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The potential of biodegradation on 1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane, based upon co-metabolism of

indigenous bacteria

Graduation thesis

Made by: Anikó Hellebrandt

Supervisor:

Dr. Emma Nehrenheim

Mälardalen University, School of Sustainable Development of Society and Technology (HST)

Consultant:

Dr. Nóra Kováts

University of Pannonia, Institute of Environmental Engineering

Västerås-Veszprém

2010

Table of Contents

Abstract .................................................................................................................................................. 8

Kivonat ................................................................................................................................................... 9

Introduction .......................................................................................................................................... 10

I. Aim ................................................................................................................................................... 11

II. Literature review .............................................................................................................................. 12

2.1. Proposition of the problem ......................................................................................................... 12

2.2. DDT, the cause of the problem .................................................................................................. 12

2.2.1. Introduction and basic properties of the DDT ..................................................................... 12

2.2.2. Occurrence in nature .......................................................................................................... 14

2.2.2.1. Sources of DDT ........................................................................................................ 14

2.2.2.2. Behavior and distribution of DDT in nature ............................................................. 14

2.2.3. The effects of the DDT ....................................................................................................... 15

2.2.3.1. Bioaccumulation and biomagnification .................................................................... 15

2.2.3.2. Effects on the ecological system and animals ........................................................... 16

2.2.3.3. Effects on human ...................................................................................................... 17

2.3. Clean up of the DDT .................................................................................................................. 18

2.3.1. International governmental action ....................................................................................... 18

2.3.2. The Stockholm Convention ................................................................................................ 19

2.3.3. DDT in Sweden .................................................................................................................. 19

2.3.4. Remediation processes, Soil remediation ............................................................................ 20

2.3.4.1. Introduction of the soil remediation methods ........................................................... 20

2.3.4.1.1. Ex situ methods ................................................................................................ 21

2.3.4.1.2. In situ methods ................................................................................................. 21

2.3.4.2. Biodegradation ......................................................................................................... 22

2.3.4.2.1. Biodegradation by microorganisms .................................................................. 22

2.3.4.2.2. Microorganisms in the soil ............................................................................... 22

2.3.4.2.3. Microbial degradation process, co-metabolism ................................................. 23

2

2.3.4.2.4. Appropriate conditions for the microorganisms ................................................ 23

2.3.4.2.5. Factors positively influencing the microbial degradation ................................ 24

2.3.4.2.5.1. Effect of Oxygen on the degradation ........................................................ 24

2.3.4.2.5.2. Effect of temperature on the degradation ................................................. 25

2.3.4.2.5.3. Effect of pH on the degradation ................................................................ 25

2.3.4.2.5.4. Effect of nutrients on the degradation ....................................................... 25

2.3.4.2.6. The advantages of bioremediation .................................................................... 26

2.3.4.2.7. The disadvantages of bioremediation ............................................................... 26

2.3.4.3. Phytoremediation ..................................................................................................... 28

2.3.4.3.1. Introduction to phytoremediation .................................................................... 28

2.3.4.3.2. Advantages of phytoremediation ...................................................................... 29

2.3.4.3.3. Disadvantages of phytoremediation .................................................................. 30

III. Materials and methods ................................................................................................................... 31

3.1. Analysis of soil .......................................................................................................................... 31

3.1.1. Determination of the Dry Weight ....................................................................................... 31

3.1.2. Determination of the Water Holding Capacity (WHC) ....................................................... 31

3.2. Spiking soil samples .................................................................................................................. 32

3.2.1.Introduction to soil spiking .................................................................................................. 32

3.2.2. Full treatment protocol ........................................................................................................ 33

..................................................................................................................................................... 33

3.2.3. Partial treatment protocol .................................................................................................... 33

3.2.4. DDT concentrations in the soil samples .............................................................................. 34

3.3. Bioreactor experiment ................................................................................................................ 34

3.3.1. Preparation and set up of the bioreactors ............................................................................ 34

3.3.2. Sampling from the bioreactors ............................................................................................ 35

3.4. Toxicity testing with Ostracodtoxkit FTM ................................................................................. 36

3.4.1. Hatching the ostracod cysts ................................................................................................. 37

3.4.2. Pre feeding the cysts ........................................................................................................... 38

3

3.4.3. Length determination of the freshly hatched ostracods ....................................................... 38

3.4.4. Preparation of the algal food suspension ............................................................................. 39

3.4.5. Preparation of the test plates .............................................................................................. 39

3.4.6. Scoring of the results- mortality scoring and length measurement ...................................... 39

3.4.7. Data Treatment ................................................................................................................... 40

3.5. Phytoremediation, pot experiment with Brassica Juncea ........................................................... 41

IV. Results ........................................................................................................................................... 42

4.1. Parameters of the soil ................................................................................................................. 42

4.1.1. Given parameters ................................................................................................................ 42

4.1.2. Measured parameters .......................................................................................................... 43

4.2. Spiking the soil .......................................................................................................................... 43

4.3. Ecotoxicology test results .......................................................................................................... 44

4.3.1. Test Sample 1. ................................................................................................................... 44

4.3.2. Test Sample 2. ................................................................................................................... 45

4.3.3. Evaluation terms of the Tests .............................................................................................. 45

4.3.4. Observations ....................................................................................................................... 46

4.4 Chemical analysis ....................................................................................................................... 50

5.1. The bioreactor experiment ......................................................................................................... 53

5.2.The phytoremediation experiment .............................................................................................. 54

VI. Conclusions .................................................................................................................................... 55

6.1. The bioreactor experiment ......................................................................................................... 55

6.2. The phytoremediation experiment ............................................................................................. 55

VII. Future work .................................................................................................................................. 56

VIII. Appendix ..................................................................................................................................... 57

8.1.Ecotoxicology test results ......................................................................................................... 57

8.1.1. Test 1. ................................................................................................................................. 57

8.1.2. Test 2. ................................................................................................................................. 62

IX. References ...................................................................................................................................... 64

4

............................................................................................................................................................. 74

X. Acknowledgements .......................................................................................................................... 75

5

THESIS ASSIGNMENT FOR

MASTER OF ENVIRONMENTAL ENGINEERING STUDENTS

Major

Radioecology

Department

Institute of Environmental Engineering

Title of thesis:

The potential of biodegradation on 1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane, based

upon co-metabolism of indigenous bacteria

Supervisors:

Dr. Emma Nehrenheim

Dr. Nóra Kováts

Task leading department:

Mälardalen University,

School of Sustainable Development of

Society and Technology (HST)

Task to be executed:

A previous project at Mälardalen University studying the degradation of TNT from soil

samples in bioreactors, showed interesting results for the potential of indigenous soil bacteria

to degrade TNT contamination. During the processing the available literature from previous

experiments should be summarized as well. In the practical part of the research project the

degradation of DDT from soil samples in bioreactors with the help of indigenous

microorganisms should be examined, together with the potential of phytoremediation. On the

basis of the experimental results the candidate should evaluate the potential of the concerned

bioremediation methods

Special requirements:

Well established knowledge of environmental engineering and of English language, the

perfection in basic laboratory practice, and use of laboratory equipment.

Deadlines of the distinct parts of the project:

1. Literature review and summary till the end of July 2010

2. Laboratory experiments till the beginning of June 2010

DIPLOMAMUNKA FELADAT

KÖRNYEZETMÉRNÖK SZAKOS HALLGATÓK RÉSZÉRE

Szakirány

Radiökológia

Tanszék

Környezetmérnöki Intézet

Diplomamunka pontos címe:

Az 1, 1, 1-triklór-2, 2-bisz(p-klórfenil)etán biodegradációs potenciáljának vizsgálata a talajban

eredetileg jelen lévő mikroorganizmusok segítségével

Témavezetők:

Dr. Emma Nehrenheim

Dr. Kováts Nóra

A kidolgozás helyszíne:

Mälardalen University,

School of Sustainable Development of

Society and Technology (HST)

Az elvégzendő feladat:

A Mälardalen University-n már előzőleg kísérleteztek TNT talajmintákból való

degradációjával bioreaktorokban. A rendelkezésre álló szakirodalom feldolgozása alatt

előzetes tanulmányok, tapasztalatok összegzésére is szükség van. A gyakorlati munka során a

DDT talajmintákból való degradációjának bioreaktorokban a talajban eredetileg jelenlévő

mikroorganizmusok segítségével való vizsgálata, illetve a fitoremediációban rejlő

talajtisztítási lehetőségek tanulmányozása is szükséges. A kísérleti eredmények alapján a

jelöltnek értékelnie kell az érintett bioremediációs eljárások alkalmazási lehetőségét.

Speciális követelmények:

Megalapozott környezetmérnöki tudás, az angol nyelv készség szintű ismerete. Laboratóriumi

munkában illetve a laboratóriumi eszközök használatában való jártasság.

Részfeladatok teljesítésének határideje:

1. A szakirodalom áttekintése és összefoglaló készítése 2010 július végéig

2. Laboratóriumi kísérletek elvégzése 2010 június elejéig

Abstract

The purpose of this project is to evaluate the potential of a bioreactor system to degrade DDT

based upon co-metabolism of indigenous bacteria. The study was performed with soil samples

spiked with four different concentrations of DDT. The prepared sludge was circulated at a

steady rate of revolution per minute in bioreactors with added M8 solution, cabbage leaf

extract and molasses. The experiment was carried out for 7 days and chemical analysis and

toxicity testing was accomplished at the beginning and the end of the experiment. The

chemical analysis was essential to support the conclusions of the ecotoxicology tests.

