 ng.   Table 3. The following CS’s were prepared for the analysis of alkylated PAHs.
Alkyl  PAHs  
Compound CS1 10 ng CS2 50 ng CS3 250 ng CS4 500 ng CS5 1000 ng
PAH/Dibenzothiophen es Mixture (2, 20ng/ul) 5 µl (10 ng) 25 µl (50 ng) 13 µl (260
ng) 25 µl (500
ng) 50 µL (1000
ng)
Metyl PAH6-mix ( 2, 20, 200 ng/µl) 5 µl (10 ng) 25 µl (50 ng) 13 µl (260
ng) 25 µl (500
ng) 50 µL (1000
ng) 2,3- Dimethylanthracene 2, 20ng/ul
5 µl (10 ng) 25 µl (50 ng) 13 µl (260 ng)
25 µl (500 ng)
50 µL (1000 ng)
25 µl (500 ng)
25 µl (500 ng)
RS Perylene D12 25 µl (500 ng)
25 µl (500 ng)
25 µl (500 ng)
25 µl (500 ng) 25 µl (500 ng)
Toluene 1185 µl 1125 µl 1161 µl 1125 µl 1050 µl
Total volume 1250 µl 1250 µl 1250 µl 1250 µl 1250 µl
Final concentration PAHs 0.008 ng/µl 0.04 ng/µl 0.2 ng/µl 0.4 ng/µl 0.8 ng/µl
The calibration curve for oxy-PAHs was already prepared, see table 4, it was a three-point calibration and it ranged from 5 to 500 ng.
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Table 4. The composition of the Oxy-PAH standards.
Oxy-­PAHs   Compound CS1 5 ng CS2 50 ng CS3 500 ng
Oxy-PAH Mix 4.5 ng 67.5 ng 450 ng
Dinaphtho[1,2-b;1',2'- d]furan; 11H- benzo[a]carbazole
5 ng 50 ng 500 ng
IS PAH16 50 ng 50 ng 50 ng
RS Perylene D12 50 ng 50 ng 50 ng
GC/MS analysis A quantification of the 16 priority EPA PAHs were done. Also the concentrations of oxy- PAHs and azaarenes were quantified but since the samples were spiked with oxy-PAHs and azaarenes after the clean up there might have been a loss of these compounds and it is not possible to see how great this loss is so the concentrations of oxy-PAHs and azaarenes that were calculated in the samples were probably underestimated. The gas chromatography-mass spectrometry (GC/MS) used was a low resolution GC/MS (LRGC-MS) with the following specifications: gas chromatography (GC) System: Agilent 7890A, mass spectroscopy (MS) System: Agilent Technologies 5975C inert XL EI/CI MSD with triple-axis detector Agilent Technologies 7693 Autosampler. See table 5 for details about the PAH16 analysis, table 6 for details about alkylated PAHs and table 7 for oxy-PAHs and azaarenes. Since no process blank were used during the experiment the limit of detection (LOD) for the compound were determined by integrating a noise peak close to each peak in the sample in MassLynx and thereafter take that concentration three times. To minimize any matrix effect the chromatograms for the samples were used when determining LOD instead of the standards. As further quality control the sample was divided into two replicates. This makes it easier to detect if something goes wrong with the analysis since the concentration in the two replicates should be equal to each other. Also a known amount of internal standards were added before the clean-up and this makes it possible to see how much sample that might have been loss during the steps of the experiment. The relative response factor (%RRF), relative standard deviation (%Dev) and r2 values for the calibration curves can be found for all the three samples in tables A2-A4 in appendix.
GC/MS-method PAH16
The conditions used for the analysis of PAH16 can be seen in table 5. The column used was a select PAH which is suitable for PAH16 analysis since it has the capacity to separate each peak. Helium was used as carrier gas and splitless injection was used. The initial temperature were 70°C which were held for 2 minutes, thereafter the temperature were risen in intervals of 40°C/min until it reached 108°C. Next the temperature were increased with 7°C/min until 230°C, this temperature were then held for 7 min. Then it reached 280°C which were held for 10 minutes by increasing the temperature by 20°C/min. In the last step it rose with 5°C/min to 325°C and this were held at 7 min.
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GC/MS  Conditions  for  PAH16   Technique GC/MS
Column Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462, Agilent, Santa Clara, USA)
Sample Concentration 0.005 - 1.25 ng/µl Injection Volume 1 µl
Temperature Program 70°C (2min), 40°C/min to 108°C (0 min), 7°C/min to 230°C (7 min), 20°C/min to 280°C (10 min), 5°C/min to 325°C (7 min)
Carrier Gas Helium, constant flow 2 mL/min Injector 250°C, Splitless mode, 1 min @ 50 mL/min
Mass Detector EI in SIM mode, ion source 230°C, transfer line 300°C
SIM Parameters (Mass, Dwell time)
(128.00, 30) (136.00, 30) (152.00, 30) (154.00, 30) (160.00, 30) (164.00, 30) (166.00, 30) (176.00, 30) (178.00, 30) (188.00, 30) (190.00, 30) (202.00, 30) (212.00, 30) (216.00, 30) (228.00, 30) (240.00, 30) (252.00, 30) (264.00, 30) (276.00, 30) (278.00, 30) (288.00, 30) (292.00, 30) (302.00, 30)
GC/MS-method Alkylated PAHs
The conditions used for the analysis of alkylated PAHs can be seen in table 6. The same column was used for alkylated PAHs as for PAH16. Helium was used as carrier gas and splitless injection was used. The initial temperature 90°C were held for 1 min then it increased with 8°C/min up to 300°C. This temperature were held for 4 minutes before further increasing it in intervals of 25°C/min until the final temperature 325°C that was held for 1 minute. Table 6. The conditions for GC/MS analysis of PAH16.
