FATE OF THE FRAGRANCE MATERIAL ACETYL CEDRENE IN SEDIMENTS INHABITED BY THE BENTHIC

MOLLUSCS MACOMA BALTHICA AND MYA ARENARIA

Kaushal BARAL**, Valentina BURDUKOVSKA*,

Minodora DAVID*, Mads KÆRHUS OLUFSEN*

Bachelor project*/Semester project**

Spring 2013

Supervisor: Prof. Henriette SELCK

Dept. ENSPAC

Roskilde University

Denmark

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Cover picture:

a) Balanidae, b) Mytilus edulis, c) Lanice conchilega, d) Lagis koreni, e) Littorina littorea,

f) Ensis americanus, g) Cerastoderma edule, h) Scrobicularia plana, i) Mya arenaria,

k) Arenicola marina, l) Hediste diversicolor, m) Macoma balthica.

(image source: Senckenberg: World of Biodiversity)

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ACKNOWLEDGEMENT

We would like to express our gratitude for all those who helped us in realizing this project.

Firstly, we would like to thank Lina Dai for providing the basis for our report and guiding us

throughout the process. We would also like to thank prof. Gary Banta, ENSPAC for helping

us identify and acclimatize the test organisms, and the technicians Anne-Grete Winding,

Klara Jensen, May-Britt Kary, for helping in the field work and getting us acquainted with the

laboratory equipment.

Last, but not least, we would like to thank our supervisor, prof. Henriette Selck, for all her

dedication and advice.

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ABSTRACT

In recent time it has come to concern that hydrophobic organic compounds, from the

relatively large group of fragrance materials, enter the aquatic environment via drainage

systems and bioaccumulate in the sediment and biota. Acetyl cedrene is one of these

compounds. In this report the fate and effects of acetyl cedrene were studied on two species of

marine molluscs – Macoma balthica and Mya arenaria – with different feeding strategies. An

experiment was conducted over a period of 14 days, after which the concentrations of acetyl

cedrene present in different compartments (sediment, water, and tissue) were analyzed with a

GC-MS machine. The results were statistically tested with a General Linear Model and a One-

Way ANOVA. The outcome suggested that the actual organisms were not efficient enough in

biotransforming the fragrance material. Additionally, the effects of acetyl cedrene on the

studied species were expressed as a delay in burrowing behavior. Mortality has been recorded

among the test organisms, but it was not a result of the presence of acetyl cedrene.

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TABLE OF CONTENTS%

1."INTRODUCTION".............................................................................................................."6%1.1"AIM"OF"REPORT"....................................................................................................................."7%1.2"HYPOTHESIS"..........................................................................................................................."7%

2."ACETYL"CEDRENE"............................................................................................................"8%2.1"PHYSICAL"AND"CHEMICAL"PROPERTIES".................................................................................."8%2.2"ROUTES"OF"ENTERING"THE"ECOSYSTEM"................................................................................."9%2.3"UPTAKE"ROUTES"BY"AQUATIC"ORGANISMS"............................................................................"9%

3."CHOICE"OF"TEST"ORGANISMS"......................................................................................."11%3.1"MYA"ARENARIA"..................................................................................................................."11%3.2"MACOMA"BALTHICA"............................................................................................................"12%

4."BIOACCUMULATION"AND"BIOTRANSFORMATION"........................................................"13%4.1"BIOAVAILABILITY"................................................................................................................."13%4.2"BIOTRANSFORMATION"........................................................................................................"15%

5."MATERIALS"AND"METHODS".........................................................................................."16%5.1"SEDIMENT:"COLLECTION"AND"HANDLING"............................................................................."16%5.2"SEDIMENT"SPIKING"WITH"ACETYL"CEDRENE"........................................................................."17%5.3"TEST"ORGANISMS:"COLLECTION"AND"HANDLING".................................................................."19%5.4"EXPERIMENT"SETFUP"............................................................................................................"20%5.5."BURROWING"BEHAVIOR"....................................................................................................."22%5.6"WATER,"TISSUE,"AND"SEDIMENT"ANALYSIS".........................................................................."22%5.7"GCFMS"ANALYSIS".................................................................................................................."27%5.8"STATISTICAL"METHODS"........................................................................................................"30%

6."RESULTS"......................................................................................................................."31%6.1"EFFECTS"ON"ORGANISMS"....................................................................................................."31%6.2"FATE"OF"AC".........................................................................................................................."36%

7."DISCUSSION"................................................................................................................."40%7.1"EFFECTS"OF"ACETYL"CEDRENE"..............................................................................................."40%7.2"FATE"OF"ACETYL"CEDRENE"...................................................................................................."43%

8."CONCLUSION"................................................................................................................"44%

9."APPENDICES"................................................................................................................."45%9.1"APPENDIX"A"–"dw:ww"measurements".................................................................................."45%9.2"APPENDIX"B"–"Burrowing"behavior"exhibited"over"a"14Fday"period"......................................"46%9.3"APPENDIX"C"–"Water"volume"in"samples"(from"overlaying"water)"........................................"47%9.4"APPENDIX"D"F"Laboratory"Techniques:"The"ASE"Machine:"Accelerated"Solvent"Extractor"......"48%9.5"APPENDIX"E"F"Sample"preparation"for"ASE"............................................................................"50%9.6"APPENDIX"F"F"Laboratory"Techniques:"The"GCFMS"Machine".................................................."51%9.7"Appendix"G"–"Overall"results"................................................................................................"53%

10."REFERENCE"LIST".........................................................................................................."54%

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1. INTRODUCTION %

Fragrance materials (FM), such as acetyl cedrene (AC), represent a large group of aromatic

compounds with varying toxicities that can be normally found in household care products.

They have been found both in the water and sediment compartments of wastewater, in the

final stages of wastewater treatment (Simonich et. al., 2000). It is especially the lipophilic

organic compounds that can pose a threat to benthic invertebrates because of their tendency to

bioaccumulate in sediment. These hydrophobic contaminants bind to the sediment particles

and can be taken up by deposit-feeding organisms that thrive on organic matter. So far, traces

of FMs have been found in clams, mussels, fish, sharks, and other marine animals (Kannan et.

al., 2005).

Even though little is known about the toxicity of acetyl cedrene in the aquatic environment, it

has been found to have a measurable concentration in sewage wastewater, it is highly

accumulative in the sediment and has low water solubility (Simonich et. al., 2000). For these

reasons, AC was chosen as the test compound of this report.

The overall aim of the report is to assess the fate and effects of the sediment-bound fragrance

material acetyl cedrene (AC) in the presence of the deposit-feeding mollusc Macoma balthica

and the suspension-feeding mollusc Mya arenaria. The above-mentioned species were

selected because of their high abundance in the local estuarine systems (Isefjord, Roskilde

Fjord, Denmark), and their reported capability of bioaccumulating and biotransforming other

organic pollutants (e.g. PAHs)(Rust et. al., 2004).

Microbial degradation has also been used as a reference point to see to what extent AC is

biodegraded in the absence of macrofauna. In order to do so, an experiment was set-up, and

burrowing behaviour and AC degradation were used as indicators of the fate and effects of the

contaminant. The article written by Dai et. al. (2012) was used as a point of departure for

methods and experimental set-up.

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1.1 AIM OF REPORT

In order to assess the fate and effects of the fragrance material acetyl cedrene, the following

questions were addressed:

1) Does the presence of Macoma balthica and Mya arenaria affect the fate of sediment-

associated AC? And if so, in what way is the fate of AC affected and which organism has the

greater impact?

2) How does the sediment-associated AC affect the studied organisms?

1.2 HYPOTHESIS

Before commencing the experiment, there were several aspects that were hypothesized on.

Firstly, it was presumed that Macoma would show a greater impact on the fate of AC, because

of its feeding strategy (deposit-feeder), compared to Mya, which was expected to have little

impact (suspension-feeder).

Secondly, it was presumed that no AC would be present in the water phase of the experiment

- because of the low water solubility and sediment-binding properties of AC.

And lastly, it was hypothesized that there will be a difference between the concentration of

AC in the sediment measured at the beginning of the exposure period (T0) and the one

measured at the end (Tend) (HA – AC conc. T0≠ AC conc. Tend), in other words, that the test

organisms would affect the fate of AC by diminishing its concentration.

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2. ACETYL CEDRENE

2.1 PHYSICAL AND CHEMICAL PROPERTIES

Acetyl cedrene (AC) is an organic compound widely used as a fragrance material (FM). FMs

are aromatic compounds that are added to care products to give a pleasing scent. They can be

characterized by the presence of aromatic rings and their volatility. These organic materials

can be extracted from nature (plants, trees) or synthesized in the laboratory. The latter are

extracted in the form of oils, concentrates or waxes by processes such as distillation, solvent

extraction. Fragrance materials are usually synthetically derived as the alternative to

macrocyclic compounds found in nature, which are relatively inexpensive compared to their

natural counterparts (Rimkus, 1999). Because of the diversity of the physical and chemical

characteristics of FMs, their ecotoxicity is variable, and their biodegradation rates can range

from readily biodegradable to non-biodegradable (Simonich et al, 2000). Acetyl cedrene has

similar chemical and physical characteristics to HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-

hexamethylcyclopenta-γ-2-benzopyran) and AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-

tetrahydronaphthalene), synthetic musk fragrances recognized as significant contaminants of

the aquatic environment, because of their lipophilic properties and persistent nature that has a

tendency to bioaccumulate in fish and other organisms (Rimkus, 1999). AC tends to

accumulate in sediment due to its high octanol-water partition coefficient (Log KOW=5.6–5.9)

and low water solubility (1.28mg/L)(Simonich et al, 2000).

Table. 1 General characteristics of acetyl cedrene

Acetyl Cedrene (CAS 32388-55-9)

Molecular Formula: C17H26O

Octanol-Water Partition Coefficient (Log Kow): 5.6 - 5.9

Water solubility: 1.28 mg/L

Vapor pressure: 0.058 Pa

Boiling point: 272 °C

Table 1 presents some additional information about the general properties of acetyl cedrene.