Ecotoxicology test was performed for the assessment of the toxicity (in terms of bioavailable

measures) of the sludge samples, and was carried out with the Ostracodtoxkit FTM sediment

toxicity test, with the freshwater benthic crustacean test species Heterocypris incongruens. As

part of the project the potential of the bioremediation method phytoremediation have been

studied. Brassica Juncea seeds have been cultivated in the soil spiked with four different

concentrations of DDT for one month, under stable circumstances. Growth of the plants was

measured at the end of the experiment, and a chemical analysis was carried out. A thorough

literature review was carried out for both the bioreactor and the phytoremediation experiments

in order to obtain information about methods and theoretical background. The ecotoxicology

tests and the chemical analysis showed increased p,p’- DDT concentrations in the bioreactors

I. and II. at the end of the 7 day experiment, the reasons of which are not known, and require

further studies.

Key words: soil contamination, DDT, Ostracodtoxkit FTM, biodegradation, bioreactors,

phytoremediation

Kivonat

A jelenlegi tanulmány célja a bioreaktor rendszer hatékonyságának bizonyítása, mely a

talajban lévő DDT degradálását az eredetileg jelenlévő mikroorganizmusok segítségével

végzi. A kísérletek során használt talajt DDT-vel szennyeztük, négy különböző

koncentrációban, majd vízzel iszapot képeztünk, és ezt bioreaktorokban cirkuláltattuk 7 napon

át. A kevertetés állandó fordulatszámon zajlott, M8 oldat, káposzta levél kivonat és melasz

hozzáadásával. A kísérlet első illetve hetedik napján a reaktorokból mintát vettünk, és a

mintákat kémiai analízisnek illetve toxikológiai tesztelésnek vetettük alá. A toxikológiai

tesztelés fontos volt, hogy reprezentáljuk a szennyező anyag ökológiai hatását (biológiailag

felhasználható mérték tekintetében), a kémiai analízis pedig az eredmények alátámasztása

érdekében. A toxikológiai teszthez az Ostracodtoxkit FTM üledék tesztet használtuk, mely a

Heterocypris incongruens héjas állatkát alkalmazza. A minták analizálva lettek a szennyező

anyag meghatározása céljából. A diploma munka célja mindezek mellett a fitoremediáció

hatékonyságának tesztelése is, mely célból Brassica Juncea magokat termesztettünk a DDT-

vel szennyezett talajban egy hónapig terjedő időtartamban állandó körülmények között. A

kísérlet végén a növényeket különféle módszerekkel analizáltuk illetve hosszukat lemértük.

Mind a bioreaktor kísérlet mind a fitoremediáció esetében alapos szakirodalmi áttekintést

hajtottam végre, az elméleti háttér és a megfelelő módszerek tanulmányozása céljából. Az

ökotoxikológiai teszt és a kémiai analízis eredménye bizonyította, hogy a minták toxikusak

voltak, illetve a kémiai analízis megnövekedett p,p’-DDT koncentrációra utaltak az I. és II:

bioreaktorban a 7 napos kísérlet végén. Az okai a megnövekedett toxikusságnak és a p,p’-

DDT koncentrációnak nem ismertek, és kiderítésükhöz további tanulmányok szükségesek.

studies.

Kulcsszavak: talaj szennyezés, DDT, Ostracodtoxkit FTM, biodegradáció, bioreaktorok,

fitoremediáció

Introduction

The application of certain pesticides, have been grown during and after the Second World

War due to the extended demand of battling insects carrying diseases, such as mosquito born

malaria, or lice born typhus appearing primarily in the Tropics. The rise of the industrial

agriculture increased the damage caused by insects, and insect-related diseases on crops. The

need for effective, cheap and easily synthesized chemical substances emerged. Besides aldrin,

dieldrin or heptachlor, DDT was one of the pesticides widely used in agriculture and

healthcare in many countries worldwide. In the early sixties, starting with Rachel Carson’s

book, Silent spring, the attention was drawn to the possible negative effects of these

pesticides, and in the seventies they were banned in the USA and most of the European

countries. In spite of these efforts, the concerned chemicals were already wide spread in the

environment due to their physical and chemical properties, and started to aggregate in the soil,

waters and flora. Once it is aggregated in the environment, DDT is ingested by representatives

of the fauna. Through bioaccumulation and biomagnification it reaches the top of the food

chain, and is stored in fatty tissues of animals and human causing varied negative effects on

health. It has been shown to cause severe ecological damage due to toxic effects such as

thinning of eggshells, which is leading to population decrease among bird species.

Traditionally, these chemical substances were eliminated from soil and waters with chemical

and mechanical methods. As treatment methods developed, there emerged more

environmentally friendly techniques, such as biological remediation processes. The desirable

method is cost, - and time- effective, producing the least amount of contaminated waste.

Under natural conditions indigenous microorganisms play a major role in soil in the

degradation of pesticides [1]. In bioreactor systems the indigenous bacteria are more effective

as the temperature, oxygen levels, and nutrients are adjusted to the optimal levels.

Phytoremediation methods are based on the natural degradation potential of the plants, and

can clean up the soil in a cost-effective way, without disrupting the soil structure or reducing

soil productivity. Nowadays research is still carried out to improve and develop the

biodegradation methods.

I. Aim

The present study is an investigation of the potential of bioremediation methods, specifically

the biodegradation in bioreactors based on co-metabolism of indigenous microorganisms and

phytoremediation with Brassica Juncea (Indian mustard) seeds. This study is the first

bioreactor project carried out at the School of Sustainable Development of Society and

Technology at the Mälardalen University focusing on the degradation of DDT. Although,

there have been experiments accomplished by Nehrenheim et al. [2], applying the same

bioreactor system. The aim of the latter study was the degradation of the explosive TNT from

soil samples in sludge phase, with the help of indigenous microorganisms using different

amendments and conditions [2]. In our case the hypothesis is that the potential of the enzymes

originating from the co-metabolism of the indigenous bacteria is effective in degrading the

pesticide, and there is no further need to use inoculums. This hypothesis is based upon

previous study (R. V. Subba-Rao and Martin Alexander, 1984) showing result of the potential

of degrading DDT with co-metabolism performed by microorganisms and fungi [3] and also

Nehrenheim et al. 2008, where the inoculums proved to be inhibiting the rate of the

degradation [2]. The aim of the phytoremediation experiment is to prove that the Brassica

Juncea plant is able to take up the contaminants from the soil, and provides a well applicable

alternative for the degradation of DDT.

II. Literature review

2.1. Proposition of the problem

Insects are harmful in two major ways, damaging crops directly, and carrying diseases. The

problem of insects harming crops increased to a great extent through the intensification of the

agriculture. Intensified agriculture includes the application of certain techniques, equipment,

and special crops which provide high yield [4]. The application of one single type of crop on

large areas of land increases the insect populations in a rampant way. Insect populations grow

unnaturally big, because they have more crops to consume than originally, and this kind of

growth is demanding pesticides to protect the plants. The traditionally used pesticides are not

efficient for treating waste areas of agricultural land and with time insects develop resistance

to them (as a result of the over use of the pesticides) , therefore emerged the need for cheap

and easily produced effective new agents [20]. Diseases transmitted by insects, which are

harmful to human health, affect most of the tropical countries, especially due to the

unfavorable circumstances such as the lack of sanitation, or high population density. In times

of natural disasters or war there is augmented need for control, which often includes the usage

of certain chemicals, just like DDT [5].

2.2. DDT, the cause of the problem

For better understanding why DDT acquired such fame and importance, it is essential to

introduce the chemical itself and explain how it affects humans and the environment.

2.2.1. Introduction and basic properties of the DDT

DDT or more specifically 1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane is a hydrophobic,

colorless, almost odorless, crystalline organic compound [6]. The melting point of the

chemical is 109°C and it is usually made from chloral hydrate, chloro-benzene, and sulfuric

acid [7]. The chemical was first produced by Othmar Zeidler in 1874, but foremost Paul

Hermann Müller synthesized [8] the subject in 1939.

Figure 2.1. The conformation of the p, p`-DDT

(Picture made by Anikó Hellebrandt based on Markó László, 2006., Szerves kémia II.,

Veszprémi Egyetemi Kiadó, Veszprém, p. 64.)

Figure 2.2. The synthesis of the DDT

(Picture made by Anikó Hellebrandt based on Markó László, 2006., Szerves kémia II.,

Veszprémi Egyetemi Kiadó, Veszprém, p. 64.)

DDT proved to be effective against insects even at low concentrations, and to be harmless to

warm blooded animals and human according to contemporary studies. The chemical was easy

and cheap to produce and it had a long lasting effect. Because of its inherent properties, the

substance was widely used during the Second World War, against nits causing typhus, and

during and after the war against mosquito and other insects, such as lice or flies. It was widely

used against moths as well, been directly applied on the clothes [7]. According to US EPA

since 1940 approximately 2 billion kilograms of DDT was produced and applied worldwide

[85].

2.2.2. Occurrence in nature

The DDT started to appear in nature straight after its application in agriculture and pest

control started. Due to the extensive usage, it was soon spread worldwide. Although the

chemical was soon banned in many countries, it was (and is) still distributed in the

environment.

2.2.2.1. Sources of DDT

The DDT present in nature is transferred as far as the Arctic and the Antarctic through the

natural boarders from countries which are still applying the pesticide [20. 21.]. The DDT is

stored and transported most likely in the atmosphere, in the ocean(s), and in the soil.

It can derive from several different sources in the environment:

• Applied straight on the ground during use

• Released in the air, during its manufacture or use

• Washed in the soil and soil water from the place of the application, or storage/disposal

sites [22].

Although the substance is usually bound to soil particles, and stored in the air and water

surfaces, it is transferred to human with the contribution of the smallest attendants of the

environment.