Conditions  for  alkylated  PAHs   Technique GC/MS Column Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462) Sample Concentration 0.008 - 0.8 ng/µl Injection Volume 1 µl
Temperature Program 90°C (1min), 8°C/min to 300°C (4 min), 25°C/min to 325°C (1min)
Carrier Gas Helium, constant flow 2 mL/min Injector 250°C, Splitless mode, 1 min @ 50 mL/min
Mass Detector EI in SIM mode, ion source 230°C, transfer line 300°C
SIM Parameters (Mass, Dwell time)
(142.00, 30) (152.00, 30) (156.00, 30) (170.00, 30) (184.00, 30) (192.00, 30) (198.00, 30) (204.00, 30) (206.00, 30) (212.00, 30) (216.00, 30) (220.00, 30) (226.00, 30) (228.00, 30) (242.00, 30) (252.00, 30) (256.00, 30) (264.00, 30) (266.00, 30)
GC/MS-method Oxy-PAHs and Azaarenes
In table 7 the running conditions for the analysis of oxy-PAHs and azaarenes are presented. The same column was used for oxy-PAHs and azaarenes as for PAH16. Helium was used as carrier gas and splitless injection was used. The initial temperature were 70°C and it was risen
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with 8°C/min up to 205°C and held for 2 minutes. Then the temperature were further increased in intervals of 8°C/min up to 250°C here the incline were lowered to 3°C/min up to 270°C which were held for 2 minutes. Next increase were 9°C/min up to 279°C and from there it were increased with 1°C/min up to 280°C and held for 2 minutes. The temperature was then increased 5°C/min to 300°C, held for 1 minute and the final increase were 25°C/min up to the final temperature 325°C which were held for 2 minutes. Table 7. The conditions for GC/MS analysis of oxy-PAHs and azaarenes.
GC/MS  Conditions  for  Oxy-­PAH  and  Azaarenes   Technique GC/MS
Column Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462)
Injection Volume 1 µl
Temperature Program 70°C, 8°C/min to 205°C (2 min), 8°C/min to 250°C, 3°C/min to 270°C (2 min), 9°C/min to 279°C, 1°C/min to 280°C (3 min), 5°C/min to 300°C (1 min), 25°C/min to 325°C (2 min)
Carrier Gas Helium, constant flow 2 mL/min Injector 250°C, Splitless mode, 2 min @ 50 mL/min
Mass Detector EI in SIM mode, ion source 230°C, transfer line 300°C
SIM Parameters (Mass, Dwell time)
Group 1: (129, 30) (132, 30) (167, 30) (175, 30) (179, 30) (180, 30) (188, 30) (204, 30) (208, 30) (216, 30) (217, 30) (222, 30) Group 2: (217, 30) (230, 30) (236, 30) (254, 30) (258, 30) (264, 30) (268, 30) (270, 30) (279, 30)
Results Fat content in freshwater snails
The fat content in the original sample were calculated to be 1.2%.
GC/MS analysis PAH16 in freshwater snails
The plan was to use a four-point calibration but since one of the calibration standards deviated from the calibration curve that point was excluded. The average concentrations for the individual PAHs can be seen in table 8. Except for PAH16 four more PAHs were analysed together with the alkylated PAHs and those four are also presented in table 8. Both the PAH concentration based on fat content and wet weight in µg/kg is presented. Four compounds were below LOD they are presented as below the calculated LOD value in table 8. It was the two low molecular weight PAHs naphthalene and acenapthylene and also the two high molecular weight PAHs dibenz(ah)anthracene and Benzo(j)fluoranthene. For the other PAHs the concentrations for the wet weight ranged between 1.8 µg/kg to 48 µg/kg, which equals to 150 µg/kg to 6100 µg/kg for the fat content. Fluoranthene contributed with the highest levels followed by pyrene and phenanthrene.
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Table 8. Average concentrations of PAH in the samples, expressed as wet weight and fat content (µg/kg).
PAH  Concentrations  (µg/kg)a  
    Wet weight (w/w)
Fat content
Naphtalene <0.93 <79 Acenaphtylene <0.31 <24 Acenaphtene 8.6 740 Fluorene 16 1400 Phenanthrene 31 2700 Anthracene 1.8 150 Fluoranthene 71 6100 Pyrene 48 4100 Benzo(a)antracene 11 910 Chrysene 24 2100 Benzo(b)fluoranthene 14 1200 Benzo(k)fluoranthene 8.9 750 Benzo(a)pyrene 3.4 290 Indeno(1,2,3-cd)pyrene 1.9 160 Dibenz(ah)anthracene <1.0 <85 Benzo(ghi)perylene 2.4 210 Benzo(c)phenanthrene 3.0 260 Benzo(j)fluoranthene <2.3 <200 Benzo(e)pyrene 5.8 510 Perylene 0.88 77 a Values below LOD are presented as lower then the calculated LOD. The measured concentrations for PAH16 expressed as wet weight can be compared with previous results from 2008 (see A6) and 2013 (see A7) presented in table 9. The values are also presented as the sum of all 16 compounds; values below LOD are not included in the sum. The values for year 2008 are before the remediation while the other two measurements are done after it. Table 9. The PAH16 concentration for the individual compounds and also the summarised value from 2008, 2013 and 2015 in µg/kg.
PAH16  concentrations  (µg/kg  w/w)a   2015 2013 2008 Naphtalene <0.93 8.2 <5 Acenaphtylene <0.31 28 17 Acenaphtene 8.6 95 290 Fluorene 16 76 290 Phenanthrene 31 240 730 Anthracene 1.8 170 51 Fluoranthene 71 580 3300 Pyrene 48 360 1900 Benzo(a)antracene 11 85 410 Chrysene 24 46 240 Benzo(b)fluoranthene 14 64 100 Benzo(k)fluoranthene 8.9 20 64 Benzo(a)pyrene 3.4 14 63 Indeno(1,2,3-cd)pyrene 1.9 2 11 Dibenz(ah)anthracene <1.0 6.3 4.1 Benzo(ghi)perylene 2.4 7.1 18 SUM PAH16 240 1800 7500 a Values below LOD are not summarised in SUM PAH16.
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Alkylated PAHs in freshwater snails
The concentrations for alkylated PAHs can be seen in table 10. Eight compounds out of twenty-seven are above LOD in the samples, see table 10. LOD for Triphenylene was higher then for the other compounds. The concentrations for the detectable compounds ranged from 0.88 µg/kg to 11 µg/kg. Table 10. Concentrations for alkylated PAHs expressed as wet weight and fat content (µg/kg).