Data related to the toxic or persistent nature of AC is scarcely available, thus more insight is

needed concerning the fate and effects of this chemical in the aquatic environment.

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2.2 ROUTES OF ENTERING THE ECOSYSTEM

When it comes to the chemicals found in household products, the main route of entering the

ecosystem is by means of sewage water. The quality of sewage water that ends up in surface

waters is closely linked to the raw sewage received, as well as the treatment applied (Benn &

McAuliffe, 1975).

Acetyl cedrene has been detected in the environment under varying concentrations. It has

been found in concentrations of 7.15"±"4.32µg/L in European wastewater influents and

4.97"±"2.27µg/L in US wastewater influents, and in between 0.071- 0.270µg/L in several

German wastewater effluents (Klaschka et. al., 2013; Simonich et. al, 2002).

AC has also been found in biota, with concentrations ranging from <10–93µg/kg fresh weight

in carp muscle tissue (Klaschka et. al., 2013).

2.3 UPTAKE ROUTES BY AQUATIC ORGANISMS

There are multiple ways through which pollutants can enter an organism. For our test

organisms the most relevant uptake routes are via the alimentary track and through respiratory

surfaces. Thus, the pollutants can be taken up in the form of food and through ambient water

(Walker et. al., 2006). If the organic pollutants are associated with particles (sediment or

suspended particles), and taken up by the respiratory system, they could be deposited in the

respiratory tract of the organisms. This is however a complex situation and as yet, the

knowledge available on the matter is scarce (Walker et. al., 2006).

A simplified model for the fate of a xenobiotic involves five types of sites: sites of uptake,

metabolism, action, storage, and excretion (Walker et. al., 2006). Once a pollutant enters an

organism, it can proceed towards the following sites and be treated in adequacy to their

function within the organism:

Sites of action. In such sites the toxic chemical interacts with the organism at a molecular or a

structural level and the resulting activity resonates at the organism-level. In other words, the

xenobiotic has a noticeable effect on the organism (Walker et. al., 2006).

Sites of metabolism. In these types of sites, enzymes capable of metabolizing xenobiotics are

acting upon the foreign chemicals, causing detoxification. Thus, the organism is acting upon

the chemical (Walker et. al., 2006). This could be seen as a protection mechanism or as

efficiency in chemical breakdown.

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Sites of storage. In this case the xenobiotic is held in an inert state in which it does not act

upon the organism, nor does the organism act upon it (Walker et. al., 2006).

Sites of excretion. The result of excretion can be either the original chemical or a

biotransformation product (Walker et. al., 2006).

Figure 1. Simplified model of xenobiotic uptake and fate within an organism

In the simple model illustrated in figure 1, the toxic chemicals are transported (after uptake) to

the different compartments of the organism’s body. The movement of the xenobiotics into the

organs and tissues could take place by diffusion through membranes, and in the case of

lipophilic chemicals (such as AC) by lipid transport. If a compound is very lipophilic, it is

transported by lipoproteins in a dissolved form. After their partial decomposition, the

fragments of the lipoproteins are transported into cells and the bound lipophilic molecules are

carried along.

2.4 EFFECTS ON BEHAVIOR

Theoretically, all behaviours exhibited by an organism can be affected, to a certain extent, by

xenobiotics. The article realized by Atchison et. al (1996) presents a review of the types of

behaviours affected in aquatic animals. As noted here, there are three main types of

behaviours that can be impaired: foraging, vigilance, and burrowing.

Dysfunctional foraging behaviour leads to reduced resource uptake, which in turn can result

in reduced production (e.g. nutrients, animal growth). Very little is known about how toxic

compounds affect the appetite. As for food handling time (time spent from capture to

ingestion), it has been shown that it is increased as a result of repetitive rejection and

recapture (Atchison et. al., 1996).

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If the vigilance behaviour is affected by the xenobiotic, the vulnerability to predators that the

animal exhibits will increase. This can further lead to an increased mortality rate.

Burrowing behaviour can also be affected in the presence of toxic contaminants. This is

mostly exhibited as a refusal of the animal exposed to the toxin to burrow in the sediment, as

it would otherwise do under normal living conditions.

3. CHOICE OF TEST ORGANISMS In this project we will investigate if the fragrance acetyl cedrene (AC) can be biotransformed

by molluscs, and to do so we will look at two abundant benthic bivalves that both exhibit

burrowing behavior but have different feeding strategies in the brackish waters of Roskilde

Fjord.

The organisms in this essay were chosen because of the close proximity to one of the major

estuarine systems in Denmark, Isefjorden and Roskilde Fjord. Both organisms are widely

abundant in coastal waters around the world, and are known to uptake and metabolize

xenobiotics (Rust et. al., 2004).

3.1 MYA ARENARIA

Easily recognized by its white elliptic shell with a grayish-brown periostracum, M. arenaria’s

distinct feature consists of two merged siphons extruding at the posterior rounding of the shell

(see figure 2). M. arenaria can grow up to 140 mm in length and have relatively large siphons

compared to its body mass, which allows it to burrow up to 250 mm into to the sediment. This

depends on the size of the individual, the larger the individual the deeper it can burrow

(Wanink & Zwarts, 1989; Zaklan & Ydenberg, 1997). M. arenaria is a suspension feeder and

is feeding strictly on the phytoplankton (maybe also other small particles) suspended in the

water column and therefore has little to no uptake of sediment. These bivalves can be found in

densities of up to 4000 individuals m-2 in estuarine areas with muddy sediment (Möller &

Rosenberg, 1983). Figure 2. Mya arenaria. This mollusc burrows deep in the sediment, extruding its

siphons to the surface of the sediment in order to feed (image source: Wheeler, J.).

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3.2 MACOMA BALTHICA

This tellinid bivalve can be recognized by its triangular oval shell, which is smooth with a few

concentric ridges and white, pinkish or orange in color (see figure 3, left). It can grow up to

35mm in length. The siphons of M. balthica are relatively long compared to the weight of the

organism and allows it to burrow up to 80mm into the sediment depending on its size. The

burrowing behavior of this species is also dependent on the seasonal change, as M. balthica

burrows deeper into the sediment in the winter (Wanink & Zwarts, 1989). M. balthica can be

found at densities of up to 1500 individuals m-2 (Olafsson, 1986). The feeding behavior of M.

balthica both relies on suspension feeding and syphoning in the surface layer of the sediment,

thereby both being a suspension and deposit feeder (see figure 3, right). In stagnant water M.

balthica almost solely (95-99% of the time) uses the deposit feeding strategy (Olafsson,

1986). This choice of feeding behavior is also dependent on the availability of organic

material in the water column (Lin & Hines, 1994). Lin and Hines also point out that the

competition for food in the water column has a strong effect on the choice of feeding strategy

of M. balthica, as the presence of high densities of other benthic organisms in the sediment

lowers the amount of food in the water and thereby forces M. balthica to change to deposit-

feeding.

Figure 3. (left) Macoma balthica. This image clearly illustrates the organism’s siphons. (right) even though M.

balthica mostly obtains its food through deposit-feeding, it can still change its strategy to suspension-feeding if

competition for food is low (image source: de Goeij & Luttikhuizen, 1998).

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As mentioned above, both molluscs exhibit burrowing behavior. The movements performed

by the organisms while burrowing increase the oxygen availability in the sediment, which

increases microbial degradation of organic compounds (Kristensen, 2000). Mya arenaria

being the least mobile of the species should have the lowest impact on the sediment (Phelps,

1989). And Macoma balthica, which is known to move more around in the sediment, could

have a larger impact on microbial biodegradation (Tallqvist, 2001).

It has been shown that though M. arenaria is better at metabolizing PAHs than M. balthica,

they are not as effective as other benthic species (Rust et. al., 2004). These results also

suggest a high bioaccumulation potential of M. balthica as a low metabolic rate combined

with a high rate of sediment ingestion and relatively long ingestion time compared to other

bivalves could add to the bioaccumulation of PAHs through the food web (Cammen, 1980;

Decho & Luoma, 1991). Bivalves have a 2-step uptake pathway; the first part involves

extracellular digestion in the intestines and is not very effective for xenobiotic uptake, though

research suggests that it differs from species to species (Decho & Luoma, 1991). The second

pathway, involving intracellular digestion by the digestive glands shows a much higher

absorption rate and takes much more time than the first one (Decho & Luoma, 1991).

4. BIOACCUMULATION AND BIOTRANSFORMATION Contaminants can accumulate in the body, tissue, and gut of organisms by means of

adsorption, absorption, diffusion, exposure to a contaminant, or through the feeding habits of

the organism. The substance is bioaccumulated when it is absorbed at a higher rate than it is

excreted.

4.1 BIOAVAILABILITY

- can be explained, as stated in Spacie’s article, as ‘the portion of the total quantity or

concentration of a chemical in the environment or a portion of it that is potentially available

for biological action, such as uptake by an aquatic organism’ (Spacie, 1995). In other words,

bioavailability describes the amount of a compound that is available for uptake and

biotransformation by an organism.

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Figure 4. The amount of readily available xenobiotic decreases with time

(image source: Semple et. al., 2003)

Hydrophobic organic compounds like PAHs are believed to have a similar bioavailability

pattern where bioavailability of the compound is lowered over time due to adsorption to and

absorption in particles in the system. The fate of these compounds is usually divided into 2

main fractions; a readily available fraction which interacts with particles in the system via

weak bonds and van der waal forces, and a more slowly absorbed recalcitrant fraction where

covalent bonds and other more complex interactions are formed with organic particles in the

sediment, thus making the compound less bioavailable (as this happens within several weeks

it is not relevant in this work).

When the compound is strongly bound to the sediment particles it becomes unavailable to the

organism as the sediment particles are not degraded or destroyed by the organism, thus the

particles are able to retain the compound and the particle bound compound is then excreted

and returned to the sediment.