2.2.2.2. Behavior and distribution of DDT in nature

As soon as DDT appears in nature, there are several important factors which will influence

the transport, storage and degradation of it. One of the most important factors is temperature,

and another is the surroundings. High temperature in air (tropical climate) is favorable for the

transport and degradation of the chemical, and accordingly at low temperatures (cold

climates) the degradation is slowed down, and it will be more likely to be stored in soil, or

natural waters [20. 23.-24.]. The surface of the land is another important influence, which will

affect the concentration of DDT in air and in soil. For instance forests will increase the DDT

amount stored in the soil [23.]. Other factors affecting the degradation and repository are pH

and organic material in soil, plant canopies, and altitude conducted condensation [26].

Typically DDT, just like other contaminants, is spreading in the environment in different

ways, which include

• Plants and animals consume contaminated water

• Plants absorb contaminants

• Animals consume the plants which previously absorbed the contaminants [27].

It is important to state the exposure routes, as the end of each route is always human. The

most important information according to our study is the amount of the chemical stored in

soil, with special consideration to Swedish soil. Although the basic properties do not change

when transferred to distinct areas, there is variant amount of DDT stored in soil in each

country according to the climate and the basic surroundings. Also several other factors affect,

just like the date the pesticide was banned in the given country, and the governmental action

done in terms of the elimination of the chemical. There are differences according to specific

areas in each country, as some locations can be more contaminated than others.

2.2.3. The effects of the DDT

The DDT has an impact on nature and human in an interconnected way, due to the basic

characters, the high chemical stability and solubility in fatty tissues. The main harmful effects

are occurring due to the bioaccumulation and biomagnifications of the substance.

2.2.3.1. Bioaccumulation and biomagnification

The main and most distressing problem is that the chemicals are gradually piling up in the

animals and human, and during years of exposure to them, they can aggregate to a level,

where they cause serious ecological and health problems. Through getting in the natural

waters, such as rivers, and groundwater, the chemicals are consumed by plankton, fishes and

birds, and accumulate in their fatty tissues. Bioaccumulation [9. 11.]

Chemicals with a bioconcentration factor (BCF) of 100 or more are susceptible of

bioaccumulation [84]. The chemical gets in the food chain and starts to aggregate in the fatty

tissues, increasing the concentration of the chemical for each step in the food chain, starting

from algae through fish to birds, and man. Biomagnification [10]

Figure 2.3. Biomagnification

(Picture made by Anikó Hellebrandt based on the website

http://www.fws.gov/contaminants/images/DDT.jpg)

Through the later effect chemicals can reach concentrations, when they cause harm on the

ecological system, and human also [84].

2.2.3.2. Effects on the ecological system and animals

http://www.fws.gov/contaminants/images/DDT.jpg

The proven ecological effect on birds (especially predatory birds as the white tailed eagle, the

bald eagle, the great horned owl, or the peregrine falcon) are egg thinning, evanescence and

damage of eggs, which can directly affect the long term population growth [18. 20. 84. 85.].

DDT can cause dysfunctions in the central and peripheral nervous systems of rats, through the

effect on the transport mechanisms in the nervous system, as it was proved by Wells et al.

(1974) [11]. The endocrine disruptor effect can cause changes in the hormonal system. These

hormonal changes induce altered sex ratio, estrogen effect on male animals, and even sterility

on rats. Studies on mice and rabbits resulted in decreased embryo implantation, irregularities

in the oestrus cycle and decreased foetal weights. It causes teratogenic effects in mice, rats

and hamsters through increased tumor production, especially in the lungs and liver. [12-17,

20.]. Another negative effect is that the chemical is non selective, this way killing the useful

insects as well. It is especially damaging for many species of the aquatic fauna [9]. Therefore

it is proven to be incontrollable, and harmful for the ecosystems [7].

2.2.3.3. Effects on human

Studies carried out in the UK found DDT, DDE and DDD residues in butter, eggs, milk, fish,

lamb and vegetables [87]. The amount of DDT found in food, and therefore the nutritional

intake is remarkably higher in developing countries, like India, or Egypt which still use the

pesticide to control malaria, and other insects carried diseases [20. 46.]. Due to fat solubility

people consuming contaminated food, will accumulate the substance in their fatty tissues, and

it will be stored for a long period of time. It takes more than 5 years to metabolize half of the

amount of the present DDT [19]. Residues (from 1.3 to 2600 ng/g lipid) of DDT and

metabolites, such as p, p’-DDE were found in most of the persons monitored during a

thorough survey carried out by WWF in 2003. During the survey blood samples were

analyzed for 78 chemicals, and the results (the metabolites were present in higher

concentration than the DDT itself) showed that most of the contamination found was coming

through indirect route (food) or from past contamination [87]. Through possible endocrine

disruptor effect, the concerned chemicals can be a reason for reproductive disorders, birth

defects, shorten the lactation period in women. Studies were showing decreased birth weights

among babies born in fisherman families, who were exposed to the chemical to a greater

extent due to the consumption of contaminated fish [84. 87.]. It has a negative effect on liver,

kidney, thyroid, blood and immune system, it is additional in causing allergies, skin disorders,

and hypersensitivity [12-17]. The US Department of Health and Human Services (DHHS),

and the US Environmental Protection Agency (EPA) determined the chemical and the

metabolites, DDE and DDD as probable human carcinogens [20]. Although the effects

depend on several various factors, as age, exposure time, dosage, state of health, the way of

contact (oral, dermal, or through the respiratory organs), personal sensitivity, therefore the

effect can be different on certain members of the population. In the following years most of

the developed/developing countries banned or restricted the usage of DDT and several other

persistent organic pollutants (POPs) [19].

2.3. Clean up of the DDT

The ban of the chemical was a compulsory step in the elimination process, but there emerged

the need for international governmental action. This kind of provision was inevitable in case

of the persistent organic pollutants, since they were affecting many countries.

2.3.1. International governmental action

The United Nations Environment Programme (UNEP) Governing Council in February 1997

decided that international action should be taken to reduce and eliminate the emissions and

production of 12 POPs including DDT, and an Intergovernmental Negotiating Committee

(INC) was found in preparation for an international agreement. The first INC meeting took

place in 1998 in Montreal, Canada, and created two assisting bodies, and carried out

immediate actions, such as fundings, evaluation of strategies, alternatives to POPS or

awareness raising workshops in developing countries [25]. The most important agreement to

defend the environment and the human health from the negative effects of the POPs and to

stop and/or regulate the production of the pesticide was the Stockholm Convention, which

will be elaborated in the next paragraph. The following years the European Commission

adopted the Soil Thematic Strategy (COM (2006) 231) and the proposal for a Soil Framework

Directive (COM (2006) 232) on the 22nd of September 2006 to protect soils in Europe from

contamination [53]. The European Community proposed a new regulation on chemicals,

called REACH (Registration, Evaluation, Authorization and Restriction of Chemical

substances) to protect the human health and the environment from the harmful effects of

chemicals, which entered to force on the 1st of June 2007 [86].

2.3.2. The Stockholm Convention

The Stockholm Convention was signed by many countries from all over the world in

Stockholm on the 22nd of May, 2001 and entered into force in 2004 [28]. The countries that

are part of the Convention should eliminate and stop (or reduce) the production of the

included persistent organic pollutants (POPs) and also clean up and remediate all the

remaining stocks of the pesticides. The production and use of the chemicals listed in Annex A

should be reduced, and many, including DDT mentioned in Annex B are banned from

production and usage. The agreement is not covering the cleaning of the polluted soils and

sites. The POPs in Annex A (Elimination) are aldrin, chlordane, chlordecon, dieldrin, endrin,

heptachlor, hexabromobiphenyl, hexabromodiphenyl ether, heptabromodiphenyl ether,

hexachlorobenzene (HCB), alpha hexachlorocyclohexane, beta hexachlorocyclohexane,

lindane, mirex, pentachlorobenzene, polychlorinated biphenyls (PCB), tetrabromodiphenyl

ether, pentatetrabromodiphenyl ether, toxaphene. The POPs in Annex B (Restriction) are the

following: DDT, perfluorooctane sulfonic acid, salts of perfluorooctane sulfonic acid,

perfluorooctane sulfonyl fluoride. Annex C (Unintentional production) contains

polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF),

hexachlorobenzene (HCB), pentachlorobenzene, polychlorinated biphenyls (PCB). Each

country taking part in the agreement should reduce or eliminate releases from intentional

production and use of the listed pollutants, from stockpiles and wastes, identify the

contaminated sites, and perform the remediation of these sites in an environmentally

satisfying way. The Convention was signed by 152 countries from all over the world, in 6

languages [28. 29. 82.].

2.3.3. DDT in Sweden

Pesticide registration system was set up in Sweden as early as 1953, and a new system

covering all the pesticides was introduced in 1963. Among aldrin and dieldrin there was a

restriction of usage for the DDT in 1970 [84]. Sweden was one of the first countries in Europe

to sign the Stockholm Convention in 2001. The Swedish EPA in 2003 estimated that

approximately there are 40.000 contaminated sites, and about ¼ of the sites are at great risk

[55]. Offshore NW Baltic proper (885 and 108 g m−2 water area yr−1, respectively) PCB and

DDT concentrations were similar in offshore and coastal areas. North Sea (Kattegat and

Skagerrak) sediment samples showed a p, p DDT mean value of 0.26 g/kg dry weight, which

is ranging from a minimum value of 0.01 g/kg dry weight to a 1.87 g/kg dry weight maximum

value. Total DDT values are mean 1.20, minimum 0.11 and maximum 3.0 g/kg dry weight

[30]. In our study, considering these values we chose the concentrations ranging from of

0.005 g DDT/kg dry weight soil to 1.0 g DDT/kg dry weight soil.