Alkylated  PAHs  Concentrations  (µg/kg)a   Wet weight Fat content 2-methylnaphthalene <0.48 <41 1-methylnaphthalene <0.37 <32 1,6-Dimethylnaphthalene 1.0 90 2,5,6-Trimethylnaphthalene 1.0 91 Dibenzothiophene 2.7 240 2-Methyldibenzothiophene <1.6 <140 2-Methylphenanthrene <2.1 <180 2-Methylanthracene <1.8 <150 2,8-Dimethyldibenzothiophene <0.54 <46 2,4-Dimethylphenanthrene <1.4 <120 2,4,7-Trimethyldibenzothiophene 1.6 140 2,3-Dimethylanthracene <1.8 <150 1,2,8-Trimethylphenanthrene <0.94 <81 1,2,6-Trimethylphenanthrene <1.4 <120 1-Methylfluoranthene 3.9 340 Triphenylene <25 <2000 7-Methylbenz(a)anthracene 11 950 3-Methylchrysene 9.0 780 2-Methylchrysene <2.4 <200 1-Methylchrysene <1.9 <170 6-Ethylchrysene <2.1 <180 7,12-Dimethylbenz(a)anthracene 1.9 170 7-Methylbenzo(a)pyrene <2.4 <210 a Values below LOD are presented as lower then the calculated LOD. In figure 6 parent PAHs and alkylated PAHs are grouped together to show the relationship between them. This can be used to determine whether the source is pyrogenic or petrogenic. For almost all compounds there is a decrease in concentration from the parent PAH to the alkylated ones.
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Figure 6. PAH profile that shows the distribution of parent PAHs and alkylated PAHs in the sample.
Oxy PAHs and azaarenes in freshwater snails
All compounds but carbazole were below LOD, they are presented as below the calculated LOD value in table 11 together with the concentration for carbazole which had a concentration of 0.48 µg/kg (w/w). Table 11. Concentrations for oxy-PAHs and azareenes, expressed as wet weight and fat content (µg/kg).
Oxy-­PAHs  and  Azaarenes  Concentrations  (µg/kg)a   Wet weight Fat content Quinoline <0.55 <47 1-Indanone <0.26 <22 Benzo(h)quinoline <1.8 <150 Acridine <0.99 <83 Carbazole 0.48 42 9-Fluorenone <1.3 <110 Anthracene-9,10-dione <1.1 <85 4H-Cyclopenta(def)phenanthrene <1.3 <110 2-Methylanthracene-9,10-dione <1.9 <160 11H-Benzo[a]carbazole <13 <990 Benzo(a)fluorenone <4.2 <350 7H-Benz(de)anthracene-7-one <1.3 <110 Benz(a)anthracene-7,12-dione <4.0 <330 Naphtacene-5,12-dione <0.26 <22 Dinaphtho[1,2-b;1',2'-d]furan <7.5 <620 6-H-Benzo(cd)pyrene-6-one <1.6 <140 9,10-Dihydrobenzo(a)pyrene-7-one <3.4 <360 Dibenz(ah)acridine <1.3 <112 a Values below LOD are presented as lower then the calculated LOD.
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QA/QC The recovery for the internal standards in the PAH16 samples can be seen in figure 7. It varied between 28-101% and the low molecular weight PAHs had the lowest recovery.
Figure 7. Recovery for all IS from the samples in the PAH16 analysis.
There were three compounds below LOD for the native PAHs. It was the two low molecular weight PAHs naphthalene and acenaphthylene and the heavier dibenz(ah)anthracene. The r2 values for the calibration curves for PAH16 were between 0.993-0.999 (see A3). According to US EPA’s methods 8000B and 8272 the r2 value must be 0.99 or higher for PAHs to be used for quantification also it says that the relative standard deviation of the relative response factors (RRF) should be less or equal to 15%. In this study the %RRF value are above that limit for all compounds but anthracene, which has a %RRF value of 13%, see A3. The recovery for internal standards for the alkylated PAHs varied between 17-47% and can be seen in figure 8. They were all quite low, below 50% for all three. The %RFF were less then 30%, which is considered acceptable for alkylated PAHs according to US EPA methods 8000B and 8272.
Figure 8. Recovery for all IS from the samples in the analysis of alkylated PAHs.
0   20   40   60   80   100   120  
Recovery  PAH16  
Sample  1  
Sample  2  
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In table 10 the calculated LOD values can be seen for the alkylated PAHs. It varied between 0.37-2.4 µg/kg wet weight for all compounds but triphenylene that has a calculated LOD of 25 µg/kg wet weight. For the alkylated PAHs the recovery varied between 33-58%, see figure 9. The r2 values for the alkylated PAHs were around 0.951-0.997, see A4. The value of 0.951 belonged to 1,2,8-trimethylphenanthrene. No guiding limits for an acceptable r2 value could be found for alkylated PAHs. However in US EPA method 8000B it says that it is not limited to the methods listed in it so the minimum of 0.99 could be applicable for these compounds as well.
Figure 9. Recovery for all IS from the samples in the analysis of oxy-PAHs and azaarenes.
The r2 values for oxy-PAHs and azaarenes were around 0.983-0.999 for almost all compounds, see A5. As for the alkylated PAHs no limit for an acceptable r2 value where found so the value in US EPA 8000B could be used as a reference here as well and all compounds but four were above 0.99, see A5. No restrictions for what is an acceptable %RFF could be found for azaarenes or oxy-PAHs. The calculated LOD values for these compounds can be seen in table 11. The values varied between 0.26-7.4 µg/kg (w/w).
