Analysis of Polycyclic Aromatic Hydrocarbons in Freshwater Snails of Family Lymnaeidae from Patholmsviken

Transkript

1 Project in Chemistry: 15 HP Analysis of Polycyclic Aromatic Hydrocarbons in Freshwater Snails of Family Lymnaeidae from Patholmsviken Malin Karlsson Supervisors: Per Ivarsson Björn Rydvall & Torbjörn Ros

2 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 woodimpregnating facility, freshwater snails were collected and analysed for PAH 16, alkylated PAHs, oxy-pahs and azaarenes using GC/MS. The concentrations of PAH 16 were compared with previous analyses and the results showed that the levels had declined since 2008 and 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. 2

3 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 träimpregneringsanläggning, har snäckor samlats in och analyserats för PAH 16, alkylerade PAHer, oxy-paher och azaarener med hjälp av GC/MS. Koncentrationerna av PAH 16 jämfördes med värden från två tidigare analyser och resultaten visade att nivåerna hade minskat sedan 2008 och 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. 3

4 Table of content ABSTRACT...2 SAMMANFATTNING...3 INTRODUCTION...5 OBJECTIVE...5 BIOINDICATORS AND BIOMONITORING...5 MOLLUSCS...6 Lymnaea Stagnalis...7 Stagnicola Sp...7 POLYCYCLIC AROMATIC HYDROCARBONS...7 Origin and Chemical Properties of PAHs...7 Metabolism of PAHs...9 HISTORY ABOUT PATHOLMSVIKEN...10 Creosote...10 METHOD...11 MATERIAL...11 FIELD WORK...11 SAMPLE PREPARATION...12 GC-MS ANALYSIS...14 GC-method PAH GC-method Alkylated PAHs...15 GC-method Oxy-PAHs and Azaarenes...15 RESULTS...16 FAT CONTENT IN FRESHWATER SNAILS...16 GC/MS ANALYSIS...16 PAH 16 in freshwater snails...16 Alkylated PAHs in freshwater snails...18 Oxy PAHs and azaarenes in freshwater snails...19 QA/QC...20 DISCUSSION...21 CONCLUSION...24 ACKNOWLEDGEMENTS...25 REFERENCES...26 APPENDIX

5 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 (PAH 16 ) according to the US Environmental Protection Agency (US EPA), alkylated PAHs and oxy-pahs and azaarenes. The PAH 16 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. 5

6 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 6

7 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). 7

8 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 8

9 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 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 + O 2 ROH + H 2 O where R is a chemical like, PAHs. Thus, when PAHs are metabolised by P450 a functional group such as OH, -NH 2, 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 9

10 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). 10

11 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). Lantmäteriet/Metria Figure 5. The red lines represent the site from where the snails were collected The snails were collected in Patholmsviken. 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: , East: , 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 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 h long but due to lack of time and travel back to Orebro the time window were extended to h. When back at Örebro University the snails were stored in a freezer. 11

12 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; m PCB weighed g, m R1 weighed g (replicate 1) and m R2 weighed 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 (Na 2 SO 4 ) (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) PAH 16 (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, 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 Na 2 SO 4 was added and then 40 ml n-hexane was used to pre wash it. When the solvent level just reached the top of the Na 2 SO 4 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 Na 2 SO 4 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 12

13 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 PAH 16 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 PAH 16. PAH 16 Compound CS 1 2 ng CS 2 20 ng CS ng CS ng PAH Mix 1 µl (2 ng) 10 µl (20 ng) 10 µl ( µl (500 ng) ng) IS PAH µl ( µl ( µl ( µl (200 ng) ng) ng) ng) RS Perylene 10 µl ( µl ( µl ( µl (200 D12 ng) ng) ng) ng) Toluene 369 µl 360 µl 370 µl 345 µl Total volume 400 µl 400 µl 400 µl 400 µl Final concentration PAHs 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 CS 1 10 ng CS 2 50 ng CS ng CS ng CS 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) 2,3- Dimethylanthracene 2, 20ng/ul IS 3 MIX 5 µl (10 ng) 25 µl (50 ng) 5 µl (10 ng) 25 µl (50 ng) 25 µl (500 ng) 25 µl (500 ng) 13 µl (260 ng) 13 µl (260 ng) 25 µl (500 ng) 25 µl (500 ng) 25 µl (500 ng) 25 µl (500 ng) 50 µl (1000 ng) 50 µl (1000 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 PAHs concentration 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. 13

14 Table 4. The composition of the Oxy-PAH standards. Oxy- PAHs Compound CS 1 5 ng CS 2 50 ng CS ng Oxy-PAH Mix 4.5 ng 67.5 ng 450 ng Dinaphtho[1,2-b;1',2'- d]furan; 11Hbenzo[a]carbazole 5 ng 50 ng 500 ng IS PAH 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 PAH 16 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 r 2 values for the calibration curves can be found for all the three samples in tables A2-A4 in appendix. GC/MS-method PAH 16 The conditions used for the analysis of PAH 16 can be seen in table 5. The column used was a select PAH which is suitable for PAH 16 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. 14