2.3.4. Remediation processes, Soil remediation

Although the affected countries stopped the production of the DDT after the signature of the

Stockholm Convention, and the use of the pesticides is controlled, many developing countries

are still producing and using the chemical, and as well it is accumulated in the soil, and

waters. The contaminated sites in Europe are approximately between 300.000 to 1.5 million

and the estimated total costs for the remediation of the sites in Europe are estimated between

59 and 109 billion euro [54]. It is inevitable to apply cost effective remediation processes in

cleaning up the pesticides, such as DDT. There is need for investigating possible remediation

techniques; within we are focusing on the bioremediation methods in this study.

2.3.4.1. Introduction of the soil remediation methods

The soil remediation methods can be different according to the place of the contamination, the

amount of the contaminant, and the endangered living beings and natural formations present

on the site. To decide which method is favorable in a given situation, there is need to make a

thorough study, which includes field studies, and chemical analysis, to determine the amount

and type of the contaminant(s), the physical, chemical, and biological properties of the

contaminants, the hydrogeological properties and the usage of the area, the state of the present

ecological system, and the microbiological state of the soil. The studies have to include risk

assessment for the area, studies about the urgency of the intervention, and the future

management of the area. After these studies are carried out, there is a wide range of

remediation methods; the choice is made based upon the cost- and time-effectiveness, and

availability of the method. There are several ways used in soil remediation, the two large

groups they can possibly be categorized into, ex-situ and in situ methods are elaborated in the

next chapters. Both the ex situ and in situ treatment methods include physical, chemical and

biological methods [31].

2.3.4.1.1. Ex situ methods

Typically ex situ methods mean that the affected soil is treated off site in all cases, usually

under laboratory conditions. The advantage of this treatment is that the soil is removed from

the actual site, and it is not affecting the local environment and residents. As well there can be

more complex methods used this way, which makes the process faster, and more effective.

For instance if there is more than one type of the pollutants, the ex situ methods are a good

alternative. Although there are several factors we cannot overlook, just like the exposure of

the personnel (transporting and handling the material) to the chemicals, or the transporting

and excavation costs, which can be especially high. With removing the soil, there is a

possibility of disturbing the natural habitat of species, and as well changing the surrounding

environment. The main physical ex-situ methods are: incineration, thermal desorption, soil

vapor extraction, mechanical separation, excavation and disposal. The chemical methods

include soil washing, solidification and stabilization, dehalogenation, solvent extraction,

chemical reduction and oxidation. The biological methods are land farming, and degradation

of the contaminants in bioreactors, the latter being used in this experiment. Other methods are

polymer adsorption, photolysis or solar detoxification, and vitrification [32].

2.3.4.1.2. In situ methods

In situ methods mean that the affected soil is being treated on site, this way avoiding the

transportation of the material, wherewith the personnel handling this operation avoid exposure

to the chemical. With this kind of technique, there isn’t possible to clear away more than one

type of pollutant, and usually a combination of several different techniques is applied. Very

precise planning and knowledge of the site is necessary, otherwise larger amount of pollutant

remains can be left on the site after the treatment. The physical methods are thermally

enhanced soil vapor extraction, containment systems and reactive walls and barriers, electro

reclamation, and landfill cap. The chemical methods are soil flushing, stabilization and

solidification, and biological are bioremediation, phytoremediation and natural attenuation.

The lasagna process is another in situ technology, which is a combination of biological and

physicochemical processes [32]. There are several other different ways to handle soils, just

like washing with surfactants, which in some cases is followed by photo-Fenton oxidation.

This process is used widely in the treatment of waste water, and specifically textile waste

water, and has 66 % efficiency in removing DDT and 80 % in removing DDE using Triton-X

100 aqueous solution [33]. The waste water after washing is going under a solar photo Fenton

process, which has 99-95 % of removing efficiencies [37]. The photo-Fenton oxidation

process is an advanced oxidation process, which is using solar irradiation and a special

catalyst reagent (Fenton reagent) to eliminate organic and inorganic pollutants [36]. Triton-X

i.e. Octyphenolpoly (ethylglykolether) x is an octylphenol ethylene oxide condensate, a

biodegradable (in liquid form) non ionic detergent [34. 35.]. Although there is a wide range of

choices from both ex- and in situ remediation methods, the main point is to find the most cost

effective and less hazardous waste producing available technology. In our study the focus was

on the biological methods, in the following the remediation method biodegradation is

presented, focusing on the contaminant degradation by indigenous microorganisms of the soil,

and phytoremediation.

2.3.4.2. Biodegradation

Biodegradation is avoiding the application of chemicals while eliminating organic

contaminants from the soil, based on the help of the natural inhabitants of the soil [38]. While

choosing the appropriate biodegradation method, we have to consider several factors, which

are the type and availability (for the microbial populations) of the pollutant, the type and

physicochemical state of the soil, the type and state of the microbial population in the soil,

and external factors, such as the temperature, pH, the presence of nutrients and oxygen [39].

2.3.4.2.1. Biodegradation by microorganisms

While performing bioremediation, typically fungi, bacteria, or plants can be used [40]. In this

project we study the degradation potential of indigenous microorganisms in bioreactors, and

the potential of plants (phytoremediation), however the focus was on microbial degradation,

therefore the next paragraph is a short description about the general residents of the soil.

2.3.4.2.2. Microorganisms in the soil

One way to classify the inhabitants of the soil is according to their size. The three big groups

according to this classification are micro-, mezo-, and macro-biota. The bacteria belong to the

micro fauna, and there can be a few thousand of species found in the soil, approximately 3-

500 millions per g soil, and around 1 µm average lengths. The soil bacteria can be categorized

as decomposers, nitrogen fixers, disease suppressors, aerob and anaerob, actino bacteria, and

sulphur oxidizers [41-43]. The bacteria suitable for the degradation of DDT includes

Escherichea Coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumonia,

Pseudomonas putida, Bacillus sp. (species), and Hydrogenomonas [42]. In a study

accomplished with activated sludge system, Pseudomonas sp., Pseudomonas aeruginosa,

Micrococcus, Bacillus pumilus, Bacillus circulans, Bacillus sp., Flavobacterium sp. were

isolated as microorganisms successfully degrading DDT (Sharma et al. 1987) [45]. In the

present study the most important are the aerob bacteria in the aspect of the degradation. The

indigenous (or isolated) microorganisms degrade the toxic pollutants. At the end of the

process, there should be mostly CO2 and water, with the residual microbial biomass and the

oxidized inorganic compounds, and finally the byproducts [46]. Implicitly the latter should be

the least amount.

2.3.4.2.3. Microbial degradation process, co-metabolism

The degradation process happens when the microorganisms use the contaminants as carbon

source, or with other words as electron donors, and with the electron donors, nutrients and the

electron acceptor more microorganisms are generated beside the oxidized end products [45.

46.]. During the co-metabolism of the microorganisms enzymes are produced, which are

accountable for the degradation of the organic chemicals. In this experiment the potential of

aerobic co-metabolism degrading DDT was studied.

2.3.4.2.4. Appropriate conditions for the microorganisms

The main point in using bioremediation systems (in the present study bioreactors) is to

increase the degradation effect of the natural inhabitants of the soil by ensuring appropriate

conditions for the best degradation rate [46].

These options are the following

• Adequate amount of oxygen (with aeration of the reactor system)

• Appropriate soil water content (sludge phase is preferable in the bioreactors)

• Neutral PH (control with the M8 stock solution)

• Appropriate amount of nutrients, both macro,- and micro nutrients (cabbage leaf

extract)

• Appropriate temperature (temperature above 10°C, room temperature) [47].

Moreover the soil should have 25-28 % water holding capacity, and low clay or silt content

and appropriate structure, to distribute air, water and nutrients adequately [39].

2.3.4.2.5. Factors positively influencing the microbial degradation

There are several different factors which influence the process in a positive way. The main

and most important attributes as mentioned in the previous paragraph are the amount of

oxygen added to the system, the appropriate temperature and the neutral pH of the soil-water

solution, and sufficient moisture, just as sufficient and appropriate nutriments [39]. According

to J. M. Aislabie et al. (1997), flooding of soil and additional organic matter can improve the

degradation of DDT [83]. In the following, the most important factors are elaborated.

2.3.4.2.5.1. Effect of Oxygen on the degradation

The Oxygen is inevitable for the aerobic degradation of the contaminants, as it works as

electron acceptor in the process. Typically the rate of oxygen can`t exceed 1 mg/L in aqueous

solution, considering that above this amount the microbial reactions are limited. This means

aerobic, minimum air-filled pore space of 10 %. The amount of the accessible oxygen will

decide that the system is aerobic or anaerobic. Air can be sprayed in the system, if the oxygen

supply is not sufficient.

The advantages of using oxygen as electron acceptor in the bioreactor systems is, that:

• Oxygen accelerates degradation

• Oxidized end products at the end of the process are non toxic

• Oxygen increases system stability

• Oxygen causes efficient system performance [39. 47.].

2.3.4.2.5.2. Effect of temperature on the degradation

The grade of the degradation is increasing with the rise of the temperature, as proved in

studies completed under tropical circumstances. At these climates most of the DDT departs

through volatilization [48]. The role of the temperature is important as well in the bioreactor

experiments; mostly the aim is keeping the temperature on approximately room temperature,

for the best results not below 10°C [49] but between 15-45°C [39]. Some studies suggest

temperatures 40°C, or higher, as the desirable temperature for the best degradation rate of

DDT [50]. The number of the biochemical reactions rise with the temperature rise, although

above a certain temperature the microbial cells decease [39].