Discussion In 2012 an environmental remediation was done of the area northeast of Patholmsviken (Karlsson & Sjöström 2008; Karlsson, 2013). Since then the concentration of PAH16 had declined with two thirds, this could indicate that the remediation has been successful and the PAH levels are declining towards the background concentration. However, there might be other reasons for this decline as well. One reason could be that the snail were collected in different seasons, this year they were collected in April when they had not had the opportunity to feed in the same extent as the ones that were collected in September 2013 or in June 2008 since they overwinter in deeper waters. The sizes of the collected snail were also quite small according to persons that had been collecting snail previously. This could mean that the snail that were analysed were younger overall and had therefore not been exposed in the same extent as the ones collected in September 2013. According to Ted von Proschwitz, biologist, most snails that could be collected this time of the years would probably be 1-2 years old. Another thing that might have had an effect on the results is the amount of analyte. It was hard to gather enough snails so it might not be representative for the site. To see if the composition of PAH16 had been altered since 2008 the concentrations for the individual PAHs
0   10   20   30   40   50   60   70  
IS  Acridi ne-­D9  
IS  Carba zole-­D8  
IS  Anthr acene-­9,
Sample  1  
Sample  2  
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were compiled in a diagram, see fig. 3.1. The concentrations had declined for almost all compounds between the three analyses. The levels for the low molecular PAHs were lower then for the other compound this is probably due to volatilisation since they evaporate more easily then the high molecular weight PAHs. Naphthalene had been below and just above LOD in the previous two analyses in 2008 and 2013 and the levels for dibenz(ah)anthracene were low the other two years as well. In both 2008 and 2013 Pelagia Miljökonsult AB also analysed freshwater snail from a reference site. The sum of PAH16 year 2008 was 7,500 µg/kg w/w and year 2012 it was 1,800 µg/kg w/w. So compared to these values the concentrations for 2015 is lower which also can be expected after the remediation. When the snails were left to depurate the time window was extended due to the travel back from Umeå, so the snails that had the longest time were left to depurate for 120 h. In the method used at Pelagia Miljökonsult AB they would depurate for 12-24 h but in the literature 24-48 h have been seen (Oehlmann & Schulte-Oehlmann, 2003). How this prolonged time window has affected the result is hard to know since PAHs are quite resistant but it is possible that some PAHs have been broken down during this phase. In 2010 Fornander analysed the PAH16 content in blue mussels in Lundåkra bay outside of Landskrona. There are a lot of different industries in that area that might have contribute to the PAH profile. The levels were highest for fluoranthene and naphthalene and also the levels of phenanthrene were quite high. The profile is comparable with Patholmsviken were also fluoranthene is the highest. The biggest difference is however the high levels of naphthalene. In an collaboration between different research centres in Europe; Umeå University, the French research institutes Bureau de Recherches Géologiques et Minières (BRGM) and the National Center for Scientific Research (CNRS) a project called PACMAN is focusing on polar PACs eg. oxy-PAHs and azaarenes. They have conducted a study at the site of the old wood treatment plant in Holmsund. Here they have analysed soil, sediment and molluscs for PAH16, oxy-PAHs and azaarenes. The profile for PAH16 in the molluscs is very similar to the one obtained in this study (Lundstedt et al, 2015.). As in this study fluoranthene has the highest concentration followed by pyrene and anthracene. The main difference is that the level of naphthalene is above detection limit. MacDonald et al. (2000) proposed probable effect concentrations (PEC) for nine PAHs, see table 15. This PEC value indicates at which levels the PAH concentrations are probable to cause an effect on sediment-living organism. These guidelines are presented in µg/kg dry weight so the wet weight concentrations in the snails had to be transformed into dry weight before a comparison could be done. According to Van Aardt, W.J. (1968) the wet content in L. Stagnalis is 91.6%, which means that the dry content in the snail could be estimated to be 8.4%. Doing this it showed that the concentrations in the snails were lower then the PEC level in the sediment. However since no PAH analysis for the sediment has been done it is possible that the PAH levels in the sediment are above these PEC values but since snails are bioaccumulators it is possible to think that the levels in the snail in fact are higher then the levels in the sediment. In a study performed by Baumard et al. (1999) both sediments and mussels were analysed for PAHs from different location among them nine spots in the Baltic Sea. Here they saw that the PAH levels in the mussels were lower then the levels in the sediment for three location, and these three locations were industrialised and/or urbanised. For the other six points, which all were offshore, the levels in the mussels were higher then the levels in the sediment. However the levels offshore were not as high as for the three first locations and they were somewhat close to each other so it is possible to believe that they represent a background level. After transforming the concentrations in the samples from Patholmsviken to dry weight it is possible to compare them with the values from the Baltic Sea.
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Table 15. Showing the PAH concentrations compared to PEC values in µg/kg (dry weight)
Patholmsviken   PEC   Anthracene 21 845 Fluorene 190 536 Napthalene 4.8 561 Phenanthrene 370 1170 Benzo(a)antracene 120 1050 Benzo(a)pyrene 38 1450 Chrysene 280 1290 Fluoranthene 840 2230 Pyrene 560 1520
The concentrations of alkylated PAHs were below LOD for most of the analysed compounds, only eight compounds could be quantified. The concentrations for these eight compounds were in the same range as for PAH16. The likely source for the PAHs at Patholmsviken is creosote from an abandoned wood-impregnation facility. According to Murphy and Brown (2005) creosote will give rise to pyrogenic PAHs and in the book “Environmental Forensics: Contaminant Specific Guide” (Murphy and Morrison, 2006) it is said that pyrogenic PAHs will contain more parent PAHs than the alkylated ones and this is also shown in this study, see figure 6. So the probable source is pyrogenic. By comparing different PAH ratios it is also possible to draw conclusion about the origin of the pollution, one common ratio is anthracene/(anthracene + phenantrene) for the sample in Patholmsviken this ratio is 0.95 and that is an indication of combustion pollution and not petroleum, (Budzinski et al., 1997). Another commonly used ratio is the one for fluoranthene/(fluoranthene + pyrene), if it is below 0.50 it is most likely a petroleum source and if it is above might be combustion or emission from cars (Yunker et al., 2002). For the sample this ratio is 0.60 so it indicates combustion or emission rather then petroleum. Also the PAH profile in figure 6 indicates a pyrogenic source rather then a petroleum one, a petroleum source would have higher levels of the alkylated PAHs. For the oxy-PAHs and azaarenes only carbazole was above LOD. In previous analyses at Örebro University it has been shown that the silica column clean up used in this experiment will have a negative effect on azaarenes since they can interact in ionic form with the silica gel. A large part might therefore be retained in the silica column. Carbazole is however often a part of creosote so it is expected to find it in the sample if the source is creosote (Hale & Aneiro, 1997). If a different clean-up technique would have been used for these compounds it is possible that more of them had been above LOD since these types of compounds often are present in creosote as well. Even the oxy-PAHs and azaarenes were studied at Holmsund by Lundstedt et al. (2015). In that study in contrary to what this study showed the oxy-PAHs were above LOD and 1-indanone was present in the highest concentration in molluscs. For the azaarenes carbazole had the highest level and this is also the only compound that could be detected in this study. However there were higher levels of 1-indanone then carbazole in the study performed by Lundstedt et al 2015. so it would have been expected to find 1-indanone in this experiment as well. When comparing the oxy-PAH and azaarenes profile between molluscs, soil and groundwater that Lundstedt et al obtained in 2015 the biggest difference between the three matrixes is for the azaarenes. In soil there is almost no azaarenes present they can instead be found in the molluscs and in even higher concentration in the ground water. The oxy-PAHs on the other hand can be found in all three matrixes but the lowest
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levels can be seen in the groundwater (Lundstedt et al, 2015). So this indicates that the more polar azaarenes can dissolve in water in higher extent then both oxy-PAHs and parent PAHs. To further improve this study a reference point should have been used to estimate the background levels that could be expected in the snails. Also it would have been interesting to analyse both sediment and water to see how the distribution of the PAHs differed in the three matrixes. More time would have been needed for the collection of snails to get more replicates for analysis or another time of season could have eased the collection phase.