15 Table 5. The conditions for GC/MS analysis of PAH 16. Technique Column Sample Concentration Injection Volume 1 µl Temperature Program Carrier Gas Injector GC/MS Conditions for PAH 16 GC/MS Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462, Agilent, Santa Clara, USA) ng/µl 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) Helium, constant flow 2 ml/min 250 C, Splitless mode, 1 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 PAH 16. 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 PAH 16. Technique Column Sample Concentration Injection Volume 1 µl Temperature Program Carrier Gas Injector Conditions for alkylated PAHs GC/MS Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462) ng/µl 90 C (1min), 8 C/min to 300 C (4 min), 25 C/min to 325 C (1min) Helium, constant flow 2 ml/min 250 C, Splitless mode, 1 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 PAH 16. Helium was used as carrier gas and splitless injection was used. The initial temperature were 70 C and it was risen 15

16 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. Technique Column Injection Volume 1 µl Temperature Program Carrier Gas Injector GC/MS Conditions for Oxy- PAH and Azaarenes GC/MS Select PAH, 30 x 0.25 mm, df = 0.15 µm (Part number CP7462) 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) Helium, constant flow 2 ml/min 250 C, Splitless mode, 2 50 ml/min Mass Detector EI in SIM mode, ion source 230 C, transfer line 300 C SIM Parameters (Mass, Dwell time) Results 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) Fat content in freshwater snails The fat content in the original sample were calculated to be 1.2%. GC/MS analysis PAH 16 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 PAH 16 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. 16

17 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 Fat content (w/w) Naphtalene <0.93 <79 Acenaphtylene <0.31 <24 Acenaphtene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)antracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenz(ah)anthracene <1.0 <85 Benzo(ghi)perylene Benzo(c)phenanthrene Benzo(j)fluoranthene <2.3 <200 Benzo(e)pyrene Perylene a Values below LOD are presented as lower then the calculated LOD. The measured concentrations for PAH 16 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 PAH 16 concentration for the individual compounds and also the summarised value from 2008, 2013 and 2015 in µg/kg. PAH 16 concentrations (µg/kg w/w) a Naphtalene < <5 Acenaphtylene < Acenaphtene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)antracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenz(ah)anthracene < Benzo(ghi)perylene SUM PAH a Values below LOD are not summarised in SUM PAH

18 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 ,5,6-Trimethylnaphthalene Dibenzothiophene 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 ,3-Dimethylanthracene <1.8 <150 1,2,8-Trimethylphenanthrene <0.94 <81 1,2,6-Trimethylphenanthrene <1.4 <120 1-Methylfluoranthene Triphenylene <25 < Methylbenz(a)anthracene Methylchrysene Methylchrysene <2.4 <200 1-Methylchrysene <1.9 <170 6-Ethylchrysene <2.1 <180 7,12-Dimethylbenz(a)anthracene 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. 18

19 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 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. 19

20 QA/QC The recovery for the internal standards in the PAH 16 samples can be seen in figure 7. It varied between % and the low molecular weight PAHs had the lowest recovery. Recovery PAH Sample 1 Sample 2 Figure 7. Recovery for all IS from the samples in the PAH 16 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 r 2 values for the calibration curves for PAH 16 were between (see A3). According to US EPA s methods 8000B and 8272 the r 2 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 Recovery alkylated PAHs Recovery (%) Sample 1 Sample 2 0 IS 1- methylnaphtalene- D10 IS Dibenzothiophene- D8 IS 9- metyhlanthracene- D12 Figure 8. Recovery for all IS from the samples in the analysis of alkylated PAHs. 20

21 In table 10 the calculated LOD values can be seen for the alkylated PAHs. It varied between 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 r 2 values for the alkylated PAHs were around , see A4. The value of belonged to 1,2,8-trimethylphenanthrene. No guiding limits for an acceptable r 2 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. Recovery oxy- PAHs and azaarenes Recovery (%) IS Acridine- D9 IS Carbazole- D8 IS Anthracene- 9,10- dione- D8 Sample 1 Sample 2 Figure 9. Recovery for all IS from the samples in the analysis of oxy-pahs and azaarenes. The r 2 values for oxy-pahs and azaarenes were around for almost all compounds, see A5. As for the alkylated PAHs no limit for an acceptable r 2 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 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 PAH 16 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 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 PAH 16 had been altered since 2008 the concentrations for the individual PAHs 21

22 were compiled in a diagram, see fig 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 PAH 16 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 h but in the literature 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 PAH 16 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 PAH 16, oxy-pahs and azaarenes. The profile for PAH 16 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. 22

23 Table 15. Showing the PAH concentrations compared to PEC values in µg/kg (dry weight) Patholmsviken PEC Anthracene Fluorene Napthalene Phenanthrene Benzo(a)antracene Benzo(a)pyrene Chrysene Fluoranthene Pyrene 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 PAH 16. 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 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 23

24 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 PAH 16 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. 24