2.3.4.2.5.3. Effect of pH on the degradation

Several studies have shown that the pH of the soil affects the degradation rate. Most studies

suggest the soil PH most competent for the best grade of degradation is around pH 7 (or

neutral pH) [49] and usually below this range the breakdown is slowed down [51]. If it is

necessary to adjust the pH, there is an opportunity to regulate it with buffer solutions [52]. In

this study the buffer M8 stock solution was used to guarantee the right pH [2].

2.3.4.2.5.4. Effect of nutrients on the degradation

The nutrients and the oxygen will stimulate the microorganism growth, which is essential to

reaching the right degradation rate. The nutrients will let the microbes produce the necessary

enzymes, which will degrade the contaminants. Carbon is the most needed nutrient, followed

by nitrogen, oxygen, hydrogen and phosphorus. The most desirable ratios are carbon to

nitrogen 10:1, and carbon to phosphorus 30:1. Sulfur, potassium, sodium, calcium,

magnesium, chloride, iron are other important elements building up the microbial cells [39].

2.3.4.2.6. The advantages of bioremediation

Bioremediation is supported by the public, as it is an option to degrade contaminants with the

help of the natural inhabitants of the environment, avoiding the use of unnecessary chemicals,

or complicated technology.

The main advantages are the following:

• The temperature and the pressure are close or equal with the environmental

temperature and pressure, accordingly no extra costs are included

• The amount of the chemicals used in the experiments is very low

• There can be water, sludge/slurry, and also gas phase treated, this means wide range of

applications

• The process is suitable for many types of contaminants, and many hazardous

compound can be transferred to harmless end products

• The amount of hazardous waste produced is low, as at the end of the process should

remain only carbon dioxide, water and cell biomass

• The remediation process can be performed on site, avoiding the costly and dangerous

transport of the contaminated soil

• Due to the favorable properties, bioremediation can be cost-effective compared to

other remediation technologies.

2.3.4.2.7. The disadvantages of bioremediation

Although bioremediation is a favorable technology, there are some necessary conditions for

the satisfactory operation of the system.

The disadvantages of bioremediation are:

• The pollutants have to be biologically degradable for this application.

• The products of the degradation can be more toxic than the original contaminant

• There are some factors, just like appropriate amount of nutrients, suitable temperature,

sufficient oxygen, without which the system is not successfully working

• The methods are not suitable to use on soil/water which contains chemicals resisting

the degradation

• The difficulty to predict the needed equipment and values for full scale operations

based on pilot-scale studies.

Bioremediation is however time consuming, therefore it is complicated to determine the end

point of the remediation process [39. 40.].

2.3.4.3. Phytoremediation

Phytoremediation is a relatively new technology, the basic idea is to contain, degrade and/or

eliminate pollutants from the soil, ground water and surface waters with the help of green

plants. The appropriate plants (hyper accumulator species) collect and turn the dangerous

chemicals into harmless secondary products. The applied species have a wide range from

forage plants to trees, and can be chosen according to the type of the contaminant, the

desirable time of the degradation process, and also aesthetical factors. The pollutants

eliminated this way can be heavy metals, radioactive material, explosives, and various

pesticides, or insecticides [56]. The necessary remediation mechanisms mainly happen in the

root system of the plant, and they are organic nutrition, water uptake, evapotranspiration,

photosynthetic production of phytochemicals, root exudation, root turnover, and bio-

metabolism, coming with processes by soil organisms such as protein and enzyme production,

bio-catalysis, and symbiosis with plant roots. The interaction happening between the root

system and the soil organisms in the rhizosphere are accountable for the degradation of the

contaminants [61].

2.3.4.3.1. Introduction to phytoremediation

Usually this type of bioremediation is applied in situ. It can be applied not only on

contaminated soil, but water, and groundwater as well. The main ways to clean the polluted

object are the following: collection, extraction, degradation, or storage of the concerned

chemicals. It can happen through phytoextraction (elimination of the pollutant and storage in

the plant tissue), rhizofiltration (the polluted water is taken up by the roots, and the plants are

disposed after), phytostabilization (the pollutants are absorbed, stabilized and stored in the

plant), phytodegradation (the pollutants are degraded to byproducts with the help of plant

enzymes), rhizodegradation (the same process as the previous, except that it is performed in

the rhizosphere of the plant), and phytovolatilization (the plants absorb the chemicals, and

release them into the atmosphere). The plant suitable for the remediation method needs to

have long roots, to reach deeper regions of the infected soil (in some cases this means the

application of tree species) in which case the long roots are not just cleaning the soil, and/or

groundwater from toxicants, but adding to fight erosion as well through ballasting the soil

particles. Hairy root cultures are degrading more effectively than plants without the structures,

or sterilized hairy roots [57]. In our case we are employing Brassica Juncea plants to

remediate DDT polluted soil samples ex situ, to approve the conformance of the technique.

The Indian mustard (Brassica Juncea) is suitable to use in this experiment because it has a

special root conformation and enzymes helping the degradation process.

Figure 2.3. Brassica Juncea plants

(Source available at http://www.botanic.jp/plants-sa/sekara.htm)

2.3.4.3.2. Advantages of phytoremediation

Phytoremediation proved to be a cost-effective and efficient soil remediation method in small

scale studies. The advantages of the phytoremediation among others are

• The high effectiveness compared to the low investment costs

• The approval of the communities because of the reasonable aesthetical properties.

• It can be used at sites with various types and concentrations of pollutants, such as

heavy metals, radioactive contaminants, scarcely hydrophobic organic pollutants, and

the contaminants can be removed at the same time with the same method.

• The plants are able to take up higher concentrations than most of the microorganisms

used in bioremediation.

• The remediation process is driven by solar energy

http://www.botanic.jp/plants-sa/sekara.htm

• The covered ground decreases the contaminant exposure to the environment and the

inhabitants of the affected area

• The vegetation decreases the effect of erosion on the site

• The useful topsoil remains undamaged

One important factor is to treat the plants appropriately after they collected the contamination.

This can mean disposal or extermination of the affected greenery. With phytoremediation the

produced hazardous waste at the end of the process is considerably less compared to other

remediation technology.

2.3.4.3.3. Disadvantages of phytoremediation

The disadvantages of the phytoremediation are the following

• It can be more time consuming than other remediation methods.

• The plants need the right conditions for their growth like light, temperature, water, soil

PH and nutrients, and in lack of these the cleanup process can be less effective or can

even fail

• The plants can typically clean up contamination from the soil layers near the surface,

to clean up deeper regions of the soil, trees have to be applied

• The contaminants can be volatilized in the air

• The method is less applicable for remarkably hydrophobic contaminants

• The method is not applicable for especially high contaminant concentrations

Furthermore the accessibility of the site must be controlled, to avoid contact with

livestock and human. The phytoremediation methods are still in an experimental phase

and therefore careful planning is needed before the full scale application is carried out.

[44. 58-60. 88.].

III. Materials and methods

3.1. Analysis of soil

There were several parameters given of the purchased soil gained by Weibulls Horto AB,

Sweden, which are elaborated in the results section of the thesis. There were, however, still

some unknown parameters, such as the dry weight and the water holding capacity, which had

to be determined.

3.1.1. Determination of the Dry Weight

The DW is important in order to calculate the proper amount of DDT needed to spike the soil.

The method of measuring the dry weight was the following, 3 hour glass were weighed (in the

following marked as I., II. and III.) Then there was 3*10 g soil weighed in the hour glasses.

The weights of the glass dishes containing the soil were measured and the values were

recorded. The dishes were placed in the oven (set to 105°C) overnight. After the soil was

taken out from the oven, the samples were placed in a desiccator (equipped without a vacuum

pump) for 1 hour and 20 minutes. This way the rest of the moisture was subtracted from the

soil samples. The soil weight was measured, and the amount of water volatilized from the

samples was calculated, from this data the dry weight was calculated.

3.1.2. Determination of the Water Holding Capacity (WHC)

The water holding capacity (WHC) is an important property of every soil showing, how much

the given soil is likely to leach. In this study it has to be determined because the plant`s

watering needs in the phytoremediation experiment will be calculated based on this property.

Usually soils with small sized particles and high organic matter content have optimum WHC.

The higher the WHC, the slower the soil reaches its saturation point (saturation point=when

no more water can be added to the soil) [62. 63.]. The method is the following, one plastic

cylinder was covered with a fine net at one end (R=3 cm), and was filled with soil (until

approximately 3 cm height). The cylinder was shaken gently for the earth to collapse. The

cylinder was placed in a beaker filled with ca. 2 cm tap water overnight.

Figure 3.1. Equipment for determination of the WHC

The beaker was picked up the next day, and the excess water was left to drain for 5 hours.

Three Petri dishes were weighed, and the soil samples were carefully transferred to the Petri

dishes, and weighed. The dry weight was determined after the samples were put in the oven

(set to 105°C) overnight. The soil samples were taken out from the oven and then placed in an

desiccator, for 1 hour. The weights of the samples were determined and dry weight and water

holding capacity was calculated.

3.2. Spiking soil samples

Originally the Structor Engineering Company was providing soil samples for the study, taken

from the southern region of Sweden, but due to the long and unusually cold winter, the

ground was frozen and not suitable for sampling in time. Therefore we provided the samples,

and spiked the previously purchased soil.

3.2.1.Introduction to soil spiking

To avoid bias originating from uneven distribution of the spiking material, the compound has

to be solved. The best available solvents for organic compounds are dichloromethane,

acetone, methanol, toluene, cyclohexane or ethyl acetate. Cyclohexanone proved to be the

most effective solvent for DDT [64-65]. During spiking the chemical (preferably previously

solved in the desirable solvent) is introduced to clean soil for determining the soil sample’s

toxicity. Soil spiking should ideally have minimal changes on the basic soil properties. The

spiking material and the soil are mixed thoroughly, and then left under fume cupboard to let

the solvent evaporate, to reduce solvent effects. The spiking material can be applied to the

entire amount of soil; or part of it, and then filled up with untreated (clean) soil.