As seen in figure 4, the more readily available fraction decreases with time as more strong

bonds are formed and degradation takes place. The time in which these bonds are formed

varies greatly dependent on mineral and organic content, temperature, pH, properties of the

xenobiotic, size and surface area and structural/spatial complexity of particles in the sediment

(Reid et. al., 2000; Semple et. al., 2003).

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4.2 BIOTRANSFORMATION

Biotransformation, also known as xenobiotic metabolism, can be defined as the chemical

process through which a xenobiotic is modified (broken down into simpler forms) by biologic

entities (micro-organisms, plants, animals) in the presence or absence of oxygen (Walker et.

al., 2006). This process often leads to a reduction in toxicity or to the transformation of the

compound metabolised into a non-toxic form. There are also cases in which the toxicity of a

xenobiotic can be enhanced or activated (Landis and Yu, 1999). A simplified model of this

process is illustrated in figure 5.

Figure 5. The different steps of biotransformation (image source: Walker et. al., 2006).

Organic contaminants are likely to be biotransformed (once they are taken up by the organism)

into a more water-soluble form is mediated by enzymes (Jørgensen et. al., 2005). The

metabolism of most lipophilic xenobiotics takes place in two phases. The initial step of

biotransformation, also called phase I, consists of processes such as oxidation (during which -

COOH, -OH, -NH2, -SH can be added), hydrolysis, hydration, or reduction, and results in

metabolites characterised by their hydroxyl groups (e.g. hydroxyl-PAHs or dihydrodiols).

These hydroxyl groups will then be used during most of the following conjugation reactions

that make up the second step of biotransformation (phase II). These metabolites are often

more reactive compounds and are easily conjugated to more easily metabolized naturally

occurring compounds such as sugar derivatives, peptides and sulphates (Selck, 2002). During

phase II of PAH biodegradation, aqueous PAH-metabolites are produced (epoxides and

phenol oxides, by means of conjugation) which are ionisable or more water soluble and can

be readily excreted. (Jørgensen et. al., 2005). Figure 5 presents a simplified model of

biotransformation.

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5. MATERIALS AND METHODS Both sediment and test organisms were collected from Roskilde Fjord (55°43'N, 11°58'E,

Gershøj, Denmark) on the 27th of February, 2013. Since the amount of sediment collected

proved to be insufficient, Isefjorden (55°67'N, 11°80'E, Munkholm, Denmark) was used as a

second collection site (see figure 6). The ratio of the sediment from each of the two sources

was roughly 0.5 and was mixed prior to the commencement of the experiment.

Figure 6. Map of collection sites. The blue star represents the first collection poin, while the red star indicates

the origin of the latter sediment collection.

5.1 SEDIMENT: COLLECTION AND HANDLING

The sediment was collected, using a 500µm sieve, by scraping the surface of the sediment.

Since the sediment surface was mostly covered my marine macroalgae, the sediment was

collected from random patches that were not completely covered by the algae. Thus, it was

tried to collect it without uptake of marine plants.

The salinity of the water was measured with a refractometer in two different places: close to

the shore, where it had a value of 4‰, and more further out, where the value measured was of

10‰. As for the temperature, the value displayed on the thermometer was of 1°C.

After sieving, the sediment was washed with distilled water. It was kept overnight for a period

of 24h to allow the sediment to settle, after which the overlaying water was removed and

replaced with seawater (10‰ salinity). Each time new water was added atop of the sediment,

the mixture was thoroughly homogenized. The homogenization was performed in order to

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assure that the seawater penetrated the sediment evenly, since the sediment has a tendency to

settle and accumulate at the bottom of the recipient in which it was kept. This procedure

(change of overlaying water and homogenization of sediment) was done twice, the purpose

being to raise the salinity level of the sediment up to 10‰.

After 48h, the overlaying water was removed and the sediment was homogenized again, it

was evenly distributed in re-sealable plastic bags, labeled, placed in the freezer at a

temperature of -20°C to destroy any organisms that might be present in the sediment, and

preserved until further use.

The next step consisted of thawing the sediment, homogenizing it thoroughly, and

determining the dry weight to wet weight ratio (dw:ww). The wet weight was determined by

weighing the wet sediment on an aluminum boat. Three samples were weighed and the

average of 6,0466g was used for the dw:ww ratio. The wet sediment from the three samples

was then introduced into a muffle furnace and kept at 105°C for 24h. After 24h, the sediment

was removed from the oven, cooled down, and re-weighed. The average of the dw (which

had a value of 4,5769g), and the average of the ww, gave a ratio of 0,7569. The ratio was

rounded up to 0.76 to ease further calculations. For complete calculations see Appendix A,

table 9.

5.2 SEDIMENT SPIKING WITH ACETYL CEDRENE

The first step in establishing the amount of AC needed for spiking was to determine

the quantity of ww sediment needed for the experiment. The amount was chosen to cover 21

beakers of 600ml (143mm height, ∅82mm, DURAN) and a depth of 4cm (this height was

chosen to fulfil the burrowing needs of the organisms studied).

The glass beaker (600ml) was weighed at 118.270g, and then sediment was added until it

reached the hight of 4cm. When weighed with sediment, the beaker gave a new mass of

292.074g. This accounted for 173.804g of ww sediment added per beaker, value that was

rounded at 180g.

After that it was decided that a 250ml beaker might be better for the experiment set-up, so the

amount of ww sediment needed per beaker was scaled down by dividing the initial amount by

2. This gave a quantity of 90g of ww sediment needed per beaker.

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At the time of the initial calculation for the amount of AC needed for sediment spiking, the

experiment set-up consisted of 21 beakers with AC treatment.

The calculations went as following:

90g ww * 21 beakers = 1890g ww sediment (total amount of ww sediment needed for spiking)

An extra of 310g ww sediment was added (to ensure that the amount spiked was enough for

the set-up).

Thus, 2200g ww * 0.76 dw:ww ratio = 1672g dw sediment

50µg AC/g dw sediment * 1672g dw sedediment = 83600µg AC = 83.6 mg AC ~84 mg AC

(amount of AC needed for spiking)

The 84 mg of AC were used to create a stock sediment (2.4828g).

Both the Control sediment (no added AC) and the sediment treated with AC were handled in

the same way. In table 2 the actual amount of AC added to the stock sediment can be seen.

Table 2. Values for spiked and Control sediment. In the Control, water was used to replace the AC.

Aluminum boat (g) Sediment (g) H2O 10% (g) AC 50µg (g)

Control 7.8118 2.3674 0.1216 -

AC treatment 7.8136 2.4828 - 0.0871

The stock sediment was then added to 2.2kg of wet sediment. After that, the spiked sediment,

as well as the Control sediment, was hand mixed in the fume hood and placed on a shaking

table for 24h. The two types of sediment were additionally hand mixed several times

throughout a two-day period. This was necessary in order to prevent the sediment from

settling on the bottom of the buckets in which it was kept and to thoroughly homogenize it.

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5.3 TEST ORGANISMS: COLLECTION AND HANDLING

All the organisms were collected from Roskilde Fjord (55°43'N, 11°58'E, Greshøj,

Denmark) on the 27th of February, 2013. Macoma balthica was obtained by removing the

sediment from the surface with a shovel and sieving it with a 2mm sieve, while Mya arenaria

was harvested by digging deeper into the sediment. Macoma balthica was mainly collected

from the areas that were more out sea (10‰ salinity), while the Myas were also collected in

the areas situated close to the shore (salinity 4‰ and 10‰). The organisms were sampled

randomly and a total of 38 Macomas and over 200 Myas were found.

It was also intended to use Cerastoderma glaucum as a test organism.182 Cersatodermas were

collected, mostly from the areas that measured a salinity level of 4‰. Unfortunately, they

were all dead by the end of the experiment, thus they were not included in the experiment set-

up section of this report.

The next step consisted of acclimatizing the organisms from a temperature of 1°C, found in

the natural environment, to 17°C, the temperature used for running the experiment.

The organisms were brought into a climate room set to run at 4-6°C and sorted. They were

then placed into plastic containers with sediment from the field, water (10‰ salinity), plastic

cover, and were linked to an air supply. Each 3-4 days the temperature was raised by several

degrees, from 4-6 to 10 to 17°C. The overlaying water was changed several times during the

acclimatization period to offer a better living environment for the organisms.

At the time of collection, all the participants in the process had a rough idea of the organisms

that were being searched for. Thus, a few days into the acclimatization period, the organisms

were identified by using Havets dyr og planter by Køie M. et. al. (2000) and The brackish-

water fauna of northwestern Europe by Barnes R. S. K. (1994).

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5.4 EXPERIMENT SET-UP

Before setting up the experiment, because most of the organisms died, due to

inadequate handling, it was decided to reduce the actual experiment size down to 18 beakers:

9 for Control (3*Macoma, 3*Mya, and 3*without organisms) and 9 beakers with 50µg AC/g

dw sediment (3*Macoma, 3*Mya, and 3*without organisms)(see figure 7).

Figure 7. Experimental set-up. The treatment groups were divided in Control (no AC) and AC-treated group. In

each of the beakers containing M. balthica and M. arenaria, 4 test organisms were added. Two additional groups

with no organisms were added to the experimental set-up to assess microbial degradation. Each group had a total

of 3 replicates.

Sediment was added to each beaker (clean sediment to the Control beakers and treated

sediment to the AC beakers) up to the level of 2 cm. Initially, the limit was 4 cm, but since the

beakers were changed from 600mL to 250mL (117mm height, ∅61mm, DURAN), the

sediment height was also diminished. Next, water (10‰ salinity, filtered with 10µm) was

added up to the 200ml mark of each beaker. The beakers were covered with parafilm (to

prevent evaporation of water), and air supply was ensured by air pumps connected through

tubes to glass Pasteur pipettes (see figure 8). This set-up was kept for two days, after which

the overlaying water was carefully removed (without disturbing the sediment) with a

volumetric pipette and replaced with new water (10‰ salinity). Changing the overlaying

water is an important step because decomposing organic chemicals enter the water phase and

can pose a threat for the test organisms.

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Figure 8. Experimental set-up. Parafilm was used to

prevent evaporation of AC, as well as to offer support for

the air pumps.