25 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. 25

26 References Agency for Toxic Substances and Disease Registry (ATSDR) (1995) Toxicological profile for polycyclic aromatic hydrocarbons (PAHs) Atlanta, Department of Health and Human Services, Public Health Service. Barnes, R. S. K., P. Calow, & P. J. W. Olive (1988). The invertebrates: a new synthesis. London, Blackwell Scientific Baumard, P., H. Budzinski, P. Garrigues, J. F. Narbonne, T. Burgeot, X. Michel, & J. Bellocq (1999) "Polycyclic aromatic hydrocarbon (PAH) burden of mussels (Mytilus sp.) in different marine environments in relation with sediment PAH contamination, and bioavailability", Marine environmental research, 47(5): Baumard, P., H. Budzinski, P. Garrigues, J.C. Sorbe, T. Burgeot, & J. Bellocq (1998) "Concentrations of PAHs (polycyclic aromatic hydrocarbons) in various marine organisms in relation to those in sediments and to trophic level", Marine pollution bulletin 36(12): Baussant, T., S. Sanni, G. Jonsson, A. Skadsheim, & J.F. Børseth (2001) "Bioaccumulation of polycyclic aromatic compounds: 1. Bioconcentration in two marine species and in semipermeable membrane devices during chronic exposure to dispersed crude oil" Environmental Toxicology and Chemistry 20(6): Brooks, K.M. (2004) Polycyclic aromatic hydrocarbon migration from creosote-treated railway ties into ballast and adjacent wetlands, Madison. Department of Agriculture. Budzinski, H., I. Jones, J. Bellocq, C. Piérard & P. Garrigues (1997) "Evaluation of sediment contamination by polycyclic aromatic hydrocarbons in the Gironde estuary", Marine Chemistry, 58(1): Coughtrey, P.J. & M.H. Martin (1977) "The uptake of lead, zinc, cadmium, and copper by the pulmonate mollusc, Helix aspersa Muller, and its relevance to the monitoring of heavy metal contamination of the environment", Oecologia 27(1): Dillon, R.T. (2000) The ecology of freshwater molluscs, Cambridge, Cambridge University Press. Meshcheryakov V. N. (1990) The Common Pond Snail Lymnaea Stagnalis in Animal Species for Developmental Studies edited by Dettlaff T. A. & S. G. Vassetzky, Springer-Verlag US. Eisler, R. (1987) Polycyclic aromatic hydrocarbon hazards to fish, wildlife, and invertebrates: a synoptic review. U.S. Fish and Wildlife Service Biological Report 85(1.11). Hale, R.C. & K.M. Aneiro, (1997) Determination of coal tar and creosote constituents in the aquatic environment, Journal of Chromatography A 774(1-2):79-95 Herod AA. (1998) Azaarenes and Thiaarenes in PAHs and Related Compounds - Chemistry edited by Neilson A.H. The Handbook of Environmental Chemistry, Vol. 3I Anthropogenic Compounds. Springer-Verlag, Berlin Heidelberg, Germany; p Jakoby W. B. (1982) Metabolic basis of detoxication: metabolism of functional groups New York, Academic Press Karlsson K. & E. Sjöström (2008) PATHOLMSVIKEN - Rapport till Ramböll AB, Pelagia Miljökonsult AB 26

27 Karlsson K PATHOLMSVIKEN - Rapport till Ramböll AB, Pelagia Miljökonsult AB Kemenes, G. & P.R. Benjamin (2009) "Lymnaea" Current Biology 19(1):R9-R11. Kemikalieinspektionen (2006) Report no. 4/06 - National Implementation Plan for the Stockholm Convention on Persistent Organic Pollutants for Sweden, Stockholm, Swedish Chemicals Inspectorate Kimbrough, K. L., W. E. Johnson, G. G. Lauenstein, J. D. Christensen and D. A. Apeti (2008) An Assessment of Two Decades of Contaminant Monitoring in the Nation s Coastal Zone Silver Spring, MD. NOAA Technical Memorandum NOS NCCOS pp. Koskinen, A.M.P. (2012) "Chapter 8 - Terpenes" in Assymetric Chichester, John Wiley & Sons. Synthesis of Natural Produts Laskowski, R. & S.P. Hopkin, (1996) "Accumulation of Zn, Cu, Pb and Cd in the garden snail ( Helix aspersa): Implications for predators" Environmental Pollution 91(3): Livingstone, D.R. 1998, The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish, New York, Elsevier Inc. Losso, C. & A.V. Ghirardini (2010) "Overview of ecotoxicological studies performed in the Venice Lagoon (Italy)" Environment international 36(1): Lundstedt S., M. Tysklind, B. Lemière, C. Mouvet, S. Colombano, A. Saada, E Fries, P. Faure, C. Lorgeoux & L. Mansuy (2015) Final report: Project No. SN Assessment and Management of polar PACs in Contaminated Soils and Remedial Processes, PACMAN Lundstedt, S., P. A. White, C. L. Lemieux, K. D. Lynes, I. B. Lambert, L. Öberg, P. Haglund & M. Tysklind (2007) Sources, Fate, and Toxic Hazards of Oxygenated Polycyclic Aromatic Hydrocarbons (PAHs) at PAH-Contaminated Sites, Ambio 36(6): MacDonald, D.D., C.G. Ingersoll, & T.A. Berger, (2000) "Development and Evaluation of Consensus- Based Sediment Quality Guidelines for Freshwater Ecosystems", Archives of Environmental Contamination and Toxicology, 39(1):21-31 Mahmoud, K.M.A. & H.M.A. Abu Taleb (2013) "Fresh water snails as bioindicator for some heavy metals in the aquatic environment" African Journal of Ecology 51(2): Menta, C. & V. Parisi (2001) "Metal concentrations in Helix pomatia, Helix aspersa and Arion rufus: a comparative study" Environmental Pollution 115(2): Murphy, B, & R. Morrison (2006) Environmental Forensics : Contaminant Specific Guide Amsterdam, Academic Press. Murphy, B. & J. Brown (2005) "Environmental Forensics Aspects of PAHs from Wood Treatment with Creosote Compounds" Environmental Forensics 6(2): Naturvårdsverket (2007) Rapport 5736 Oavsiktligt bildade ämnens hälso- och miljöegenskaper, Bromma, CM Gruppen AB 27