3.2.2. Full treatment protocol

The solved contaminant was added to the required amount of soil, and the beakers containing

the mixture were left in fume cupboard for 5 days in order to let the contaminant spread

evenly in the samples and the excess solvent to evaporise. This way the solvent influences the

microorganisms the least. For performing the protocol, the DDT was measured on an

analytical scale, and then solved in cyclohexanone (20-5 ml according to the amount of

DDT). After this step 0.5 kg soil was measured in a beaker, and stirred together thoroughly

with the solved DDT for approximately 2-3 minutes. Finally 0.5 L tap water was added to the

soil, and mixed together thoroughly. The sludge was in fume cupboard for 6 days [67. 68.].

This method was used for spiking the soil samples treated in the bioreactors.

3.2.3. Partial treatment protocol

The solved contaminant is added to 25 % of the soil (carried out as in the Full treatment

protocol), and after 6 days waiting time, the spiked soil is mixed with the rest of the soil (75

%). The partial treatment protocol method was used for preparing the soil applied in the

phytoremediation process. In both cases it is important to mix the soil substantially with the

contaminant for 2-3 minutes with a metal spatula, and afterwards, creating the sludge, mix the

spiked soil thoroughly with water for ca. 5 minutes [67. 68.] this way avoiding bias deriving

from uneven distribution of concentration.

3.2.4. DDT concentrations in the soil samples

In soil samples taken from the Kattegat and Skagerrak in Sweden, DDT concentrations

usually are between 0.11 up to 3.0 g/kg dry weight [69]; we would like to represent values

present in Swedish soil, therefore the chosen concentrations for the experiment were the

following:

• 1 beaker with a concentration of 0.005 g DDT/kg dry weight soil




3.3. Bioreactor experiment

Bioreactor experiments were earlier accomplished at the department, therefore the bioreactor

system was assembled in the laboratory. The previously spiked soil and the four reactors were

prepared for the experiment, which included the transfer of the soil, the nutrients, the M8

solution, and water into the reactors.

3.3.1. Preparation and set up of the bioreactors

For the set up of the bioreactors, four clean beakers were prepared, each to be filled with soil

previously spiked with different concentrations of DDT (more detailed explanation is

included in the chapter soil spiking). Approximately 250 ml soil was measured in each plastic

beaker. M8 stock solution was prepared, 13.8 g Na2HPO4*H2O, 6.0 g KH2PO4, 1.0 g NaCl

was measured and thoroughly solved in 200 ml deionised water. 40 ml of M8 stock solution

was then measured with a pipette in four glass beakers. 16 ml of molasses were measured in

four glass beakers. 20 ml of previously prepared cabbage leaf extract (CLE) was measured

with pipette in four plastic beakers. Each reactor was filled up with the so prepared 250 ml

soil, 40 ml M8 stock, 16 ml molasses, and 20 ml CLE. After this step approximately 600 ml

tap water was added to compose ca. 1.0 liter sludge in the reactors. The stirrer was started up

around 250-500 rpm, and the top was placed on the reactor cylinder, finally the air extractors

were switched on, and placed above the reactors.

Figure 3.2. Basic features of a slurry phase bioreactor

3.3.2. Sampling from the bioreactors

After 30 minutes of stirring, from each reactor ca. 25 ml sample was taken and transferred in

plastic containers. Four days after the set up another sampling followed, and seven days later

the final sampling happened. The samples were dry frozen and kept in dark, before they were

sent to ALS Laboratory Group (Analytical Chemistry and Testing Services) for analysis.

Figure 3.3. The bioreactor system, filled with sludge containing DDT in four different concentrations.

3.4. Toxicity testing with Ostracodtoxkit FTM

Ecotoxicological tests are very important in the evaluation process of the experiment. These

tests will show the impact the substance has on the ecological system, in terms of bioavailable

measures. The testing material used for the ecotoxicological evaluation in this study is the

Ostracodkit FTM. This test kit is originally developed for sediment testing, and has been

widely used for measuring the toxicity of sediment samples contaminated with heavy metals

[70], and toxicity assessment of sewage sludge containing polycyclic aromatic hydrocarbons

and heavy metals [71]. In the present study it has been applied for testing the toxicity of the

sludge samples taken from the bioreactors. The test is applying the aquatic crustacean specie

Heterocypris incongruens.

Figure 2.4. Heterocypris incongruens consuming algae

(Source: http://www-eve.ucdavis.edu/stachowicz/mutualism.shtml)

Ostracod species are widely found in fresh and salt water habitats worldwide, and in the seas

and fjords of Sweden [72]. The test animals lay eggs (cysts) in the interest of survival when

the conditions are not suitable. The test kit contains these cysts, which can be stored under

appropriate conditions, and hatched according to the start of the test. The Heterocypris

incongruens and other ostracods are generally consumed by smaller fish species [73]. The test

species are members of the benthic fauna and represent an ecologically significant group. The

advantage of the test is that not only the bioavailable, but the stored contaminants are

measurable as well [74]. Every step of the test was carried out according to the Standard

Operational Procedure of the Ostracodtoxkit FTM. The preparation for the test included

hatching of the ostracod cysts, and pre-feeding after two days of incubation, which was

followed by the length determination. Subsequently the toxicity test was started.

3.4.1. Hatching the ostracod cysts

The cysts were hatched in plastic Petri dishes containing standard fresh water. The dishes

were kept under invariant circumstances in the incubator room at 25°C, for 52 hours, under

continuous illumination (light source of 3000-4000 lux).

http://www-eve.ucdavis.edu/stachowicz/mutualism.shtml

3.4.2. Pre feeding the cysts

After 48 hours hatching time a pre feeding step was performed, to provide the newly hatched

animals with food immediately. Vial containing Spirulina powder was filled with standard

fresh water, and mixed on a vortex thoroughly in order to disperse the particles. Spirulina

suspension was poured in the Petri dish, and the dishes were placed back in the incubator

room for 4 hours in the same circumstances.

3.4.3. Length determination of the freshly hatched ostracods

Shortly after the incubation of the ostracods, their length must be determined with dissection

microscope and the help of a micrometer slip. Therefore 10 ostracods were picked up from

the hatching Petri dish with a glass micropipette and put in a multiwell plate for measurement.

The ostracods tend to attach to solid surfaces when disturbed (thigmotactic property) [75],

therefore it is recommended to use glass pipette. The length figures (the newly hatched

ostracods are about 200 µm) were recorded on the result sheet included in the toxkit.

Figure 3.4. Length measurement of the freshly hatched ostracods

3.4.4. Preparation of the algal food suspension

To avoid the starvation of the test subjects during the 6 days exposure period, algal food

supplement was added to the test wells, as a preceding step of the testing. The vials containing

algal beads were shaken on a vortex so the algal beads were dissolved, their matrix were

broken up. Then the vials were centrifuged at 3000 rpm for 10 minutes, washed with distilled

water, and centrifuged again at the same rate and time. The solution was poured in a

volumetric flask and filled up with standard fresh water. Our aim was to get a homogenous

algal suspension.

3.4.5. Preparation of the test plates

2 ml standard fresh water, 1000 µl soil sample and 2 ml algal food suspension were

transferred in each test plate. The test plates were then gently shaken, in order to evenly

distribute the mixture of soil, algal food and water. After 5 minutes waiting time, while the

sediment settled at the bottom of each cup; the test was started. 10 newly hatched ostracod

were transferred in each previously prepared well (6 wells per test plate) with the

micropipette, under a dissection microscope (while picking up and replacing the test subjects

in the test cups, great attention is needed, because other than appropriate number of ostracods

will lead to distorted mortality results). The test plates were then covered with parafilm in

order to avoid evaporation of the water, and put in the incubator room at 25°C in darkness

(the tray containing the test wells was covered with a cardboard box top), for 6 days.

3.4.6. Scoring of the results- mortality scoring and length measurement

The soil was overlaying the ostracods, therefore all the contents of the wells were transferred

in a micro sieve, and washed under the tap, and then the test animals were transferred to a

Petri dish filled with standard fresh water. The live animals were counted, and scored on a

result sheet, and transferred in a multiwell plate with a glass micropipette for length

measurement. One drop of Lugol fixative solution (iodine solution) was added to each cup of

a multiwell plate to immobilize the ostracods. After a few minutes of waiting time, the length

was determined by the help of a micrometer slip, and scored on the results sheet. From the

length values the growth inhibition rate was calculated, which was showing the contaminant`s

sub lethal toxicity effect on the organisms.

3.4.7. Data Treatment

The number of dead ostracods was calculated by subtracting the number of live ostracods

from the original number (10) of ostracods in each cup. The mean mortality, mean percentage

mortality, mean length of 10 freshly hatched ostracods at the start of the experiment, mean

length of the live ostracods in each test cup containing the reference sediment, mean length

for all the cups with the test sediments (samples from bioreactors; I.,II.,III.,IV.) was also

calculated.

The mean length increment of the ostracods in the reference and test sediments was calculated

based on the following equation (1):

(1)

(The mean length increment drawn by Anikó Hellebrandt based on the Ostracodtoxkit F

Standard operational procedure, Micro Bio Test Inc., Belgium, Gent, 2010, page 26.)

The % growth inhibition was calculated according to the formula (2).

(2)

(The % growth inhibition drawn by Anikó Hellebrandt based on the Ostracodtoxkit F

Standard operational procedure, Micro Bio Test Inc., Belgium, Gent, 2010, page 26.)