The overall sizes of the organisms collected were in between 1.0 – 2.2cm for Mya arenaria

and 0.8 – 1.7cm for Macoma balthica. Upon dissection it was determined that the Macomas

with a size of 1.2cm were equal in biomass (~0.081g) with the Myas of size 1.4cm.

Due to restrictions in organism numbers (only 38 Macomas were available), it was decided to

add 4 organisms per beaker (corresponding to a density of 1,428 organisms/m2) and the sizes

selected for each of them were: 0.8 – 0.9cm; 1.0 – 1.1cm; 1.1 – 1.2cm; 1.3 – 1.4cm for

Macoma, and 1.0 – 1.1cm; 1.2 – 1.3cm; 1.5 – 1.6cm; and 1.7 – 1.9cm for Mya (one organism

from each range specified)(see figure 7). These sizes were selected because they had

matching biomass and were the most representative for the sampled populations. Next, the

organisms were gently added to the beakers. The experiment was conducted for a 14-day

period with the climate room set at the temperature of 17°C.

During the preparation phase all the components of each beaker (Control and treatment) -

sediment, water, and test organisms - were handled in precisely the same manner to avoid

errors that can arise from reasons unrelated to the actual effects of the chemical.

Additionally, 3 beakers (T0 1, T0 2, T0 3) with AC and no organisms were made for determining

the initial conditions (i.e. the initial AC levels). They were kept for two days (under same

conditions as the experiment set-up), and taken down, the sediment and water being

separately stored in the freezer at -20°C.

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5.5. BURROWING BEHAVIOR

The burrowing behavior of the organisms was monitored and noted down half an hour, 1 hour,

and 8 hours after the exposure start. During the 2-week period in which the experiment was

running, the set-up was checked upon once a day to make sure the oxygen supply was

functioning properly. Any dead organisms found were removed and noted down. The

burrowing behavior and mortality can be seen in Appendix B.

The burrowing behavior was monitored in order to make sure that the test organisms accept

the environment they were settled in and to see if they behave as expected. Under normal

conditions, both species are normally burrowed completely in the sediment, having only their

siphons extruding from it. %

5.6 WATER, TISSUE, AND SEDIMENT ANALYSIS

After the 14-day period of exposure ended, the water, sediment, and organisms were prepared

for further analysis.

WATER. The first step consisted of removing the overlaying water with a 10ml pipette,

determining the amount of water existent per beaker, and storing it in labeled blue-cap bottles

(100ml, VWR). The volume of water existent per beaker ranged 130-140ml (see Appendix C,

table 11 A and B). After, the water samples were placed in labeled plastic bags and frozen at -

20℃.

TISSUE. Next, the organisms were taken out of the experimental beakers, rinsed with miliQ

water, and left to clean their guts overnight (in new beakers with clean water, 10‰ salinity,

and air supply). They were then dissected, the tissue being placed in labeled and re-sealable

test tubes, and frozen at -80℃.

SEDIMENT. The sediment was homogenized, placed in labeled and re-sealable plastic bags,

and frozen at -20℃ until further analysis.

PREPARATION OF SEDIMENT SAMPLES FOR GC-MS

The sediment corresponding to Mya (sample 1, 2, and 3), Macoma (sample 1, 2, and 3),

microbial degradation (sample 1, 2, and 3) with AC addition, and Mya (sample 1, 2, and 3),

Macoma (sample 1, 2, and 3) without AC addition were thawed in warm water.

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Aluminum trays were used to weigh 5g of wet sediment (from each individual sample), while

a corresponding 5g of Diatomaceous Earth (Hydromatrix) were weighed separately (for exact

measurements, see Appendix E, table 12). The sediment was mixed with the hydromatrix and

homogenized using plastic spoons.

The ASE extraction cells were prepared by adding two cellulose filters. 2.90 – 3.10g of silica

gel were then added to the extraction cells, as a safety measure for avoiding water, and

compressed with the insertion tool. A metal funnel was used to ease the process. Half of the

mixture of sediment and hydromatrix belonging to every sample was then added to their

corresponding ASE cell and compressed with the insertion tool. Next, 100!l of internal

standard was added. The internal standard chosen was phenanthren-d10 (1500!g/ml toluene).

For safety reasons, this step was performed under the fume hood. The remaining half of the

sediment and hydromatrix mixture was added on top of the internal standard and compressed

with the insertion tool, hydromatrix being used to fill the cell up to 1cm from the top.

The cell was closed and placed on the Cell Tray of the ASE machine for sample extraction.

Labeled glass vials were placed on the Vial Tray in the places corresponding to the extraction

cells (see figure 9, left).

In preparation of the ASE, the EPA-type VOA glass vials were kept in an oven at 550℃!to

make sure there were no organic contaminants and the dichloromethane (CH2Cl2) was placed

on an ice bath 2h prior to using the ASE machine.

The program selected for the ASE machine was:

a. Preheat (0 min)

b. Heat (5 min)

c. Static (15 min)

d. Flush (50 vol)

e. Purge (60 sec)

f. Cycles (2)

g. Pressure (2000 psi)

h. Temperature (60℃) i. Solvent (Dichloromethane, 100%)

More details on the function of the ASE200 machine are presented in Appendix D.

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After all the cells were processed, the glass vials were taken to evaporate on the Evaporator

SE 500 (see figure 9, right). Prior to that, 1 ml of toluene was added to each vial.

Figure 9. (left) The ASE extraction cells being loaded on the Cell Tray.

(right) Sample evaporation on the SE 500 machine.

The program used on the SE 500 machine was:

a. Entry 1: Direction = CW

b. Entry 2: Time ON (Secs.) = (do not select any value)

c. Entry 3: Time ON (Mins.) = (do not select any value)

d. Entry 4: Time ON (Hrs.) = (do not select any value)

e. Entry 5: Pulse/Min = 70 (which was later reduced to 50)

f. Entry 6: Duty Cycle = 90%

g. Entry 7: Setpt 1 = 15 deg. C (Top Manifold)

h. Entry 8: Setpt 2 = 75 deg. C (Bottom block)

The remaining samples were filtered by using a glass pipette, glass wool, and anhydrous

sodium sulfate (Na2SO4) to prevent any water from ending up in the GC-MS machine, and

then washed with toluene into a 5ml volumetric flask. The samples were then transferred to

GC-MS vials, closed with a cap, and stored at -20℃.

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The T0 samples, that were removed at the beginning of the experiment, were also prepared for

the GC-MS analysis in the same way as the rest of the samples. Details on preparation for the

ASE machine and on dw:ww measurements can be seen in Appendix A and E (table 10 and

12).

Separately, the dw:ww ratio was determined. Aluminum trays were weighed, and 2g of

sediment was added in order to determine the ww. Next, the sediment trays were added into

an oven and kept overnight at 105℃. The sediment treated with AC, and the one without,

were added to the oven at different times: 13:45, respectively 15:42, and were extracted from

the oven at the same time, 14:00. The results can be seen in Appendix A, table 10.

PREPARATION OF TISSUE SAMPLES FOR GC-MS

The tissue sample tubes were taken out of the freezer, defrosted and placed on an ice bath.

2ml of MeOH and 2ml of water were added to each centrifuge glass containing tissue samples.

The contents of the centrifuge glass were mixed with a Homogenizer and the tubes were

placed back on the ice bath.

The method used for packing of the ASE extraction cells with the tissue sample was mostly

similar to the one used for packing the sediment samples. The difference consists of 4g of

hydromatrix being mixed with the tissue. The entire homogenized mixture was then added to

the extraction cell, followed by the internal standard that was added last.

The preparation for the ASE200 machine was exactly the same (i.e. the glass vials kept in an

oven at 550℃, the dichloromethane kept on ice, the program used by the machine).

During the removal of the EPA-type VOA glass vials, the Macoma 3 sample treated with AC

was broken due to human error.

The program used for the evaporation on the SE 500 machine was the same as used during the

sediment preparation. After the evaporation, the remaining samples were filtered by using a

glass pipette, glass wool, and anhydrous sodium sulfate to prevent any water from ending up

in the GC-MS machine, and then washed with toluene into a 5ml volumetric flask. The

samples were then transferred to GC-MS vials, closed and stored at -20℃.

26"|"P a g e "

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PREPARATION OF WATER SAMPLES FOR GC-MS

The volume of each sample was measured and transferred to conical flasks. The values

measured ranged between 66-77ml. All the samples had precipitate on the bottom and it was

tried as much as possible to pour without disturbing it. In microbial degradation (1) all of the

precipitate ended up in the conical flask. Note: In the lower section of the samples treated

with AC, colloids could be observed. It was tried as much as possible to avoid taking them up

while pouring the samples.

The extractions were done in two rounds to ease handling:

Round 1: Microbial degradation 1, 2, and 3 + Mac 1 and 2;

Round 2: Mac 3 + Mya 1, 2, and 3.

The compounds from the water were removed through Solid Phase Extraction (SPE), by using

Strata-XL tubes from Phenomenex (500mg/6ml, 100u Polymeric Reversed Phase). The SPE

consists of a number of 6 steps.

1. Condition. A vacuum manifold was used, and 3 ml of methanol (MeOH) were flushed over

each column to wash and activate the filter. The vent was adjusted to secure the constant flow

of 3ml/minute.

2. Equilibration. To rinse the tube from MeOH, an additional 3ml of water were used. Again, the

vent was adjusted to secure the constant flow of 3ml/minute.

3. Load sample. The AC-water samples were loaded on the vacuum manifold. The vacuum pump

was turned on and the vent on the vacuum manifold was adjusted at a flow of 3-10 ml/minute.

4. Wash. After all the samples went through the filter, each flask was flushed with 4 ml of water

and added to the column. It is important to flush the conical flasks so that all of the potential

compounds will go to the filter (this step was omitted for the Macoma 3 and Mya 1 samples

due to human error).

5. Dry. The vacuum pump was kept on for 2 minutes, time in which the samples were dried out.

After this step, the vacuum pump was turned off.