28 Oehlmann, J. & U. Schulte-Oehlmann (2003) "Chapter 17 Molluscs as bioindicators" in Bioindicators and biomonitors. edited by Markert B.A., A.M. Breure & H.G. Zechmeister. Oxford, Elsevier. Ortmann A. E., 1909, The Destruction of the Fresh-Water Fauna in Western Pennsylvania. Proceedings of the American Philosophical Society 48(191): Phillips, D. J. H. (1980) Quantitative aquatic biological indicators: their use to monitor trace metal and organochlorine pollution. London, Applied science. Ruppert, E.E. & R.D. Barnes (1994) Invertebrate zoology Fort Worth, Saunders College Pub. Salánki, J., A. Farkas, T. Kamardina, & K. S. Rózsa (2003) "Molluscs in biological monitoring of water quality", Toxicology letters 140: Sette, C., T. Pedrete, J. Felizzola, A. Nudi, A. Scofield & A. Wagener (2013) "Formation and identification of PAHs metabolites in marine organisms", Marine environmental research 91:2-13. Stegeman, J.J. & J.J. Lech (1991) "Cytochrome P-450 monooxygenase systems in aquatic species: Carcinogen metabolism and biomarkers for carcinogen and pollutant exposure" Environmental health perspectives 90: Umeå Kommun (2014) Accessed :47 United Nations (1998) Protocol to the 1979 Convention on Long Range Transboundary Air Pollution on Persistent Organic Pollutants (POPs), Aarhus 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 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 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)

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 PAH 16 Tr. Naphthalene Methylnaphthalene 142 Quinoline 129 Naphtalene- D8 136 Acenaphthylene Methylnaphthalene Indanone 132 Acenaphthylene- 160 D8 Acenaphthene 154 1,6-156 Carbazole 167 Acenaphtene- D Dimethylnaphthalene Fluorene 166 2,3,5-170 Benzo(h)quinoline 179 Fluorene- D Trimethylnaphthalene Phenanthrene 178 Dibenzothiophene 184 Acridine 179 Phenanthrene- 188 D10 Anthracene Fluorenone 180 Anthracene- D Methyldibenzothiophen e Fluoranthene Methylphenanthrene 192 4H- 204 Fluoranthene- D Cyclopenta(def)phenanthrene Pyrene Methylanthracene 192 Anthracene- 9,10- dione 208 Pyrene- D Benzo(a)anthracene 228 2,8- Dimethyldibenzothiophe ne Chrysene 228 2,4- Dimethylphenanthrene Benzo(b)fluoranthene 252 2,4,7,- Trimethylphenantrene Benzo(k)fluoranthene 252 2,4,7- Trimetyldibenzothiophe ne Benzo(a)pyrene 252 1,2,8- Trimethylphenantrene Indeno(1,2,3- cd)pyrene 276 1,2,6- Trimethylphenantrene H- Benzo[a]carbazole 217 Benzo(a)anthrace ne- D Methylanthracene- 9,10- dione 222 Chrysene- D Benzo(a)fluorenone 230 Benzo(b)fluoranth 264 ene- D H- Benz(de)anthracene- 7- one 230 Benzo(k)fluoranth 264 ene- D Benz(a)anthracene- 7,12- dione 258 Benzo(a)pyreneD Naphtacene- 5,12- dione 258 Indeno(1,2,3- cd)pyrene- D12 Dibenz(ah)anthracene Methylfluoranthene H- Benzo(cd)pyrene- 6- one 254 Dibenz(ah)anthrac ene- D12 Benzo(ghi)perylene 276 Benzo(c)phenantrene 228 9,10- Dihydrobenzo(a)pyrene Benzo(ghi)perylen one e- D12 Triphenylene 228 Dibenz(ah)acridine 279 IS 3 Mix 7- Metylbenz(a)anthracene 242 Dinaphtho[1,2- b;1',2'- d]furan 268 Dibenzothiphene- D8 3- Methylchrysene Methylnaphthalen e- D10 2- Methylchrysene Methylantracene- D12 1- Methylchrysene 242 IS Mix 3 6- Ethylchrysene 256 Carbazole- D , Acridine- D9 188 Dimethylbenz(a)anthrac ene Benzo(j)fluoranthene 252 Anthracene- 9, dione- D8 Benzo(e)pyrene 252 Perylene 252 Recovery Standard 7- Methylbenzo(a)pyrene 266 Perylene- D

30 A2. Solutions with grade and supplier used for making all standards. Name Purity Supplier 1- Indanone > 99% 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 PAH/Dibenzothiophenes Mixture 20 >96.5% to analytes >99.5% Dinaphtho[1,2- b;1',2'- d]furan >96% Dibenzothiophene- D atom% D Carbazole- D8 98.9% Anthraquinone- D8 99.4% Acridine- D9 98.7% 9- Methylanthracene- D atom% D 2,3- Dimethylanthracene 99.8% 1- Methylnaphthalene- D atom% D Anthracene- 9,10- dione 99.8% Naphthacene 99.5% Benzo[a]fluorenone BCR- 342; 99.8% 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% Alfa Aesar, Ward Hill, USA Chiron, Trondheim, Norway Fluka, Sigma- Aldrich, Steinheim, Germany Institute for Reference Materials and Measurements, Geel, Belgium Dibenzo[a,c]anthracene 97.5% Labor Dr. Ehrenstorfer Schäfers, Teddington, Dibenzo[a,j]anthracene 99.8% Middlesex, UK 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% 9,10- Dihydrobenzo[a]pyren- 7(8H)- 97% one 7,12- Dimethylbenz[a]anthracene 99.9% 9- Fluorenone 98% 2- Methylanthracene 97.0% 7- Methylbenz[a]anthracene n/a Sigma- Aldrich, Steinheim, Germany 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% Naphtho[2,3- a]pyrene 99.00% Ultra Scientific, North Kingstown, USA PAH mixture 16 analytes n/a 30