Conclusion The concentrations of PAH16 have declined since previous analyses; this indicates that the remediation that has been done was successful. Since the concentrations declined between 2013 and 2015 it is possible that they still have not reached the background levels. The concentrations of alkylated PAHs were generally lower then for the native PAHs and that coincide with the assumption that the PAHs originate from a pyrogenic source like creosote. Also the PAH ratios indicates that the source is pyrogenic. Oxy-PAHs and azaarenes were below LOD in the samples for all compounds except for one, but for some of them a hint of a peak could be seen. This could either be an indication that oxy-PAHs and azaarenes are present in low concentrations or it could be due to interfering compounds since the response were low. However, since both oxy-PAHs and azaarenes were detected in another study from the same area (Lundstedt et al. 2015) there is reason to conclude that the sample contains these compounds. Freshwater snails seem to be suitable organisms for biomonitoring, but because of their overwintering it would be preferable to gather them under the same period of time. My suggestion would be during late summer or early autumn when they have been active after the winter, this makes it possible to gather snails of different ages if that would be of interest. Since they are present almost all over the world it could be a good tool to use for comparing pollutions in different regions. Also they fulfil many of the desired criteria for a good biomonitoring organism. More information about their uptake and excretion rates are needed to get a better understanding of what happens with the organic pollutants in their bodies. It would also be good to now more about how different types of environment possibly could affect uptake and excretion rates. These areas may be objects of future research.
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Acknowledgements I would like to thank my supervisors Per Ivarsson, Björn Rydvall and Torbjörn Ros for all the help during the project and also everyone at Pelagia Miljökonsult AB for the warm welcome and all the help during the fieldwork. I also would like to thank my wonderful classmates for all the support and company during long hours of work and of course everyone at MTM for good advises and guidance during my project.
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US EPA method 8000B (1996). “Determinative chromatographic separations”. US EPA method 8272 (1996). “Parent and alkyl polycyclic aromatics in sediment pore water by solid-phase microextraction and gas chromatography/mass spectrmetry in selected ion monitoring mode”. Van Aardt, W.J. (1968) Quantitative Aspects of the Water Balance of the Water Balence in Lymnaea Stagnalis (L.). Leiden, Brill Archive. von Proschwitz, T., biologist, Göteborgs Naturhistoriska Museum. Personal communication 2015-05- 06 Walker, C. H. & D. R. Livingstone (1992). Persistent pollutants in marine ecosystems. Oxford, Pergamon Pr. Wenning R.J. & L. Martello (2014) ”Chapter 8 POPs in Marine and Freshwater Environments” in Environmental Forensics for Persistent Organic Pollutants, edited by O'Sullivan G. & Sandau C., pp. 357 - 390 Elsevier
Yunker, M.B., R.W. Macdonald, R. Vingarzan, R. H. Mitchell, D. Goyette & S. Sylvestre (2002) "PAHs in the Fraser River basin: a critical appraisal of PAH ratios as indicators of PAH source and composition", Organic Geochemistry, 33(4)489-515.
29
Appendix A1. Quantification standards (QS), internal standards (IS) and recovery standard used throughout the experiment. Also the trace (Tr.) for each compound is listed next to it.
QS  PAH  Mix   Tr.   QS  Alkyl  mix   Tr.   QS  Oxy-­PAHs   Tr.   IS  PAH16   Tr.  
Naphthalene   128   2-­Methylnaphthalene   142   Quinoline   129   Naphtalene-­D8   136  
Acenaphthylene   152   1-­Methylnaphthalene   142   1-­Indanone   132   Acenaphthylene-­ D8  
160  
Fluorene   166   2,3,5-­ Trimethylnaphthalene  
Phenanthrene   178   Dibenzothiophene   184   Acridine   179   Phenanthrene-­ D10  
188  
198   9-­Fluorenone   180   Anthracene-­D10   188  
Fluoranthene   202   2-­Methylphenanthrene   192   4H-­ Cyclopenta(def)phenanthrene  
204   Fluoranthene-­D10   212  
Pyrene   202   2-­Methylanthracene   192   Anthracene-­9,10-­dione   208   Pyrene-­D10   212  
Benzo(a)anthracene   228   2,8-­ Dimethyldibenzothiophe ne  
212   11H-­Benzo[a]carbazole   217   Benzo(a)anthrace ne-­D12  
240  
222   Chrysene-­D12   240  
Benzo(b)fluoranthene   252   2,4,7,-­ Trimethylphenantrene  
264  
206   7H-­Benz(de)anthracene-­7-­one   230   Benzo(k)fluoranth ene-­D12  
264  
264  
220   Naphtacene-­5,12-­dione   258   Indeno(1,2,3-­ cd)pyrene-­D12  
288  
292  
270   Benzo(ghi)perylen e-­D12  
D8   152  
192  
204  
    1-­Methylchrysene   242       IS  Mix  3       6-­Ethylchrysene   256       Carbazole-­D8   175  
    7,12-­ Dimethylbenz(a)anthrac ene  
216  
    Perylene   252       Recovery  Standard       7-­Methylbenzo(a)pyrene   266       Perylene-­D12   264  
30
A2. Solutions with grade and supplier used for making all standards. Name   Purity     Supplier   1-­Indanone   >  99%  
Alfa  Aesar,  Ward  Hill,  USA    
2-­Methylanthracene-­9,10-­dione   97%   7H-­Benz[de]anthracene-­7-­dione   99%   Acridine   >  98%   Benz[a]anthracene-­7,12-­dione   >  98%   Benzo[h]quinoline   98%   11H-­Benzo[a]carbazole    
Chiron,  Trondheim,  Norway    