6. Elute Analyte. 3 ml MeOH were added to the tubes to elute the compounds collected by the

filter. Collection flasks were placed under the SPE tubes.

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Because the GC-MS machine’s column can be damaged in the presence of water, sodium

sulphate was used to remove any trace of it from the samples. Glass wool was added to glass

pipettes. Separately, a minute amount of Na2SO4 (on the tip of a small metal spoon) was

placed in each sample glass. The Na2SO4 binds to H2O, impeding its access through the filter.

The sample microbial degradation (3) was the only one that was handled slightly different, by

adding Na2SO4 in the glass pipette, on top of the glass wool. The sample was then filtered

through it. The microbial degradation (3) was the first sample to be processed and the

procedure used on it did not seem to be efficient enough, thus the change in method was made.

The small flasks with the eluted compounds in MeOH were each added 500µl of toluene and

100µl (at first only 50µl was added and halfway through the evaporation another 50µl was

added) of the internal standard, phenanthren-d10 (1500µg/ml toluene) and placed on the

Evaporator (SE500). Toluene is lipophilic and it will capture AC and keep it from evaporating.

When the samples are evaporating, only the MeOH will evaporate while the toluene and

lipophilic compounds will remain.

Most of the samples were almost dry after evaporation due to inadequate monitoring (they

were in the machine for half an hour and there was still MeOH left in the glasses; after

another hour in the machine, they were almost completely dried out). To recover the samples,

1000µl of toluene was added to all of the vials, after which they were sonicated for two hours.

Because of precipitate on the bottom, the samples were filtered one more time. Sodium

sulphate was added to the samples and Pasteur pipettes with glass wool and sodium sulphate

atop were used. The rinsed samples were collected in the GC-glasses and stored in the freezer

until further use.

5.7 GC-MS ANALYSIS

The GC-MS instrument used for the AC analysis consisted of a 6890N Network GC System,

MS 5975, and a 7683B Series Injector, from Agilent Technologies (for a general description

of the function of the GC-MS, see Appendix F).

The method ran was called SIM (single ion monitoring) and the sub-method chosen was AC

with Phenanthrene-d10. SIM is used if the purpose of the analysis is to quantify something (in

our case AC).

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For the analysis of AC, a blank containing dichloromethane was placed on the autosampler to

commence the sequence. The purpose of this first blank (Blank1) was to rinse the needle of

the injector before proceeding to the samples treated with AC. Next followed the water,

tissue, and sediment samples. Seven more blanks were added in between the samples at a 5-7-

step interval, the final blank (Blank8) following the last sample. At the end of the sequence, 4

GC-MS-vials with different concentrations of AC (30, 100, 150, 250 !g) were mounted on the

autosampler and used to calibrate the machine.

The T0 sediment samples were ran separately on the GC-MS, the loading sequence being

T0(1), T0(2), T0(3), AC 30, AC 100, AC 150, AC 250. They were treated in the same way as

all the other samples.

As the GC-MS is started, the needle of the injector is firstly washed with CH2Cl2. Only after

this step has been performed, the first sample is taken for analysis (in our case Blank1). It

takes 28.95 minutes for the machine to process one sample, and some cooling time in between

samples is also required.

The parameters used for running the GC-MS instrument were:

Inlet-F Temperature – 250℃ (the temperature required for changing phase from liquid to gas);

Column Flow – 1.0 ml/min (represents the speed of carrying the analyzed compound with

helium. Helium is chosen in our case because it doesn’t interact with AC);

Inlet-F Pressure – 8.1 Pa;

Oven Temperature - 80℃;

Inlet-F Total Flow – 23.8 ml/min (this parameter illustrates the flow of gas used to rinse the

machine);

MS Quad – 150℃!(the temperature in the quardupole);

MS Source – 250℃; HiVac – 6.20e-006 (the vacuum inside the MS);

These numbers represent the parameters existent at the beginning of the analysis (t = 0

minutes). It should be noted that some parameters change over time (e.g. the oven

temperature). The program used for the oven can be seen in table 3.

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Table 3. Program used for the temperature increase in the oven

Step ℃/min Next ℃ Hold min

Initial 80 2.00

Round 1 20.00 165 11.00

Round 2 50.00 200 2.00

Round 3 20.00 280 5.00

As the first sample is harvested by the injector, 1!l of the mixture of AC and phenanthrene-

d10 is injected into the GC unit. The mixture is carried further by helium and is vaporized as

the temperature reaches 250℃. Helium was chosen because it does not react with AC. In the

oven, the chemicals are guided by the helium along the column (length 30m, ∅0.25, DB-1701,

Agilent Technologies), and the temperature is slowly increased from 80 to 280℃. Inside the

column the compounds are sorted by boiling point and polarity, so that the compound with the

lowest boiling point is lead through first and the heavier compounds last. After passing

through the column, the chemicals enter the MS unit. In the MS there is a vacuum and

therefore no other molecules, except the ones lead there by the column. Here the compounds

are bombarded with electrons, thus making them lose electrons and create positive ions. Most

positive ions of organic compounds are charged with a surplus of energy and in the absence of

other ions the molecule will split into ion fragments. These ion fragments represent the

specific fingerprint of the molecular ion. Each ion has a specific mass to charge ratio (m/z)

and is lead through a small hole in a negatively charged plate and into the quadrupole. The

charge between the poles in the quadrupole changes with a frequency of 1 MHz (1.000.000

times/sec). At a set current and frequency, only ions with a specific mass to charge ratio will

pass through. The current, produced by the ions, is amplified by an electron multiplier

HED/EM (High Energy Dynode/Electron Multiplier). In the HED, the flow of ions from the

quadrupole hits a dynode that releases electrons. Each of these electrons hits the inner walls of

the EM and releases more electrons. This electron flow is shown as a peak on the GC graph,

where the area under the curve is an expression of the amount of ions. When running the SIM

programme, the current and frequency is changed over time so that the ions will be “sorted”

by their mass to charge ratio. This change can occur between 1 and 10 times/sec. If the change

in current and frequency is set too fast, the sensitivity will be lowered. A strong current

increases the sensitivity, but shortens the lifespan of the machine.

Three types of reference ions were selected for both AC and the internal standard:

30"|"P a g e "

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Group 1 – the AC ions – Consisted of the target ion 246.00 m/z (Tgt), and two qualifying

ions: 161.00 m/z, 231.00 m/z (mass/charge)(Q1 and Q2).

Group 2 – Deuterium phenantherene-d10 ions – Consisted of the target ion 188.00 m/z (Tgt),

and the qualifying ions: 80.00 m/z and 189.00 m/z (Q1 and Q2).

5.8 STATISTICAL METHODS

In order to test the hypothesis of this report, the General Linear Model Test and One-Way

ANOVA were used. The Tukey Test was also used to assess the differences between

individual samples. The program used for computing all the statistical data was SYSTATS.

The analysis of variance (ANOVA) is used to investigate the differences between the means

of two or more samples. A prerequisite of ANOVA is that the samples that undergo analysis

must be evenly distributed. The reason for choosing One-Way ANOVA is because we were

dealing with only one parameter (AC concentration).

The General Linear Model Test was used merely because it offered more options than

ANOVA, while still including the analysis of variance.

While performing the tests, the confidence interval chosen was always 0.95. As additional

options, the normality test, Kolmogorov-Smirnov, and the Equality of variance test, Levene,

were chosen.

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6. RESULTS

During the exposure of the organisms to AC and after the analysis of the water, tissue, and

sediment samples on the GC-MS machine, two different sets of effects have been monitored.

They can be grouped into effects of AC on the test organisms (these have been observed

through behavioral changes and assessment of mortality levels) and fate of AC (change in AC

concentration in water, sediment, and soft tissue).

6.1 EFFECTS ON ORGANISMS

MORTALITY

Table 4 offers an overview of the number of organisms that were alive at the end on the

experiment exposure and their biomass.

Table 4. Tissue weight and corresponding number of organisms for:

A. Mya arenaria, and B. Macoma balthica

AC TREATMENT AC TREATMENT

Weight (g) Nr. of org. Weight (g) Nr. of org.

Mya 1 0.1865 1 Mac 1 0.2683 1

Mya 2 0.2800 2 Mac 2 0.3088 2

Mya 3 0.4481 3 Mac 3 0.4182 4

CONTROL CONTROL

Mya 1 0.0998 2 Mac 1 0.0628 4

Mya 2 0.1368 2 Mac 2 0.1395 3

Mya 3 0.0753 2 Mac 3 0.2039 2

During the 14-day exposure period, a total number of 5 organisms died from the Mya AC

groups, while 4 organisms died from the Macoma AC group

From the mortality graphs (figure 10 and 11), we can see that under similar conditions,

Macoma balthica has a higher resistance to environmental stress than Mya arenaria, since

less organisms from the Macoma group died, compared to the Mya group.

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Figure 10. Cumulative comparison of mortality of Mya between Control and samples treated with AC.

From comparing the two graphs we can see that the Myas began dying already from the

second day of exposure, whereas the Macomas - on the 5th day.

Figure 11. Cumulative comparison of mortality of Macoma between Control and samples treated with AC.

BURROWING BEHAVIOR

During the exposure period, the burrowing behavior of the test organisms was observed. This

was done in order to see how the organisms perceive the sediment they were introduced to, if

they accepted the conditions (i.e. AC treatment, no treatment), and if they behave as predicted.

Under normal circumstances, the organisms would instantly start burrowing once placed atop

of the sediment. In the presence of AC, this behavior might be altered as the organisms might

consider the new environment ‘unfit’ for burrowing.

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To obtain a better overview of the burrowing behavior exhibited by Macoma balthica and

Mya arenaria during the 14-day exposure interval, the burrowing-table from Appendix B was

used to see how often (in %) the organisms would be inside, outside, or halfway in the

sediment.