31 A3. Table showing the %RRF, %RDS and r 2 for PAH 16 Compound %RRF %RSD r 2 Naphtalene Acenaphtylene Acenaphtene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)antracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3- cd)pyrene Dibenz(ah)anthracene Benzo(ghi)perylene IS Naphthalene- D IS Acenaphthylene- D IS Acenaphthene- D IS Fluorene- D IS Phenanthrene- D IS Anthacene- D IS Fluoranthene- D IS Pyrene- D IS Benzo(a)anthracene- D IS Chrysene- D IS Benzo(b)fluoranthene- D IS Benzo(k)fluoranthene- D IS Benzo(a)pyrene- D IS Indeno(1,2,3- c,d)pyrene- D IS Dibenz(a,h)anthracene- D IS Benzo(g,h,i)perylene- D RS Perylene- D

32 A4. Table showing the %RRF, %RDS and r 2 for alkylated PAHs. Compound %RRF %RSD r 2 2- methylnaphthalene methylnaphthalene ,6- Dimethylnaphthalene ,5,6- Trimethylnaphthalene Dibenzothiophene Methyldibenzothiophene Methylphenanthrene Methylanthracene ,8- Dimethyldibenzothiophene ,4- Dimethylphenanthrene ,4,7- Trimethyldibenzothiophene ,3- Dimethylanthracene ,2,8- Trimethylphenanthrene ,2,6- Trimethylphenanthrene Methylfluoranthene Benzo(c)phenanthrene Triphenylene Methylbenz(a)anthracene Methylchrysene Methylchrysene Methylchrysene Ethylchrysene ,12- Dimethylbenz(a)anthracene Benzo(j)fluoranthene Benzo(e)pyrene Perylene Methylbenzo(a)pyrene IS 1- Methylnaphtalene- D IS Dibenzothiophene- D IS 9- Methylanthracene- D RS Perylene- D

33 A5. Table showing the %RRF, %RDS and r 2 for oxy-pahs and azaarenes. Compound %RRF %RSD r 2 Quinoline Indanone Carbazole Benzo(h)quinoline Acridine Fluorenone H-Cyclopenta(def)phenanthrene Anthracene-9,10-dione H-Benzo[a]carbazole Methylanthracene-9,10-dione Benzo(a)fluorenone H-Benz(de)anthracene-7-one Benz(a)anthracene-7,12-dione Naphtacene-5,12-dione Dinaphtho[1,2-b;1',2'-d]furan H-Benzo(cd)pyrene-6-one ,10-Dihydrobenzo(a)pyrene-7-one Dibenz(ah)acridine IS Carbazole-D IS Acridine-D IS Anthracene-9,10-dione-D RS Perylene-D

34 A6. Report Patholmsviken 2013, Pelagia Miljökonsult AB PATHOLMSVIKEN 2013 Rapport till ÅF konsult AB 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 (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, Umeå, Sweden Telefon ( ) Fax ( ) Organisationsnummer E-post info@pelagia.se, 34

35 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. 2/18

36 Sammanfattning Liksom vid 2008 års undersökning så visar analyserna från undersökningen år 2013 att de högsta halterna av PAH 16 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 PAH 16 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. 3/18

37 Innehållsförteckning Sammanfattning... 3 Innehållsförteckning Inledning Material och metod Snäckor Resultat och diskussion Fältresultat snäckor Analysresultat snäckor Referenser Bilaga /18

38 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 (PAH 16). 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 En omfattande sanering nordväst om Patholmsviken utfördes under Kompletterande undersökningar görs under 2013 för att ge underlag för riskbedömning, åtgärdsutredning och riskvärdering inför eventuellt fortsatt saneringsarbete i området. Patholmsviken Figur 1. Patholmsvikens läge i Holmsund, Umeå kommun, Lantmäteriet. 5/18

39 2 Material och metod I Patholmsviken insamlades snäckor under perioden , 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. 6/18

40 Figur 3. Radix baltica Figur 4. Stagnicola sp 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 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 Analys av PAH 16 är utförd av ALS Scandinavia AB som är ett av Swedac ackrediterat laboratorium (ackrediteringsnummer 2030) för analys av PAH 16. 7/18

41 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. 8/18

42 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. 9/18

43 3.2 Analysresultat snäckor Liksom vid 2008 års undersökning så visar analyserna från undersökningen år 2013 att de högsta halterna av PAH 16 uppmättes vid Lokal 1 i Patholmsvikens innersta del (Tabell 1 och 2). Halterna av PAH 16 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 PAH 16 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 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 PAH 16 i snäckor från Patholmsviken och den lokala referensen (Lokal 5) vid Långsmaludden 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 PAH * 442* 93* 75* 11* *I summaberäkningarna ingår ej värden under rapporteringsgräns (<), se Bilaga 1. Vid 2008 års beräkning av summa PAH 16 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 PAH 16 i snäckor från Patholmsviken och referensen i Kylören Område Patholmsviken Patholmsviken Patholmsviken Kylören Lokal Lokal1 Lokal 2 Lokal 3 Referens Provtyp Snäckor Snäckor Snäckor Snäckor Enhet µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt µg/kg våtvikt Summa PAH *I 2008 års summaberäkningarna har värden under rapporteringsgräns (<) ersatts med rapporteringsgränsens värde. 11/18