Dinaphtho[1,2-­b;1',2'-­d]furan   >96%   Dibenzothiophene-­D8   98.7  atom%  D   Carbazole-­D8   98.9%   Anthraquinone-­D8   99.4%   Acridine-­D9   98.7%   9-­Methylanthracene-­D12   98.0  atom%  D   2,3-­Dimethylanthracene   99.8%   1-­Methylnaphthalene-­D10   98.8  atom%  D   Anthracene-­9,10-­dione   99.8%  
Fluka,  Sigma-­Aldrich,  Steinheim,  Germany   Naphthacene   99.5%   Benzo[a]fluorenone   BCR-­342;  99.8%  
Institute  for  Reference  Materials  and   Measurements,  Geel,  Belgium  
 
6H-­Benzo[cd]pyren-­6-­one   BCR-­339;  98.8%   4H-­Cyclopenta[d,e,f]phenanthrenone   BCR-­338;  99.5%   Fluorenone   BCR-­342;  99.8%  
Dibenzo[a,c]anthracene   97.5%   Labor  Dr.  Ehrenstorfer–Schäfers,  Teddington,   Middlesex,  UK  
  Dibenzo[a,j]anthracene   99.8%   PAH  mix  9  deuterated  (16  IS)   97.1%   Dibenz[ah]acridine   99.6%   LGC  standards,  Teddington,  Middlesex,  UK   Cyclopenta[d,e,f]phenathrene   97.0%  
Sigma-­Aldrich,  Steinheim,  Germany    
97%  
7,12-­Dimethylbenz[a]anthracene   99.9%   9-­Fluorenone   98%   2-­Methylanthracene   97.0%   7-­Methylbenz[a]anthracene   n/a   7-­Methylbenzo[a]pyrene   98.0%   1-­Methylchrysene   99.1%   2-­Methylchrysene   99.3%   3-­Methylchrysene   99.3%   Naphthacene-­5,12-­dione   97%   Perylene-­D12   n/a   Quinoline   98%   1,4-­Chrysenequinone   >  93%   Tokyo  Chemical  Industry,  Tokyo,  Japan   Benzo[a]fluorene   98.0%  
Ultra  Scientific,  North  Kingstown,  USA    
Naphtho[2,3-­a]pyrene   99.00%   PAH  mixture  16  analytes   n/a  
31
 IS  Acenaphthylene-­D8   17.3336   11.9726    IS  Acenaphthene-­D10   23.3617   3.00213    IS  Fluorene-­D10   2.35792   2.6911    IS  Phenanthrene-­D10   5.05821   3.42927    IS  Anthacene-­D10   11.6912   8.2246    IS  Fluoranthene-­D10   4.91453   2.92625    IS  Pyrene-­D10   5.84676   3.45693    IS  Benzo(a)anthracene-­D12   5.98281   4.83256    IS  Chrysene-­D12   8.46954   6.18805    IS  Benzo(b)fluoranthene-­D12   0.687429   0.642267    IS  Benzo(k)fluoranthene-­D12   15.1832   10.1999    IS  Benzo(a)pyrene-­D12   6.47402   6.67104    IS  Indeno(1,2,3-­c,d)pyrene-­D12   7.13401   6.65672    IS  Dibenz(a,h)anthracene-­D14   4.11868   6.04622    IS  Benzo(g,h,i)perylene-­D12   4.35512   4.88932    RS  Perylene-­D12   17.859   6.86585    
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 IS  Dibenzothiophene-­D8   7.28237   8.29677    IS  9-­Methylanthracene-­D12   5.28611   8.76081    RS  Perylene-­D12   8.84074   7.53246    
33
 IS Acridine-D9 31.3848   23.4698    IS Anthracene-9,10-dione-D8 10.8821   20.0355    RS Perylene-D12 13.7115   12.9817    
34
2013-11-28
RAPPORT Utfärdad av ackrediterat laboratorium REPORT issued by an Ackreditated Laboratory
Laboratorier ackrediteras av Styrelsen för ackreditering och teknisk kontroll (SWEDAC) enligt svensk lag. Den ackrediterade verksamheten vid laboratorierna uppfyller kra ven i SS-EN ISO/ IEC 17 025 (2005).
Denna rapport får endast återges i sin helhet, om inte utfärdande laboratorium i förväg skriftligen godkänt annat.
Pelagia Miljökonsult AB, Sjöbod 2, Strömpilsplatsen 12, 90743 Umeå, Sweden Telefon 090-702170 (+46 90 702170) Fax 090 702179 (+46 90 7021 79) Organisationsnummer 556643-3917
E-post info@pelagia.se, www.pelagia.se
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Författare: Kenneth Karlsson, Pelagia Miljökonsult AB
Foto: Lokal 2 vid vägbanken till Umeå Hamn. Pelagia Miljökonsult AB.
Kartor är publicerade med tillstånd av SeSverigeavtal, Metria AB.
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Sammanfattning Liksom vid 2008 års undersökning så visar analyserna från undersökningen år 2013 att de högsta halterna av PAH16 i snäckor uppmättes vid Lokal 1 i Patholmsvikens innersta del. Halterna minskar med ökat avstånd från vikens innersta del och är betydligt högre längs vikens västra strandområde än det östra.
Vid Lokal 1 år 2013 var halterna av PAH16 i snäckor betydligt lägre än vid 2008 års undersökning. Även vid Lokal 2 kan en minskning urskiljas, men inte i samma omfattning. Den lokala referensen (Lokal 5) vid 2013 års undersökning uppvisade jämförbara halter som i referensen (Kylören) vid 2008 års undersökning.
Under 2012 utfördes en omfattande sanering av markområdet nordväst om Patholmsviken som avvattnas till den inre delen av viken. Avvattningen sker dels diffust genom markskiktet och dels genom den trumma som mynnar vid Lokal 1, längst in i viken. Eventuellt kan den minskande halten i snäckor från Lokal 1 kopplas till denna sanering.
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1 Inledning .......................................................................... 5 2 Material och metod ......................................................... 6
2.1 Snäckor ....................................................................... 6 3 Resultat och diskussion ................................................ 8
3.1 Fältresultat snäckor ..................................................... 8 3.2 Analysresultat snäckor ............................................. 10
Referenser ........................................................................................... 12 Bilaga 1 ................................................................................................ 13
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1 Inledning Pelagia Miljökonsult AB har på uppdrag av ÅF konsult AB utfört en undersökning av organiska miljögifter i biota (snäckor) i Patholmsviken (Figur 1) belägen i Holmsund inom Umeå kommun.
Undersökningens syfte var att beskriva hur föroreningssituationen ser ut i biota i anslutning till det förorenade markområdet norr om Patholmsviken. Den djurgrupp som undersöktes var snäckor vilka analyserades med avseende på halter av organiska miljögifter (PAH16).