3.5. Phytoremediation, pot experiment with Brassica Juncea

The soil was diluted with the remains of the spiked soil after filling up of the bioreactors. 1

part (1 liter soil) spiked soil was mixed with 2 parts (2 liter) clean soil. The soil was

thoroughly mixed for approximately 1-3 minutes with a metal spatula. The concentration of

the DDT in the pots therefore was 1/3 of the originally spiked soil. The same amount of soil

was transferred in 3 pots for each concentration, and 3 pots as control (with untreated soil), 15

pots together. The seeds were sown in the pots, and covered with ca. 1 cm clean soil, for the

best growth result. The pots including the seeds were incubated at 25°C, 3-4000 LUX, and

grown for approximately 30 days. After the half of the growth period, and at the end of the

growth period the height of the plants was measured. Then the plants were completely

removed from the pots, and sent to analysis, to determine the type and amount of DDT

residues in the soil, with the help of HPLC and DAD techniques. The total weight of the

plants was measured as well. The different weights of the plants would represent the different

DDT concentrations of the soil samples.

The test started on the 28th of April, and the plants were watered every third day with 100 ml

tap water (amount based on the water holding capacity measurements)

Figure 3.5.The phytoremediation experiment with 3*5 pots, one reference pot and four different DDT concentrations

IV. Results

4.1. Parameters of the soil

Several parameters, like mineral compounds, PH, particle size were known, displayed on the

packaging of the purchased soil. Although the dry weight and the water holding capacity was

measured in order to calculate the right amount of soil needed for the spiking, and the

appropriate amount of water needed for the plants .

4.1.1. Given parameters

Table 4.1.Soil components

Element Concentration (g/m3 ) Element Concentration (g/m3 )

Total Nitrogen (N) 112 Iron (Fe) 0.7Phosphorus (P) 56 Manganese (Mn) 1.3

Potassium (K) 120 Boron (B) 0.2

Magnesium (Mg) 256 Copper (Cu) 1.0

Sulphur (S) 63 Zinc (Zn) 0.3

Calcium (Ca) 2175 Molybden (Mo) 1.6

PH: ranging from 5.5 to 6.5.

Fine partition (particle size): 0-20 mm (fine)

Density: 400 kg/m3

Conductivity: 150-300mSiemens/L

Compounds: 95% peat and 5 % gravel

Additive: 7 kg lime per m3 and 0.8 kg dung (manure) per m3 soil.

4.1.2. Measured parameters

Table 4.2.Dry weight

Hour glass 1. (g)

Hour glass 2. (g)

Hour glass 3. (g)

Weight of the hour glass 45.492 46.200 54.872Weight of the soil and the hour glass 55.509 56.401 65.089

Weight of the soil 10.017 10.201 10.216

Weight of the soil after drying (with dish) 49.062 49.811 58.539

Amount of water volatilized from the samples 6.447 6.589 6.549

Dry weight 3.569 3.611 3.667

Mean dry weight 3.616

Table 4.3.Water holding capacity

Hour glass 1. (g)

Hour glass 2. (g)

Hour glass 3. (g)

Weight of the Petri dish 45.876 46.202 45.628Weight of the soil and the Petri dish 60.90 61.184 60.370


Weight of the samples after drying 48.535 48.896 48.337


Actual dry weight 2.687

4.2. Spiking the soil

To represent a relatively wide range of DDT concentrations present in the soil samples, we assembled the following:

1. 1 beaker with a concentration of 0.005 g DDT/kg dry weight soil (0.002 g DDT)




2, 4`-DDT was used to spike the soil, produced by Sigma Aldrich, Sweden AB.

The cyclohexanone solvent had a purity of 99. 5 %, and density of 0.95 g/cm3 and was

produced by Fisher Scientific.

4.3. Ecotoxicology test results

The complete tables containing the results can be found in the Appendix (chapter VIII.).

4.3.1. Test Sample 1.

Table 4.4.Mortality and growth inhibition in samples taken from the bioreactor on the 26th of

April 2010, 1ST day of the experiment

Reference sediment

Test sediment I.

Test sediment II.

Test sediment III.

Test sediment IV.

Mortality

Mean % mortality for all replicates

8.3 56.6 31.6 33.3 33.3

Standard deviation of mean % mortality

6.8 14.9 15.6 18.86 9.4

Variation coefficient of mean % mortality

85 26.1 48.75 57.2 28.5

Growth inhibitionMean length for all replicates 1044.36 498.7 591.95 741.43 802.5

Mean length increment for all replicates

837.36 291.7 384.95 534.43 595.5

Mean growth inhibition (in µ) for all replicates

- 545.66 452.41 302.93 241.86

Mean % growth inhibition in test sediment

- 65.16 54.03 36.17 28.88

4.3.2. Test Sample 2.

Table 4.5.Mortality and growth inhibition in samples taken from the bioreactor on the 3rd of

May 2010, 7TH day of the experiment

Reference sediment

Test sediment I.

Test sediment II.

Test sediment III.

Test sediment IV.

Mortality


5 100 100 60 100


7.64 - - 20.81 -


153 - - 35 -

Growth inhibitionMean length for all replicates

1097.5 - - 409 -


887.5 - - 199 -


- - - 688.5 -


- - - 77.6 -

4.3.3. Evaluation terms of the Tests

The tests are valid, if

• the mean % mortality of the ostracods in the control sample (reference sediment) is

less than 20 % (2 out of 10)

• the mean length increment in the of the ostracods in the control sample is at least 400

µm

4.3.4. Observations

Test number 1.: The test samples II.-IV. were showing a relatively high mortality rate of

average 30-33 %, except the sample number I., which was showing a mortality rate of 56 %.

The mean % growth inhibition is 65-28 %, and is representing the DDT concentrations well,

the highest growth inhibition (sample number I. contained the highest DDT concentration) in

sample number I., and decreasing in the following samples (such as the concentration of the

chemical is decreasing).

Figure 4.1.Variation coefficient of mean % mortality for the samples taken on the 1st day of

the experiment

Figure 4.2.Mean % mortality for the samples taken on the 1st day of the experiment

Figure 4.3.Mean % growth inhibition for the samples taken on the 1st day of the experiment

Test number 2: the sediment samples number I and II contained no ostracods that is a

mortality rate of 100 %, and is showing an obvious result, the samples were highly toxic.

Sediment sample IV contained 1 ostracod in one cup (6 cups per test plate), length 400 µm,

which also gives a mortality rate of 100 %. The top of the sludge samples in the cups

contained a silver colored thin film. The ostracods in the sample number III, were brown

colored, and slower moving than the ones in the Reference sample. According to the chemical

analysis, the concentrations of p, p-DDT were significantly high in samples (bioreactors)

number 1., and 2., compared to the other samples from the bioreactors number 3. and 4. The

possible reasons for the high toxicity rate are elaborated in the Discussion (chapter V.) part,

under bioreactor experiment title. It is interesting, that samples I-IV had 100 % mortality

rates, while sample III: only 60 %, and that there have been significant difference between the

p, p- DDT concentrations according to the chemical analysis. The growth inhibition was 77,6

% in the sediment sample number III, and was not possible to measure in samples number I, II

and IV, because of the 100 % mortality rate. Further studies and analysis are necessary; to

investigate what caused the high concentrations, and therefore the high toxicity in the

samples.

Figure 4.4.Variation coefficient of mean % mortality for the samples taken on the 7th day of

the experiment

Figure 4.5.Mean % mortality for the samples taken on the 7th day of the experiment

Figure 4.6.Mean % growth inhibition for the samples taken on the 7th day of the experiment

4.4 Chemical analysis

Table 4.6.Chemical analysis of the samples taken from the bioreactor on the 1st, 4th and 7th day of the experiment

Date: Samples taken from the bioreactor:

1.day of the experiment 2010 04.26.

4.day of the experiment 2010. 04.30.

7.day of the experiment 2010.05.03.

Abbreviations:

DW: dry weight

HCH: hexachlorocyclohexane

Figure 4.1.Diagram of the chemical analysis

V. Discussion

5.1. The bioreactor experiment

Biological degradation methods are favorable and therefore are getting more important, as

using these methods the usage of unnecessary chemicals is avoided, and minor amount of

hazardous wastes is produced. To prove the applicability and to improve the methods, there is

need to perform studies with different types and measures of amendments and added

nutrients. The present study was the first bioreactor experiment to degrade DDT from sludge

phase samples performed at the Mälardalen University. The test samples show 30-57 %

mortality and 30-60 % growth inhibition from samples taken on the first day of the bioreactor

experiment. The test plates from the 7th day of the experiment did not contain any ostracods at

the end of the experiment, therefore there a 100 % mortality rate is applied (except the sample

number III, where mortality rate was 60 %). The reasons for such high mortality rate in the

samples from the end of the experiment can be various. On the top of each test cup in the test

plates from the 7th day of the experiment, there was a thin silver colored film appearing. This

silver film could possibly inhibit the oxygen transfer from the air to the sludge surface, and so

providing an unfavorable anaerobic environment for the gill-breathing test organisms. The

contaminant could as well degrade in anaerobic conditions [76], but the microorganisms need

time to adapt to this condition. In anaerobic circumstances the byproduct DDD is formed [77].