44 Vid lokal 1 i Patholmsviken var halten av PAH 16 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 PAH 16 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 Jmf Lokal 1/Lokal 2 Ca 4 ggr Snäckor Jmf Lokal 2/Lokal 3 och 4 Ca 5 ggr Pelagia Miljökonsult AB har på ett antal andra områden längs Norrlandskusten analyserat PAH 16 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). Seskaröfjärden Rånefjärden Storfjärden (Piteå) Lokala referenser Recipientlokaler Figur 6. Halter av PAH från ett antal områden längs Norrlandskusten år De olika färgerna i diagrammet anger olika arter av snäckor. 11/18

45 Referenser Björklund, I., 1985: Regional kartering av metallinnehåll i mjukdelar hos Lymnaea utmed Bottenvikskusten Naturvårdsverket rapport 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 /18

46 Bilaga 1 Analysresultat 13/18

47 PAH i snäckor från Patholmsviken 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 < ,0058 < acenaftylen mg/kg 0,028 0,0053 < < < acenaften mg/kg 0,095 0,094 0,012 0,0037 < fluoren mg/kg 0,076 0,036 0,0037 0,0073 < fenantren mg/kg 0,24 0,07 0,0086 0,024 0,0034 antracen mg/kg 0,17 0,02 0,028 0,0036 < 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 < < krysen mg/kg 0,046 0,011 0,0024 < < bens(b)fluoranten mg/kg 0,064 0,015 0,0049 0,003 < bens(k)fluoranten mg/kg 0,02 0,0048 < < < bens(a)pyren mg/kg 0,014 0,0029 < < < dibenso(ah)antracen mg/kg 0,002 < < < < benso(ghi)perylen mg/kg 0,0063 0,0021 < < < indeno(123cd)pyren mg/kg 0,0071 0,0027 < < < 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 14/18

48 15/18

49 16/18

50 17/18

51 A7. Report Patholmsviken 2013, Pelagi Miljökonsult AB PATHOLMSVIKEN Rapport till Ramböll AB 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 (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, Umeå, Sweden Telefon ( ) Fax ( ) Organisationsnummer E-post info@pelagia.se, 18/18

52 PATHOLMSVIKEN Ansvarig utgivare: Torbjörn Johnson, Pelagia Miljökonsult AB Författare: Kenneth Karlsson, Pelagia Miljökonsult AB Erik Sjöström, Pelagia Miljökonsult AB 2/18

53 PATHOLMSVIKEN Innehållsförteckning Innehållsförteckning Inledning Material och metod Snäckor Abborre Resultat Snäckor Fältresultat snäckor Analysresultat snäckor Abborre Analysresultat abborrmuskel Diskussion Referenser Bilaga /18

54 PATHOLMSVIKEN 1 Inledning Pelagia Miljökonsult AB har på uppdrag av Ramböll AB utfört en undersökning av metaller och organiska miljögifter i biota i Patholmsviken (Figur 1) i Holmsund inom Umeå kommun. Undersökningens syfte var att beskriva hur föroreningssituationen ser ut i biota i anslutning till det förorenade markområdet. De djurgrupper som undersöktes var snäckor och abborre vilka analyserades med avseende på metallhalter och halter av organiska miljögifter (PAH16). Kort historik om 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 Kompletterande undersökningar görs under 2008 för att klarlägga föroreningarnas utbredning och för att utgöra underlag för kommande åtgärder r Figur 1. Patholmsviken med snäcklokaler och nätstationer. 4/18

55 PATHOLMSVIKEN 2 Material och metod I Patholmsviken plockades snäckor på tre lokaler , två i själva viken och en i vattensamlingen öster om Blå vägen (Figur 1). I referensen Kylörsviken plockades snäckor från en lokal Kylörsviken som ligger söder om Norrbyn, ca 40 km söder om Patholmsviken, valdes ut som lokalt referensområde. Abborre insamlades från Patholmsviken till 22 och i Kylörsviken till 16. Ett samlingsprov från vardera område och enskilda lokaler analyserades. 2.1 Snäckor På varje lokal insamlades Radix baltica, Lymnaea stagnalis och Stagnicola sp. Insamlingen i Patholmsviken utfördes av Kenneth Karlsson, Pelagia Miljökonsult och Stefan Falemo, Ramböll. I Kylörsviken utfördes insamlingen av Björn Rydvall och Mats Uppman, Pelagia Miljökonsult AB. Fältarbetet genomfördes i första veckan av juni. Plockade snäckor förvarades under insamlingsarbetet uteslutande i glaskärl för att undvika kontaminering av snäckorna under insamlingsarbetet. I Figur 2 4 visas bilder på tre av de vanligen förekommande arterna i undersökningsområdet. Figur 2. Radix baltica Figur 3. Stagnicola sp Figur 4. Lymnaea stagnalis Insamlingsarbetet genomfördes längs stränderna på de aktuella lokalerna. De allra flesta snäckorna plockades strandnära på grunt vatten (< 0,3 m) och företrädelsevis satt de på undersidan av stenar, block, träföremål eller direkt på sedimentytan. 5/18