Kort historik om området vid Patholmsviken Området norr om Patholmsviken har sedan 1944 använts för träimpregnering av virke. Från början användes den så kallade Bolidenmetoden med arseniksalt och 1953 utvidgades anläggningen så att även kreosotimpregnering kunde utföras. Fram till 1976 användes dessa metoder växelvis ca 2 månader i taget. Från 1976 fram till 1981, när anläggningen lades ned, användes uteslutande arseniksalt (WSP Samhällsbyggnad 2007). Marken för anläggningen har undersökts och delvis sanerats 1983. En omfattande sanering nordväst om Patholmsviken utfördes under 2012. Kompletterande undersökningar görs under 2013 för att ge underlag för riskbedömning, åtgärdsutredning och riskvärdering inför eventuellt fortsatt saneringsarbete i området.
Figur 1. Patholmsvikens läge i Holmsund, Umeå kommun , © Lantmäteriet.
Patholmsviken
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2 Material och metod I Patholmsviken insamlades snäckor under perioden 2013-09-06 – 2013-09-10, från fem lokaler. Fyra lokaler i Patholmsviken och en referenslokal vid Långsmaludden sydost om Patholmsviken (Figur 2).
Figur 2. Patholmsviken och lokaler där snäckor insamlats år 2013, © Lantmäteriet.
2.1 Snäckor På varje lokal insamlades Radix baltica, Lymnaea stagnalis och Stagnicola sp. Insamlingen i Patholmsviken utfördes av Kenneth Karlsson och Anja Rubach, Pelagia Miljökonsult AB. Insamlade snäckor förvarades under insamlingsarbetet uteslutande i glaskärl för att undvika kontaminering av snäckorna. I Figur 3 – 5 visas bilder på tre av de vanligen förekommande arterna i undersökningsområdet.
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Figur 5. Lymnaea stagnalis
Insamlingsarbetet genomfördes längs stränderna på de aktuella lokalerna. De allra flesta snäckorna insamlades strandnära på grunt vatten (< 0,5 m) och företrädelsevis från stenar eller block.
Efter fältarbetet sumpades snäckorna i 12-24 h i stora glasbehållare. Samtliga snäckor sumpades i vatten från den lokal de insamlats från. Sumpningen syftar till att djuren skall tömma tarmen innan de infryses för vidare behandling. Samtliga snäckor infrystes därefter i glaskärl med plastlock/aluminiumfolie.
På laboratoriet, efter upptining, plockades mjukdelarna ut från skalen med hjälp av metallpincett för prov till organiska miljögifter. Vid urplockningen valdes, så långt det var möjligt, snäckor av samma storleksordning för samtliga lokaler. Pelagia Miljökonsult AB är ett av Swedac ackrediterat organ för insamling och preparering av snäckor, ackrediteringsnummer 1846. Analys av PAH16 är utförd av ALS Scandinavia AB som är ett av Swedac ackrediterat laboratorium (ackrediteringsnummer 2030) för analys av PAH16.
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3 Resultat och diskussion
3.1 Fältresultat snäckor Nedan presenteras resultaten från insamlingsarbetet av snäckor uppdelat mellan de olika lokalerna. För varje lokal ges en kortare beskrivning av lokalen och från vilka substrat snäckorna insamlades.
Patholmsviken, Lokal 1 Strandzonen på lokalen dominerades av grova block och sprängsten. Snäckorna insamlades främst från stenar och block i strandzonen. Vid lokalen mynnar en vägtrumma som avvattnar området nord/nordväst om lokalen.
Patholmsviken, Lokal 2 Strandzonen (vägbanken till Blå vägen) på lokalen dominerades av grova block och sprängsten. Vid denna lokal var det en omfattande påväxt på de stenar och block från vilka snäckorna insamlades.
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Patholmsviken, Lokal 3 Liksom vid lokal 2 dominerades strandzonen (vägbanken till Blå vägen) på lokalen av grova block och sprängsten från vilka snäckorna insamlades.
Lokal 4 Strandzonen på lokalen dominerades av sten och block varifrån snäckorna insamlades.
Lokal 5 Liksom vid lokal 4 dominerades strandzonen på lokalen av sten och block varifrån snäckorna insamlades.
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3.2 Analysresultat snäckor Liksom vid 2008 års undersökning så visar analyserna från undersökningen år 2013 att de högsta halterna av PAH16 uppmättes vid Lokal 1 i Patholmsvikens innersta del (Tabell 1 och 2). Halterna av PAH16 i snäckorna minskar med ökat avstånd från vikens innersta del, halterna är betydligt högre längs vikens västra del än den östra (jämför lokal 2 och 4). I referensen (Lokal 5) uppmättes den lägsta halten av PAH16
där den uppgick till 11 µg/kg våtvikt
Vid jämförelse mellan de två undersökningarna så var halterna vid Lokal 1 år 2013 betydligt lägre än vid 2008 års undersökning (Pelagia Miljökonsult AB, 2008). Även vid Lokal 2 kan en minskning urskiljas, men inte i samma omfattning. Den lokala referensen (Lokal 5) vid 2013 års undersökning uppvisade jämförbara halter som i referensen Kylören vid 2008 års undersökning. Det bör noteras att en viss mellanårsvariation förekommer beroende på omgivningsfaktorer som exempelvis vattentemperatur. Observera att Lokal 3 år 2013 inte är lokaliserad till samma plats som år 2008.
Under 2012 utfördes en omfattande sanering av markområdet nordväst om Patholmsviken som avvattnas till den inre delen av viken. Avvattningen sker dels diffust genom markskiktet och dels genom den trumma som mynnar vid Lokal 1, längst in i viken. Eventuellt kan den minskande halten i snäckor från Lokal 1 kopplas till denna sanering.
Tabell 1. Halter av PAH16 i snäckor från Patholmsviken och den lokala referensen (Lokal 5) vid Långsmaludden 2013. Lokal Lokal1 Lokal 2 Lokal 3 Lokal 4 Lokal 5, ref Provtyp Snäckor Snäckor Snäckor Snäckor Snäckor Enhet µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt
Summa PAH16 1801* 442* 93* 75* 11*
*I summaberäkningarna ingår ej värden under rapporteringsgräns (<), se Bilaga 1.