1) Macoma AC and Macoma Control

After summing up all the instances in which the organisms would be in, out, or halfway in the

sediment, it was found that in the groups treated with AC, Macoma would be burrowed in

~64,5% of all the possible cases, halfway burrowed in ~18,5% of the cases, and atop of the

sediment in 17% of the cases. As for the Control group, Macoma was burrowed in ~70% of

the cases, halfway in ~10,5%, and out in ~19,5% of the cases.

Macoma’s burrowing behavior shows that in most of the cases they burrow well and stay in

the sediment. This applies for both Control and AC treated samples. There were however

some Macoma that did not burrow fully or stayed above the sediment. That might be due to

individual preferences. In general, the behavior from both types of treatments follows a

similar pattern. Figures 12 and 13 offer a better view of the behavior exhibited by the

organisms form the Control and AC groups, during the 14-day exposure period.

%%%%%%%%%%%%%%%%%% %

Figure 12. Burrowing behavior exhibited by Macoma in the Control groups.

From figure 12 we can see that 8 out of 12 organisms burrowed in the sediment as soon as the

exposure began. This is a good sign of organism fitness and shows that the organisms were

not affected (stressed) too much during the acclimatization and exposure period.

0%

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From figure 13 we can see that it took a one full day for the Macomas from the samples

treated with AC to burrow in the sediment, compared to the ones from the Control samples

that burrowed fully within the first 1/2h. Also, the number of organisms that chose to not fully

burrow or not burrow at al was slightly higher in the AC treated group compared to the

Control group.

%%%%%%%%%%%%%%%%%%

Figure 13. Burrowing behavior exhibited by Macoma in the groups treated with AC.

2) Mya AC and Mya Control

The burrowing of Mya was not as successful as that of Macoma. It was expected that they

would go in the sediment but they mainly stayed partially burrowed and close to the surface

of the sediment.

In the AC treatment, the organisms were burrowed in 25,5% of the cases, halfway in the

sediment in ~52% of the cases, and out ~22,5% of the time. In the Control group, ~22% of the

time the Myas were burrowed, they were halfway in the sediment ~18% of the cases, and the

remaining ~60% of the time they were atop of the sediment.

Figures 14 and 15 illustrate the burrowing behavior of Mya in the Control samples and in the

samples treated with AC. From these graphs we can see that Mya has a chaotic distribution of

organisms, both in the Control and AC group.

0%

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Figure 14. Burrowing behavior exhibited by Mya in the Control groups.

In both the Control and AC group there is a tendency for the organisms to burrow in the

sediment, after which the tendency slowly turns towards going out of the sediment. In the

Control it can be seen that in day 4 there are just 4 organisms out of the sediment, one day

later the number increasing to 10, after which, on the 9th day the number goes down to 3. This

could be explained by the fact that the organisms that went out of the sediment, after being

previously burrowed, simply died and were removed.

Figure 15. Burrowing behavior exhibited by Mya in the groups treated with AC.

0%

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No.of%organism

s%

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3) Comparison between species

The burrowing behavior of the two organisms gives us a clear insight on the different

responses that the two species have when dealing with the same stress factors.

When comparing the two organisms, we can observe that Macoma burrowed more readily

into the sediment compared to Mya and this may indicate that Macoma is more resistant to

external stress (the burrowing graphs from Macoma are stable and the mortality is lower

compared to Mya, that have fluctuating burrowing behavior and higher mortality).

6.2 FATE OF AC

The results obtained from the GC-MS machine, which are relevant to our study, can be seen

in table 5. These numbers were used to determine whether the AC added to the different

samples was biodegraded during the 14-day period in which the experiment was held.

Table 5. Amount of AC measured in sediment, water, and tissue on day 14, and initial amount (T0)

Treatment( Sediment(AC( Body(burden( Water(

,, (ug/g), (ug/g), (ug/ml)%Mya,ac,1, 23.1147, J% J%Mya,ac,2, 23.1214, J% J%Mya,ac,3, 25.0069, J%% J%Mac,ac,1, 24.3754, 92.1729, J%Mac,ac,2, 22.0136, 76.2953, J%Mac,ac,3, 22.5651, DAMAGED, J%T0,initial,ac,1, 37.1318, >, J%T0,initial,ac,2, 35.6810, >, J%T0,initial,ac,3, 40.8549, >, J%Microbial,degradation,1, 28.1599, J, J%Microbial,degradation,2, 22.1222, J, J%Microbial,degradation,3, 21.2805, J, J%No,organisms,1,(control), >, J, J%No,organisms,2,(control), >, J, J%No,organisms,3,(control), >, J, J%

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ACETYL CEDRENE IN SEDIMENT

As expected, all the sediment samples spiked with AC showed the presence of AC on the GC-

MS mass-spectrum. The presence of AC is showed in the form of a peak in the region where

the selected target ion, and the qualifying ions are found (see figure 16 and 17).

Figure 16. The small peak to the left represents the abundance of AC found in the sample. The peak to the right

represents the mass-ion spectrum for the internal standard, phenanthrene-d10.

Figure 17. The ions used for identifying AC were the target ion 246.00 m/z (Tgt), and the qualifying ions 161.00

m/z, 231.00 m/z (Q1 and Q2).

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Both One-Way ANOVA and the General Linear Model were used to test if our alternative

hypothesis (HA – AC conc. T0!≠ AC conc. Tend) is accepted or rejected. In other words, to see

if there is a notable difference between the amount of AC found in the T0 samples, and the

amount found in the Mya, Macoma, and microbial degradation samples, at the end of the

exposure period.

A bar chart graph was selected to better illustrate the differences in AC amount (!g/g) found

in the T0 samples, and the ones found in the no-AC/no-organism sample (Control), and the

Mya, Macoma, and microbial degradation samples treated with AC (see figure 18).

Figure 18. (left) Comparison between AC concentrations in T0 and Tend samples. (right) Comparison between

AC concentrations within the the Tend samples.

From the bar chart we can see, as expected, that the AC concentration in the T0 samples is

greater than that of the Mya, Macoma, and microbial degradation samples. It should be noted

that the amount of AC present in the T0 samples is slightly lower (avg. 38!g/g dw sediment)

than the nominal concentration of AC in the sediment (50!g/g dw sediment). As for the

Control samples, no trace of AC has been detected.

According to Levene’s Homogeneity Test (table 6), the data obtained is evenly distributed,

with a p-value of p = 0.078.

Table 6. Homogeneity of variances

Levene's Test for Homogeneity of Variances Test Statistic p-Value Based on Mean 3.314 0.078 Based on Median 0.459 0.719

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We proceeded by presuming that the alternative hypothesis is accepted. By running the

General Linear Model test on the T0, Macoma AC, and Mya AC groups, a p-value of p =

0.000 was obtained (see table 7). This means that there is a significant difference between the

AC level in the T0 samples and the one from the other samples (p<0.05).

Table 7. ANOVA results

Analysis of Variance Source Type III SS df Mean Squares F-Ratio p-Value TREATMENT$ 465.346 3 155.115 25.939 0.000 Error 47.840 8 5.980

To further test which of the samples are different (or if all the samples are different) from the

T0 sample, the Tukey HDS test was performed. From the results presented in table 8, we can

observe that the AC concentration in T0 is significantly different from that of the Macoma AC

(p = 0.000), Mya AC (p = 0.000), and microbial degradation AC (p = 0.001) samples. On the

other hand, when compared among each other, the end samples (Tend) did not show a

significant difference among AC levels: Macoma AC vs Mya AC – p = 0.980, Macoma AC

vs microbial degradation AC – p = 0.971, and Mya AC vs microbial degradation AC – p =

1.000.

Table 8. Results obtained from the Tukey Test.

Tukey's Honestly-Significant-Difference Test TREATMENT$(i) TREATMENT$(j) Difference p-Value 95% Confidence Interval Lower Upper Mac ac Mya ac -0.763 0.980 -7.157 5.631 Mac ac Micro degr ac -0.869 0.971 -7.264 5.525 Mac ac T0 initial ac -14.905 0.000 -21.299 -8.510 Mya ac Micro degr ac -0.107 1.000 -6.501 6.288 Mya ac T0 initial ac -14.142 0.000 -20.536 -7.747 Micro degr ac T0 initial ac -14.035 0.001 -20.429 -7.641

ACETYL CEDRENE IN TISSUE

Traces of AC have been found in the Macoma samples treated with AC, with a value of

92!g/g for replicate 1, and 76!g/g for replicate 2. These results were displayed in the form of

a small peak on the mass-spectrum graph. Even though the peaks themself were under the

3xbackground noise value needed for qualifying the samples, we proceeded in determining

the AC concentration. We decided to do this as none of the other samples showed a similar

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trend (thus we can exclude the possibility of contamination from other samples).

No traced of AC were detected in any of the Mya samples.

Figure 19. AC peak on the mass-spectrum in the Macoma AC 2 sample.

ACETYL CEDRENE IN WATER

No detectable traces of AC have been found in the water phase of the samples.

7. DISCUSSION

7.1 EFFECTS OF ACETYL CEDRENE

During the experimental period, both burrowing behavior and mortality have been monitored.

From the mortality graphs presented in section 6.1 (figure 10 and 11), we could observe that

several organisms from both the Mya and Macoma samples have died. This was a trend that

was not expected, as the concentration chosen for AC was not high enough to be considered

lethal. We believe that the mortality might not be due to the presence of AC, as both the

Control and AC treated samples of each of the test organisms have a similar number of dead

organisms (i.e. 5 organisms in Control and 5 in AC treated samples for Mya; 3 in Control and

4 in AC treated samples for Macoma).

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From the graphs we can also see that M. arenaria began dying earlier in the exposure period

(day 2) compared to M. balthica (day 5). Also, by the end of the experiment, a larger number

of Myas (10 in total) was reported dead, compared to Macoma (7 in total).

We presume that the reason why some of the organisms died was because of handling and

constant stress brought to their environment (e.g. acclimatization, frequent movement of the

organisms, defective air pumps). Another explanation for the increasing numbers of dead

organisms per beaker might be that once one of the organisms died, decomposing organic

matter was released, hence contaminating the water and making the other organisms more

susceptible to dying.