56 PATHOLMSVIKEN Efter fältarbetet sumpades snäckorna i 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 plastpincett till metallprover och metallpincett för prov till organiska miljögifter. Vid urplockningen valdes, så långt det var möjligt, snäckor av samma storlek. I standarden för undersökning av bioackumulation av metaller i mjukdelar hos Lymnaea (BIN BR21) rekommenderas att snäckorna delas in i storleksklasser för att minska risken för skillnader i bioackumulation beroende av storlek. Undersökningar har dock visat att skillnaderna mellan bioackumulationen i små respektive stora individer av Lymnaea är relativt liten. Björklund (1985) visade i sin undersökning att halterna för ett antal metaller var mellan 7 och 12 % högre i den större storleksklassen. För andra metaller (bly) noterades istället ca 15 % lägre halter i den större storleksklassen. Vid låga metallhalter var dock avvikelsen procentuellt större än inom mediana eller höga haltintervall. 2.2 Abborre Abborre insamlades med hjälp av nät i slutet av maj i både Patholmsviken och Kylörsviken. Näten lades ut på kvällen och togs upp nästföljande morgon. Abborrarna lösgjordes från näten och mättes och styckefrystes i uppmärkta plastpåsar. Fiskarna längdmättes och vägdes samt åldersbestämdes på lab. Åldersbestämningen utfördes på gällock. Från 20 st. abborrar från Patholmsviken och 18 st. abborrar från Kylörsviken (framförallt i storleksintervallet mm) togs muskel och leverprover. Från varje område togs ett samlingsprov av lever och ett samlingsprov från muskel för analys av metall respektive PAH16. Utpreparerad lever och muskel vägdes för kontroll av levererad mängd till analyserande laboratorium. 6/18

57 PATHOLMSVIKEN 3 Resultat 3.1 Snäckor Fältresultat snäckor Nedan presenteras resultaten från insamlingsarbetet av snäckor uppdelat mellan de olika lokalerna. Från varje lokal ges en kortare beskrivning av lokalen och från vilka substrat snäckorna plockades från. Det kan noteras att vattenståndet är högre vid fotograferingstillfället än vid plockningstillfället. Patholmsviken, Lokal 1 Strandzonen på lokalen dominerades av grova block och sprängsten. Bottensedimentet var mycket löst och oljefilm bildades när sedimentet rördes upp. Snäckorna plockades främst från i vattnet liggande trädgrenar och stenar/block. Patholmsviken, Lokal 2 Strandzonen (vägbanken till Blå vägen) på lokalen dominerades av grova block och sprängsten från vilka snäckorna plockades. 7/18

58 PATHOLMSVIKEN Patholmsviken, Lokal 3 Strandzonen på norra delen av lokalen dominerades av i vattnet liggande föremål som snäckorna plockades ifrån. Bottensedimentet var löst och dyigt. På den södra delen av lokalen dominerades strandzonen av sten och block varifrån snäckorna plockades. Kylörsviken Strandzonen på lokalen dominerades av sten och block. Snäckorna plockades främst från dessa substrat. 8/18

59 PATHOLMSVIKEN Analysresultat snäckor PAH Samtliga analysresultat redovisas i Bilaga 1. Analyserna visar att de högsta halterna återfinns på Lokal 1 i Patholmsvikens innersta del där halten uppgick till hela 7516 µg/kg våtvikt (Tabell 1). I referensen uppmättes den lägsta halten av PAH16 till 16 µg/kg våtvikt. Tabell 1. PAH 16 halter i snäckor från Patholmsviken och Kylören (referens). Område Lokal Provtyp Enhet Patholmsviken Lokal1 Snäckor µg/kg våtvikt Patholmsviken Lokal 2 Snäckor µg/kg våtvikt Patholmsviken Lokal 3 Snäckor µg/kg våtvikt Kylören Referens Snäckor µg/kg våtvikt Summa PAH Halten av PAH16 i snäckor är fallande från lokal 1 till den lägsta i referensen. På lokal 1 i Patholmsviken var halten av PAH16 ca 500 ggr högre än i referensen Kylören (Tabell 2). Lokal 2 längs vägbanken till Blå vägen och lokal 3 i gölen på Blå vägens västra sida uppvisar ca 40 gånger respektive 7 gånger högre halt än i referensen. Halten av PAH16 i snäckor på Lokal 1 var ca 12 gånger högre än på lokal 2 och ca 6 gånger högre på lokal 2 än på lokal 3 (Tabell 2). Tabell 2. Jämförelse av PAH 16 -halt i snäckor mellan lokalerna i Patholmsviken och referensen. Snäckor Snäckor Snäckor Jmf Lokal 1/ref Jmf Lokal 2/ref Jmf Lokal 3/ref 482 ggr 40 ggr 7 ggr Snäckor Snäckor Jmf Lokal 1/Lokal 2 Jmf Lokal 2/Lokal 3 12 ggr 6 ggr 9/18

