Analysis of Polycyclic Aromatic Hydrocarbons in Freshwater Snails of Family Lymnaeidae

68 Pages·2015·6.08 MB·English

Project
 in
 Chemistry:
 15
 HP

Analysis
 of
 Polycyclic
 Aromatic

Hydrocarbons
 in
 Freshwater
 Snails
 of

Family
 Lymnaeidae
 from

Patholmsviken

Malin Karlsson
2015-05-29
Supervisors: Per Ivarsson
Björn Rydvall & Torbjörn Ros
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 PAH , alkylated
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PAHs, oxy-PAHs and azaarenes using GC/MS. The concentrations of PAH were compared
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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 PAH , alkylerade
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PAHer, oxy-PAHer och azaarener med hjälp av GC/MS. Koncentrationerna av PAH
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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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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 ..........................................................................................................................14

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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 in freshwater snails .............................................................................................................16

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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 ............................................................................................................................................29

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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 (PAH ) according to the US Environmental Protection Agency (US
16
EPA), alkylated PAHs and oxy-PAHs and azaarenes. The PAH content will be compare
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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 + O → ROH + H O where R is a chemical like, PAHs.
2 2
Thus, when PAHs are metabolised by P450 a functional group such as –OH, -NH , and -
2
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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