Vid 2008 års beräkning av summa PAH16 ingick värden under rapporteringsgränsen med rapporteringsgränsens värde, vilket innebär ett ”worst case” scenario. Vid jämförelse mellan åren har detta betydelse endast för referensen Kylören där en summaberäkning skulle ge ett värde på 9,7 µg/kg våtvikt istället för 16 µg/kg våtvikt. Endast ett värde för Lokal 1-3 understeg rapporteringsgränsens värde.
Tabell 2. Halter av PAH16 i snäckor från Patholmsviken och referensen i Kylören 2008. Område Patholmsviken Patholmsviken Patholmsviken Kylören
Referens Lokal Lokal1 Lokal 2 Lokal 3 Provtyp Snäckor Snäckor Snäckor Snäckor Enhet µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt
Summa PAH16 7516 616 103 16
*I 2008 års summaberäkningarna har värden under rapporteringsgräns (<) ersatts med rapporteringsgränsens
värde.
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Vid lokal 1 i Patholmsviken var halten av PAH16 år 2013 ca 160 ggr högre än vid referenslokalen (Lokal 5) (Tabell 3). Lokal 2 och Lokal 3 längs vägbanken till Blå vägen uppvisar ca 40 gånger respektive 8 gånger högre halt än i referensen. Halten av PAH16 i snäckor på Lokal 1 var ca 4 gånger högre (Tabell 3) än på lokal 2 och ca 19 respektive 24 gånger högre på lokal 3 respektive lokal 4. Om en jämförelse görs mellan lokal 2 och lokal 3 och 4 där halterna är jämförbara så är det en faktor 5 högre halt vid Lokal 2 (Tabell 3).
Tabell 3. Jämförelse av PAH16 -halt i snäckor mellan lokalerna i Patholmsviken och
referensen.
Snäckor Snäckor Snäckor Snäckor Jmf Lokal 1/ref Jmf Lokal 2/ref Jmf Lokal 3/ref Jmf Lokal 4/ref 161 ggr 39 ggr 8.3 ggr 6,7 ggr Snäckor Snäckor
Jmf Lokal 1/Lokal 2
Jmf Lokal 2/Lokal 3 och 4
Ca 4 ggr Ca 5 ggr
Pelagia Miljökonsult AB har på ett antal andra områden längs Norrlandskusten analyserat PAH16 i snäckor och de högsta halter som uppmätts i dessa områden är ca 200 µg/kg våtvikt. Dessa halter uppmättes i Luleås skärgård med närhet till SSAB (Figur 6).
Figur 6. Halter av PAH från ett antal områden längs Norrlandskusten år 2007. De olika färgerna i diagrammet anger olika arter av snäckor.
Se sk
ar öf
jä rd
Referenser Björklund, I., 1985: Regional kartering av metallinnehåll i mjukdelar hos Lymnaea utmed Bottenvikskusten 1980-82. Naturvårdsverket rapport 3047.
Naturvårdsverket 2007: Rapport 5736, Oavsiktligt bildade ämnens hälso- och miljöegenskaper. – en kunskapsöversikt.
Naturvårdsverket 1999: Bedömningsgrunder för miljökvalitet, Kust och hav. Rapport 4914
WSP Samhällsbyggnad 2007: Holmsund 2:52 m fl., Umeå kommun. Fd träimpregneringsområdet i Holmsund.
Pelagia Miljökonsult AB, 2008: Patholmsviken, Rapport till Ramböll AB. 2008-12- 05.
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PAH i snäckor från Patholmsviken 2013.
ELEMENT SAMPLE Lokal 1 Lokal 2 Lokal 3 Lokal 4 Lokal 5 ref TS vikt-% 18,2 18,5 17,1 16,7 naftalen mg/kg 0,0082 0,013 <0.0050 0,0058 <0.0050 acenaftylen mg/kg 0,028 0,0053 <0.0020 <0.0020 <0.0020 acenaften mg/kg 0,095 0,094 0,012 0,0037 <0.0020 fluoren mg/kg 0,076 0,036 0,0037 0,0073 <0.0020 fenantren mg/kg 0,24 0,07 0,0086 0,024 0,0034 antracen mg/kg 0,17 0,02 0,028 0,0036 <0.0020 fluoranten mg/kg 0,58 0,1 0,019 0,018 0,0049 pyren mg/kg 0,36 0,054 0,012 0,0099 0,0029 bens(a)antracen mg/kg 0,085 0,011 0,0024 <0.0020 <0.0020 krysen mg/kg 0,046 0,011 0,0024 <0.0020 <0.0020 bens(b)fluoranten mg/kg 0,064 0,015 0,0049 0,003 <0.0020 bens(k)fluoranten mg/kg 0,02 0,0048 <0.0020 <0.0020 <0.0020 bens(a)pyren mg/kg 0,014 0,0029 <0.0020 <0.0020 <0.0020 dibenso(ah)antracen mg/kg 0,002 <0.0020 <0.0020 <0.0020 <0.0020 benso(ghi)perylen mg/kg 0,0063 0,0021 <0.0020 <0.0020 <0.0020 indeno(123cd)pyren mg/kg 0,0071 0,0027 <0.0020 <0.0020 <0.0020 summa 16 EPA-PAH mg/kg 1,80 0,442 0,093 0,0753 0,0112 PAH cancerogena mg/kg 0,24 0,047 0,0097 0,003 <0.007 PAH, summa övriga mg/kg 1,55 0,39 0,083 0,072 0,011
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PATHOLMSVIKEN
RAPPORT Utfärdad av ackrediterat laboratorium REPORT issued by an Ackreditated Laboratory
Laboratorier ackrediteras av Styrelsen för ackreditering och teknisk kontroll (SWEDAC) enligt svensk lag. Den ackrediterade verksamheten vid laboratorierna uppfyller kraven i SS-EN ISO/IEC 17 025 (2005).
Denna rapport får endast återges i sin helhet, om inte utfärdande laboratorium i förväg skriftligen godkänt annat.
Pelagia Miljökonsult AB, Sjöbod 2, Strömpilsplatsen 12, 90743 Umeå, Sweden Telefon 090-702170 (+46 90 702170) Fax 090 702179 (+46 90 7021 79) Organisationsnummer 556643-3917
E-post info@pelagia.se, www.pelagia.se
PATHOLMSVIKEN
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Författare: Kenneth Karlsson, Pelagia Miljökonsult AB Erik Sjöström, Pelagia Miljökonsult AB
PATHOLMSVIKEN
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Innehållsförteckning
Innehållsförteckning .................................................................................... 3 1 Inledning .....................