60 PATHOLMSVIKEN Pelagia Miljökonsult AB har på ett antal andra områden längs Norrlandskusten analyserat PAH16 i snäckor och de högsta halterna som återfunnits i dessa områden är ca 200 µg/kg våtvikt. Dessa halter uppmättes i Luleås skärgård med närhet till SSAB (Figur 5). 250 PAH16 i L ymnea, µg /kg fä rskvikt utl M l i l ll l l l i Figur 5. Halter av PAH:er från ett antal områden längs Norrlandskusten. De olika färgerna i diagrammet anger olika arter av snäckor. Metaller De flesta (5 av 7) av metallerna uppvisade högst värden på lokal 3 i Patholmsviken (Figur 6 och 7). Däremot var halten av kadmium lägst på lokal 3 och kadmiumhalterna var likvärdiga mellan lokal 2 och referensen (Figur 6). mg/kgts 1,6 1,4 1,2 1,0 0,8 0,6 0,4 Metaller i snäckor Lokal 1 Lokal 2 Lokal 3 Referens 0,2 0,0 Bly Kadmium Krom Nickel Figur 6. Halter av bly, kadmium, krom, och nickel i snäckor från Patholmsviken och referensen (Kylören). 11/18

61 PATHOLMSVIKEN Det är framförallt zink och koppar på lokal 3 som avviker från referensens värden (Figur 7). Det är endast för koppar som halten är lägst i referensen. Metaller i snäckor mg/kgts Lokal 1 Lokal 2 Lokal 3 Referens Arsenik Koppar Zink Figur 7. Halter av arsenik, koppar och nickel i snäckor från Patholmsviken och referensen (Kylören). 3.2 Abborre I Patholmsviken provtogs 20 abborrar i storleksintervallet mm och 18 abborrar från Kylören i intervallet mm (15 av 18 under 148 mm). Åldersfördelningen i Patholmsviken var i medeltal 2 år och varierade mellan 1+ till 3+ (Bilaga 1). I Kylören var medelåldern 2,1 år med samma intervall som i Patholmsviken Analysresultat abborrmuskel PAH Halten av PAH16 i abborrmuskel från respektive område var ca 3,6 gånger högre i Patholmsviken i jämförelse med Kylören (referens). Däremot var halten i abborrmuskel från Patholmsviken bara något högre än halten i snäckor från Kylören (Tabell 1 och 3). Tabell 3. PAH 16 halt i muskel från abborre från Patholmsviken och Kylören (referens) samt jämförelse mellan dessa områden. Område Provtyp Enhet Patholmsviken Muskel abborre µg/kg våtvikt Kylören Muskel abborre µg/kg våtvikt Abborre Jmf P-viken/ref Summa PAH ,6 ggr 11/18

62 PATHOLMSVIKEN Metaller Metallhalterna i Patholmsviken och referensen var likvärdiga för samtliga metaller (Figur 8 och 9). mg/kgts 0,6 Metaller i abborre Patholmsviken Referens 0,5 0,4 0,3 0,2 0,1 0,0 Bly Kadmium Krom Nickel Figur 8. Halter av bly, kadmium, krom, och nickel i abborre från Patholmsviken och referensen (Kylören). Metaller i abborre mg/kgts Patholmsviken Referens Arsenik Koppar Zink Figur 9. Halter av arsenik, koppar och nickel i abborre från Patholmsviken och referensen (Kylören). 12/18

63 PATHOLMSVIKEN Halterna av kadmium, koppar och zink i abborrlever uppvisar generellt tydlig avvikelse från jämförvärdet för Östersjön. Endast kopparhalterna i referensen uppvisar stor avvikelse från jämförvärdet för Östersjön (Tabell 4). För de övriga metallerna överstiger rapporteringsgränsen bakgrundsvärdet och klassificerad därför inte men anges i Tabell 4 som mindre än (<) värden. För krom kan det ses att mindre än värdet ligger vid ingen eller liten avvikelse. För bly och nickel däremot ligger rapporteringsgränsen betydligt högre än bakgrundsvärdet vilket ger högre avvikelse. Tabell 4. Resultat från metallanalys i lever från abborre samt jämförvärde, avvikelse och avvikelseklassning för dessa. Område Ämne Resultat mg/kg TS Jämförvärde mg/kg TS Avvikelse från jämförvärde Patholmsviken Arsenik As 4,2 Referens Arsenik As 4,1 Patholmsviken Bly Pb < ,04 < 2,28 Referens Bly Pb <0.10 0,04 < 2,50 Patholmsviken Kadmium Cd 0,5 0,2 Referens Kadmium Cd 0,49 0,2 Patholmsviken Koppar Cu 16 7 Referens Koppar Cu ,50 2,45 2,29 2,86 Patholmsviken Krom Cr <0,091 0,1 < 0,91 Referens Krom Cr <0,10 0,1 < 1,00 Patholmsviken Nickel Ni <0,14 0,06 < 2,33 Referens Nickel Ni <0,15 0,06 < 2,50 Patholmsviken Zink Zn ,69 Referens Zink Zn ,54 Tabell 5. Avvikelseklassning enligt Naturvårdsverket (1999). Klass Benämning Bly Kadmium Koppar Krom Nickel Zink 1 Ingen/obetydlig avvikelse < 1,0 < 1,0 < 1,0 < 1,0 < 1,0 < 1,0 2 Liten avvikelse 1,0-1,7 1,0-1,7 1,0-1,6 1,0-1,4 1,0-2,0 1,0-1,4 3 Tydlig avvikelse 1,7-2,8 1,7-3,0 1,6-2,4 1,4-2,1 2,0-4,0 1,4-1,9 4 Stor avvikelse 2,8-4,6 3,0-5,0 2,4-3,7 2,1-3,1 4,0-8,0 1,9-2,7 5 Mycket stor avvikelse >4,6 >5,0 >3,7 >3,1 >8,0 >2,7 13/18