If multiple toxic compounds are present in the environment, the toxicity of the mixture will

approximately sum up the values of toxicity of the individual components (Walker et. al.,

2006). This means that every individual chemical would roughly have the same toxicity when

measured in a mixture or measured alone. The concentration of each of the chemicals that

take part in a mixture dictates the levels of toxicity that each of the chemicals will have. This

information might be relevant to our study because we do not know if the sediment used for

the experiment set-up was contaminated with any other compounds. Thus, if any other toxic

compound was present in the sediment, it could explain why organisms from both the Control

and AC treatment groups died.

When observing the burrowing behavior, two different trends were noticed, a stable

burrowing behavior for Macoma and a fluctuating one for Mya.

M. balthica behaved as expected, burrowing in the sediment by the end of the first day, both

in the Control and treatment group (see section 6.1, figure 12 and 13). This is generally a sign

of good fitness. If comparing the Control group to the AC treated one, it can be seen that most

of the organisms in the Control were fully burrow within half an hour, whereas it took an

entire day for the organisms in the treatment group to do the same. This might be because

they sensed the presence of AC and it took time for them to adjust to the new environment.

Also, the number of organisms that did not fully burrow (halfway in) or not burrow at all (out)

was slightly higher in the AC treated group compared to the Control group. This might also be

due to the presence of AC in the former.

M. arenaria’s burrowing behavior shows a totally different distribution pattern, as the data is

more chaotically dispersed. This fluctuation in burrowing behavior numbers seen in the first

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half of the graphs (for both the Control and AC samples) might be explained by the fact that

the monitoring was performed by different people in different days. No specific method of

assessing the burrowing behavior was established. Thus, what one person might have noted

down as halfway in the sediment, another might have perceived as out of the sediment (the

same applies for burrowed vs halfway burrowed). The way in which a person saw in what

state the organisms were was especially affected by the angle from which the observer looked

at them (see figure 20). However, it might be argued that if this was the case, why didn’t the

same pattern appear in the Macoma samples as well. In the case of Macoma, it was easy to

say, without any problem, if the organism was inside, halfway, or outside of the sediment.

However, Mya was more problematic as one could not be always sure of the actual position in

which they were (the delimitations between in/halfway and halfway/out, were hard to

establish). When looking at the second half of the two graphs, the fluctuation in patterns was

also a result of mortality.

Figure 20. The burrowing behavior results might be slightly

biased by the angle at which the observation was made (i.e.

viewing the organisms from atop of the beaker or from side-

view through the glass).

Research states that M. arenaria has greater difficulty in reburrowing after disturbances. This

is due to the size its foot, which remains the same as the clam grows, therefore the older the

clam, the less successful it is at re-burrowing (Checa & Cadee, 1997; Pfitzenmeyer &

Drobeck, 1967). In our experiment this could imply that the larger organisms were not able to

burrow. If the clam was placed horizontally, it might have difficulties to turn itself vertically

and commence burrowing.

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7.2 FATE OF ACETYL CEDRENE

When assessing the concentration level of AC in the T0 sample, it was observed that the initial

concentration was lower than expected (~38!g/g dw sediment, instead of the aimed value of

50!g/g dw sediment). This might be due to loss during handling, evaporation from the system,

and degradation mediated by microbes.

From the results obtained from the one-way ANOVA, we can see that there is a significant

difference between the AC concentration of the T0 sample and that of the microbial

degradation sample (p = 0.000). We believe that this is mainly a result of microbial activity.

When comparing the AC level of the microbial degradation samples to that of the Macoma

AC and Mya AC samples, the statistical data showed that there was no significant difference

(microbial degradation vs Macoma, p = 0.971; microbial degradation vs Mya, p = 1.000).

This implies that the two test organisms were not efficient at taking up and metabolizing AC.

Furthermore these results show that the movement of the organisms in the sediment had no

effect on microbial degradation.

We were expecting to see a lower level of AC in the samples containing M. balthica, due to

its feeding strategy and because the water was filtered, forcing Macoma to obtain its nutrients

solely from the sediment.

Traces of AC have been found in the tissue of the organism, suggesting that Macoma did take

up the contaminant from the sediment. The actual concentration of the xenobiotic in the tissue

was higher than the initial AC concentration added to the system. Since this did not

significantly affect the overall concentration present in the sediment, we could presume that

the organism was accumulating and storing it, but the rate at which they did so was too slow

to have an impact. This could be supported by research on M. balthica with respect to other

organic contaminants. For example, it has been shown that Macoma is efficient in

accumulating PAHs, but not biortansforming them (Rust et. al., 2004).

As for Mya arenaria, it was expected that it would be less efficient at taking up AC from the

sediment, due to its suspension-feeding habits. In general, Mya has been shown to be capable

of metabolizing some PAHs (e.g. benzo[∝]pyrene) at a higher rate than M. balthica, however

this doesn’t seem to be the case with AC.

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8. CONCLUSION

From the result obtained from ANOVA, we could conclude that the alternative hypothesis is

accepted because there is a significant difference between the T0 samples and the Macoma

AC and Mya AC samples (T0 vs Macoma AC, T0 vs Mya AC, p = 0.000). However, because

the microbial degradation was not significantly different when compared to the samples with

organisms, we can safely state that any difference in AC concentration detected in the

Macoma and Mya samples was due to the presence of microbial degradation. Thus, we have

to reject our alternative hypothesis and accept the null hypothesis; the presence of Macoma

balthica and Mya arenaria does not affect the fate of AC, but microbial degradation does.

Since there is no significant difference between the amount of AC found in the Macoma and

Mya samples, we can not state which of them has the greatest impact on AC, or if there is any

impact at all.

When looking at the effect of sediment-associated AC on the test organisms, the mortality

graphs provide no evidence that mortality was caused by AC. As for the burrowing behavior,

we believe that the organisms were aware of its presence, and therefore they were not

particularly keen to burrow (at least in the case of Mya). It should be also mentioned that the

AC ingested by Macoma might have had an effect on the actual organisms, but we were not

able to tell how exactly they were affected.

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9. APPENDICES

9.1 APPENDIX A – dw:ww measurements

Table 9. dw:ww measurements for determining the amount of sediment needed for experimental set-up.

Aluminum boat - WW

(g) WW sediment (g)

Aluminum boat –

DW (g) DW sediment (g)

7,3448 5,6864 5,9651 4,3067

10,3840 8,7218 8,2511 6,5889

5,3954 3,7315 4,4989 2,8351

Table 10. Measurements for dw:ww

AC TREATMENT

Aluminum

tray (g)

Sediment –

wet weight (g)

Sediment + tray –

dry weight (g)

Sediment –

dry weight (g)

Mac 1 1.6653 2.1590 3.2652 1.5999

Mac 2 1.6067 2.1118 3.2152 1.6085

Mac 3 1.6168 2.1509 3.2340 1.6172

Mya 1 1.6051 2.1212 3.1968 1.5917

Mya 2 1.6186 2.1324 3.2447 1.6261

Mya 3 1.6103 2.1303 3.2436 1.6333

Micro. degr. 1 1.6087 2.1030 3.1896 1.5809

Micro. degr. 2 1.6075 2.1440 3.2586 1.6511

Micro. degr. 3 1.6140 2.1305 3.2463 1.6323

NO AC TREATMENT

Mac 1 1.6300 2.0730 3.2231 1.5931

Mac 2 1.6180 2.0840 3.1949 1.5769

Mac 3 1.6156 2.0580 3.1948 1.5792

Mya 1 1.6062 2.1601 3.2653 1.6591

Mya 2 1.6040 2.1560 3.2580 1.6540

Mya 3 1.5990 2.1445 3.2167 1.6177

No organisms 1 1.6054 2.0517 3.1749 1.5695

T0 SAMPLES (AC TREATMENT)

T0 1 1.6060 3.7511 4,4629 2.8569

T0 2 1.6129 2.8668 3,8033 2.1904

T0 3 1.6010 3.2387 4,1359 2.5349

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9.2 APPENDIX B – Burrowing behavior exhibited over a 14-day period

%% Mya"AC% Mya"Control%Time/beaker%no.% 1% 2% 3% 1% 2% 3%after"1/2h" 1i,%2h,%1o% 2i,%2o% 1i,%2h,%1o% 1i,%3h% 1i,%1h,%2o% 1i,%3o%after"1h" 2i,%2h% 1i,%3o% 2i,%2o% 2i,%1h,%1o% 2i,%1h,%1o% 1i,%3o%after"8h" 2i,%2h% 2i,%2o% 2i,%1h,%1o% 2i,%1h,%1o% 2i,%2o% 2i,%1h,%1o%26.03.%Tue% 2i,%2h% 2h,%2o% 2i,%1h,%1o% 2i,%1h,%1o% 2i,%2o% 2i,%1h,%1o%27.03.%Wed% 4h% 2h,%2o% 2i,%1h,%1o% 2i,%1h,%1d% 1i,%1h,%2o% 2i,%2o%28.03.%Thu% 4h% 3h,%1o% 2i,%1h,%1o% 1i,%2h,%1d% 1i,%1h,%2o% 1i,%2h,%1o%29.03.%Fri% 2i,%2h% 4h% 2i,%1h,%1o% 1i,%2h,%1d% 1i,%1h,%2o% 1i,%1h,%2o%30.03.%Sat% 4h% 4h% 2i,%1h,%1d% 3o,%1d% 1i,%3o% 4o%31.03.%Sun% 4h% 4h% 2i,%1h,%1d% 3o,%1d% 1i,%3o% 4o%01.04.%Mon% 1h,%3d% 4h% 2i,%1o,%1d% %%%%%3o,%1d"% 2i,%2o% 4o%02.04.%Tue% 1h,%3d% 4h% 2i,%1o,%1d% 3o,%1d% 1i,%2o,%1d% 3o,%1d%03.04.%Wed% 1h,%3d% 4h% 2i,%1o,%1d% 3o,%1d% 1i,%2o,%1d% 3o,%1d%04.04.%Thu% 1h,%3d% 4h% 2i,%1o,%1d% 1h,%2o,%1d% 1i,%2o,%1d% 3o,%1d%05.04.%Fri% 1h,%3d% 3h,%1d% 2i,%1o,%1d% 1h,%2o,%1d% 1i,%2o,%1d% 3o,%1d%06.04.%Sat% 1o,"3d% 2h,%1o,%1d% 2i,%1o,%1d% 1h,%2o,%1d% 1h,%1o,%2d% 3o,"1d"%07.04.%Sun% 1o,%3d% 2h,%1o,"1d% 1h,%2o,%1d% 1h%2o,%1d% 1h,%1o,%2d% 3o,%1d%08.04.%Mon% 1o,%3d% 1h,%2o,%1d% 1h,%2o,%1d% 1h,%2o,%1d% 1h,%1o,%2d% 1h,%2o,%1d%Dissection% % 1d" " 1d" " 1d"

LEFT% 1" 2"" 3" 2" 2" 2"

! Mac"AC" Mac"Control"

Time/beaker%no.% 1" 2" 3" 1" 2" 3"

after"1/2h! 2i,%2h" 2i,%2o" 1i,%3h" 3i,%1h" 3i,%1o" 2i,%2o"after"1h! 2i,%2h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1o" 2i,%2o"after"8h! 2i,%2h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1o" 2i,%2o"26.03.%Tue% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1o" 2i,%2o"27.03%Wed% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1o" 2i,%2o"28.03%Thu% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1o" 2i,%2o"29.03%Fri% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1h" 2i,%2o"30.03%Sat% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"31.03%Sun% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"01.04%Mon% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"02.04%Tue% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"03.04%Wed% 3i,%1h" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"04.04%Thu% 3i,%1d" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%2o"05.04%Fri% 3i,%1d" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%1o,%1d"06.04%Sat% 3i,%1d" 2i,%2o" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%1o,%1d"07.04%Sun% 2i,%2d" 1i,%2o,%1d" 3i,%1h" 3i,%1h" 3i,%1d" 2i,%1o,%1d"08.04%Mon% 1i,%1h,%2d" 1h,%1o,%2d" 3i,%1h" 3i,%1h" 2h,%1o,%1d" 1h,%1o,%2d"Dissection% 1d" " " " " "

LEFT% 1" 2" 4" 4" 3" 2"

Legend:" i%=%inside,%h%=%halfway,%o%=%out%(of%sediment),%d%=%dead%

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9.3 APPENDIX C – Water volume in samples (from overlaying water)

Table 11. A. Overlaying water volume from the AC treatment

AC Treatment

Time Organisms (beaker nr.) Overlaying water (ml)

15:02

Mya (1) 130

Mya (2) 130

Mya (3) 130

15:18

Macoma (1) 130

Macoma (2) 130

Macoma (3) 130

15:53 Microbial degradation (1) 140

Microbial degradation (2) 130

Microbial degradation (3) 130

Table 11. B. Overlaying water volume from the Control samples

CONTROL

Time Organisms (beaker nr.) Overlaying water (ml)

14:32 – 14:54

Mya (1) 130

Mya (2) 140

Mya (3) 130

14:08 – 14.29

Macoma (1) 130

Macoma (2)* 140

Macoma (3) 130

14:00

No organisms (1) 140

No organisms (2) 100

No organisms (3) 140

*in the Macoma (2) beaker, there were fine particles of sediment suspended in the water

column.

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9.4 APPENDIX D - Laboratory Techniques: The ASE Machine: Accelerated Solvent Extractor

Accelerated Solvent Extraction (ASE) is a method used for the extraction of test compounds

from soil or other solid matrices. The process itself is fully automated.

The machine is made up of a static part (consisting of the Electronics Area, Oven Area, and

the Solvent Reservoir Compartment) and a mobile part (consisting of the Needle Mechanism,

the Cell Tray – where the cells are loaded –, and the Vial Tray – where the glass-vials are

positioned –). The Cell Tray can support a number of 24 cells and 4 rinse-tubes, while the

Vial Tray can support 26 vials (60ml, clear glass) and 4 rinse-vials (60ml, amber glass)(vial

type: EPA-type VOA) (DIONEX, 1999).

Figure 21. ASE machine components (image source: DIONEX, 1999)

Prior to using the ASE machine, the Dichloromethane is placed on an ice bath to avoid the

formation of bubbles (Dichloromethane has a low boiling point) and the Nitrogen pump is

turned on. As mention earlier in the report, the program selected for the ASE machine was:

a. Preheat (0 min)

b. Heat (5 min)

c. Static (15 min)

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d. Flush (50 vol)

e. Purge (60 sec)

f. Cycles (2)

g. Pressure (2000 psi)

h. Temperature (60℃) i. Solvent (Dichloromethane, 100%)

Before running any of the loaded cells, the machine will first rinse itself with

Dichloromethane and deposit the waste into the amber rinse-vials. After this step, the samples

are processed.

The standard procedure for running a sample-cell consists of preparing the cells and loading

them on the Cell Tray (the same is done with the glass-vials). Once the machine is started, the

cells and glass vials are rotated to the initial positions specified by the running method. The

machine’s needle then perforates the glass-vial corresponding to the processed cell. The cell is

picked-up and moved to the oven. Dichloromethane is then used to wash the cell. The next

step consists of heating the cell up to 60℃ (so the cell reaches thermal equilibrium) and

pressurizing it to 2000 psi. Static extraction then takes place, after which the contents of the

cell are re-washed with fresh solvent. As Dichloromethane passes through the sample, it

attaches to and collects the chemicals of interest, washing them into the glass vials situated on

the Vial Tray. The process ends with the unloading of the cell.

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9.5 APPENDIX E - Sample preparation for ASE

Table 12. ASE guideline. This table records the amount of sediment and hydromatrix used for

each sample per ASE cell. The far right column illustrates the number of sample corresponding

to each position on the ASE machine.

AC TREATMENT

Sediment

(g)

Hydromatrix

(g)

Corresponding cell

(nr.)/(ASE nr.)

Mac 1 5.79 5.05 1%(2)

Mac 2 5.34 5.02 2%(3)

Mac 3 5.11 5.12 3%(4)

Mya 1 5.04 5.10 4%(5)

Mya 2 5.74 4.99 5%(6)

Mya 3 5.73 5.00 6%(7)

Micro. degr. 1 5.72 5.00 7%(8)

Micro. degr. 2 5.51 4.97 8%(9)

Micro. degr. 3 5.67 5.03 9%(10)

NO AC TREATMENT

Mac 1 5.23 5.02 10%(11)

Mac 2 5.16 5.08 11%(12)

Mac 3 5.04 5.14 12*(13)

Mya 1 5.69 5.03 13%(14)

Mya 2 5.28 5.04 14%(15)

Mya 3 5.15 5.13 15%(16)

No organisms 1 5.61 5.20 20%(1)

T0 SAMPLE

T0 1 5.3260% 5.0094% 22%

T0 2 5.1060% 5.0677% 23%

T0 3 5.1855% 5.0531% 24%

*cell nr 12 (Mac 3) – the internal standard was added twice (200 µl instead of 100 µl)

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9.6 APPENDIX F - Laboratory Techniques: The GC-MS Machine

The Gas Chromatography – Mass Spectrometer (GC-MS) instrument is used for separating

chemicals based on the ease with which they evaporate into a gas (change state from liquid to

gas), and identifying them based on the structure they possess.

The instrument is composed of two parts: the gas chromatography (GC) part that separates the

chemical compounds into pulses of pure chemicals, and the mass spectrometer (MS) part that

is used for identifying and quantifying the chemicals (OSU, 2013).

Figure 22. The different components of the GC-MS machine

The Gas chromatography unit of the machine is made up of three parts:

- Injector – extracts the solvent from the GC-MS-vials and sends it to the GC. The needle of

the injector extracts one microliter (1 !l) of the compound and injects in the GC from where

the sample is propagated further by a non-reactive gas. The injector can be heated up to a

temperature of 300℃ to change the phase of the solvent from liquid to gas.

- Oven – represents the outer part of the GC unit. It contains the column, which is heated to

enable the molecules of the analyzed chemicals to move through it. The oven can normally

reach temperatures from 40℃ to 320℃ (OSU, 2013).

- Column – is situated inside the oven and consists of a thin tube of varying lengths and

thicknesses that is coated on the inside with a special polymer (polymer coating, length and

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thickness depend on the type of chemical analyzed). The chemicals that undergo analysis are

separated based on their volatility and are transported through the column with the help of the

non-reactive gas. Chemicals that have high volatility propagate faster through the column than

the ones that are characterized by low volatility (OSU, 2013). The volatility of any given

substance is linked to the size of its molecules, as small molecules tend to propagate at higher

speeds compared to larger molecules.

The Mass Spectrometer unit consists of:

- Ion source – once the chemical analyzed has passed through the GC, the resulting pulses go

to the MS unit. The molecules are then broken into pieces, as they are blasted with electrons.

This determines the molecules to lose, themselves, electrons and become positively charged

particles (ions). These charged particles then continue their way to the filter.

- Filter – the charged particles then pass through an electromagnetic field that uses the mass

of the ions as a base for filtration. The desired range of masses (that will be able to pass

through the filter) is chosen prior to the commencement of analysis.

- Detector – quantifies the ions with the desired mass (target ions) and sends the collected

information to a computer, which reinterprets it in the form of a mass spectrum. The mass

spectrum is illustrated in the form of a graph that consists of a number of ions of different

masses that have passed through the filter.

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9.7 Appendix G – Overall results

Table 13. Overall results. From left to right: Treatment type, Initial AC concentration (added in the sediment), Measured AC concentration (at the end of the exposure period), Calculated AC concentration (based on dw),

Dry weight, Wet weight, dw:ww ratio, Amount of sediment measured used for analysis (ww), Amount of sediment used for analysis (dw)

Figure 23. Additional General Linear Model graphs: (left) Overall view of the To vs Tend samples; (right) Comparison between Tend samples.

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