And also the formation of the DDD is speeded up when the soil is flooded [76]. DDD is

carcinogen and suspected endocrine disruptor, and highly toxic for crustaceans [78]. DDE is

the major product during aerobic degradation [77]. This way we assume that during the 7 days

of the experiment, toxic substances was formed, such as byproducts of the DDT, which can be

even more toxic than the substance itself. Traces of the solvent, cyclohexanone, (which is a

clear oily liquid, insoluble in water) [79], can also affect the microorganisms in a negative

way. The concerned chemical proved to be toxic on aquatic animals [80]. Although after the

spiking, the soil samples were left in the fume cupboard for 5 days period, which time should

have been enough for the cyclohexanone to volatilize [81]. There have been 20 ml

cyclohexanone added to the highest concentration of DDT (which was then spiked into the

soil and marked as sample I.), and 10 ml to the sample II., both 5 ml to soil sample III., and

IV. After ca. 4 days of the bioreactor experiment, there appeared a white-and also grey

colored substance on top of the sludge, and on the inside of the bioreactor itself. This white-

grey material did not appear on the reactor number I. (which reactor contained the sample

number I. containing the highest concentration of DDT, and also the largest amount of

cyclohexanone as solvent). Nevertheless the second bioreactor contained some substance, and

there was quite a notable amount in the third and fourth bioreactors. We assume that the white

and gray colored substance implies the presence of indigenous bacteria and fungi. The fungi

can be both useful and harmful to the degradation, because it can produce useful metabolites,

but toxic material also.

5.2.The phytoremediation experiment

This experiment is the first phytoremediation attempt to degrade DDT from soil samples. The

plants were showing normal growth, and there was no significant difference between the soil

samples (which contained four variant DDT concentrations). It has to be mentioned, that the

soil used in the phytoremediation experiment was spiked using the partial treatment method,

which means that already spiked soil was mixed with clean soil. This step can reduce the

possible negative effect of the solvent on the microorganisms, and also reduces the

concentration of the DDT in the soil to 1/3 of the originally spiked soil. These can be the

reason of the satisfactory plant growth.

VI. Conclusions

6.1. The bioreactor experiment

The results of the ecotoxicology tests with Heterocypris incongruens showed that the samples

exerted a toxic effect on the test species, and the results of the chemical analysis are showing

increased concentrations of p, p-DDT in samples 1 and 2. At the same time the soil samples

were not inhibiting the plant growth.

Furthermore, there appears to be a great diversity of the type bacteria and fungi forming in the

different bioreactors. Further studies and analysis are necessary; to investigate what caused

the high DDT concentrations, and therefore the high toxicity in the samples.

6.2. The phytoremediation experiment

The results of sufficient plant growth are demonstrating that the DDT had no toxic effect on

the Brassica Juncea plants when added to the soil. Either the plants did not take up significant

amounts of DDT even though the plant itself is lipid rich, or DDT posed little acute toxic

effect to the plant growth.

Although it is important to state that the crops used in the a full scale application must be

energy crops, and not plants for feeding stock, or crops for human use, until a full scale risk

assessment has been performed. Cultivation of e.g. the energy crop Salix, common in

Sweden, could be argued to pose little risk to ecosystems and humans.

VII. Future work

The future work includes continuous experiments with bioreactors, with longer operational

times. Also there is potential in studying the presence of the indigenous fungi present in the

soil, and the effect of the fungi on the degradation of organic contaminants (vide DDT).

Phytoremediation should be henceforth studied, especially focusing on phytoextraction and

phytodegradation. The procedures in the plant`s rhyzosphere should be examined and

regarding literature thoroughly studied. This would include experiments with the biological

processes present in the ominous root zone.

VIII. Appendix

8.1.Ecotoxicology test results

8.1.1. Test 1.

Table 8.1.: Sample taken from the bioreactor on the 26th of April, 2010 (the 1ST day of the experiment) Reference sediment

Length

Day 0

Reference sediment

(µm) Day 6

Replicate

Test organism 1 2 3 4 5 6

1 200 1150 1150 1100 1100 1100 600

2 250 1100 1100 1050 1000 1150 550

3 200 850 400 1050 800 1100 850

4 210 1000 1100 1150 1050 1100 1050

5 200 1050 1100 1100 1100 850 1100

6 200 1100 1100 1150 850 1150 1150

7 200 1050 1050 1000 1050 1100 1200

8 200 1100 1000 1050 1050 1000 1150

9 200 1200 - 1000 800 1100 1150

10 210 - - - 1100 - 1000

Mortality

Number of dead ostracods per replicate

1 2 1 0 1 0

Mean % mortality per replicate 10 20 10 0 10 0


8.3


6.8


85

Growth inhibition

Mean length of ostracods per replicate

207 1066.6 1087.5 1072.2 990 1072.2 977.7

Mean length for all replicates 1044.36

Mean length increment per replicate

859.6 880.5 865.2 783 865.2 770.7


837.36


_


_

Table 8.2.: Sample taken from the bioreactor on the 26th of April, 2010 (the 1ST day of the experiment) Test sediment I.

Length

Day 0

Test sediment I.(µm) Day 6

Replicate


1 200 450 400 300 400 850 8002 250 600 400 350 450 900 850

3 200 550 300 - 500 450 600

4 210 400 350 - - 600 600

5 200 - 400 - - 450 800

6 200 - 300 - - 600 -

7 200 - - - - - -

8 200 - - - - - -

9 200 - - - - - -

10 210 - - - - - -

Mortality


6 4 8 7 4 5



56.6


14.9


26.1

Growth inhibition


207 487.5 358.3 325 450 641.6 730



280.5 151.3 118 243 434.6 523


291.7


545.66


65.16

Table 8.3.: Sample taken from the bioreactor on the 26th of April, 2010 (the 1ST day of the experiment) Test sediment II.

Length

Day 0

Test sediment II.(µm) Day 6

Replicate


1 200 400 650 900 450 400 600

2 250 450 850 600 600 550 650

3 200 600 600 600 450 600 400

4 210 550 600 850 650 600 600

5 200 400 600 - 400 850 400

6 200 450 600 - 600 550 -

7 200 550 600 - 900 - -

8 200 550 650 - 600 - -

9 200 - - - 400 - -

10 210 - - - 500 - -

Mortality


2 2 6 0 4 5



31.6


15.6


48.75

Growth inhibition


207 493.8 643.8 737.5 555 591.6 530



286.8 436.8 530.5 348 384.6 323


384.95


452.41


54.03

Table 8.4.: Sample taken from the bioreactor on the 26th of April, 2010 (the 1ST day of the experiment) Test sediment III.

Length

Day 0

Test sediment III.(µm) Day 6

Replicate


1 200 600 600 800 600 900 8502 250 600 550 800 600 600 900

3 200 600 800 850 900 850 600

4 210 800 800 800 900 800 850

5 200 600 850 800 400 850 -

6 200 - 600 800 600 850 -

7 200 - 800 800 - 850 -

8 200 - - 850 - 850 -

9 200 - - 800 - 800 -

10 210 - - - - - -

Mortality


5 3 1 4 1 6



33.3


18.86


57.2

Growth inhibition


207 640 714.3 811.1 666.6 816.6 800



433 507.3 604.1 459.6 609.6 593


534.43


302.93


36.17

Table 8.5.: Sample taken from the bioreactor on the 26th of April (2010, the 1ST day of the experiment) Test sediment IV.

Length

Day 0

Test sediment IV.

(µm) Day 6

Replicate


1 200 900 600 850 800 800 8502 250 850 750 800 850 1200 850

3 200 1150 850 850 800 500 800

4 210 850 900 900 800 600 850

5 200 850 600 800 850 900 800

6 200 850 600 850 - 850 650

7 200 850 800 850 - 1200 -

8 200 - - 850 - - -

9 200 - - - - - -

10 210 - - - - - -

Mortality


3 3 2 5 3 4



33.3


9.4


28.5

Growth inhibition


207 900 728.6 843.75 820 864.3 658.3



693 521.6 636.7 613 657.3 451.3


595.5


241.86


28.88

8.1.2. Test 2.

Table 8.6.: Sample taken from the bioreactor on the 3rd of May 2010 (7th day of the experiment) Reference sediment

Length

Day 0

Reference sediment(µm) Day 6

Replicate


1 200 1200 1150 1000 1200 1100 1100

2 200 900 750 1100 1150 1200 1150

3 250 1150 1150 1150 1150 1250 1100

4 200 1100 1150 1100 1100 1100 1100

5 200 1200 1150 1150 900 1200 1150

6 200 1200 1200 1100 1200 1050 1200

7 200 1200 1150 800 800 1100 1100

8 250 1200 1150 1100 850 1100 850

9 200 - 1200 1150 900 1150 1100

10 200 - - 800 1200 1100 1150

Mortality


2 1 0 0 0 0



5


7.64


153

Growth inhibition


210 1144 1116.6 1045 1045 1135 1100



934 906.6 835 835 925 890


887.5


_


_

Table 8.7.: Sample taken from the bioreactor on the 3rd of May 2010 (7th day of the experiment) Test sediment III.

Length

Day 0

Test sediment III.

(µm) Day 6 Replicate


1 200 600 500 - 600 450 450

2 200 450 450 - 400 400 450

3 250 600 550 - 450 400 400

4 200 600 - - 600 400 450

5 200 700 - - 400 - 500

6 200 650 - - 500 - -

7 200 - - - - - -

8 250 - - - - - -

9 200 - - - - - -

10 200 - - - - - -

Mortality


4 7 10 4 6 5



60


20.81


35

Growth inhibition


210 600 500 - 492 413 450

Mean length for all replicates 409


390 290 - 282 203 240


199


688.5


77.6

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X. Acknowledgements

This thesis project would not have been possible without the help and support of Silvia

Waara, Emma Nehrenheim, Veronica Ribé, and Monica Odlare from the School of

Sustainable Development of Society and Technology. I would like to thank Elisabet Aulenius

for the advice she gave me regarding the analysis of the soil. I am grateful for Ann-Sofie

Magnusson, and Jamal Saywan, that they were helping with the technical difficulties I faced

during my experiments. I am especially thankful for Dr Nóra Kováts, from Pannon University

for her help and advice. I would like to thank Puposné Bertalan Mónika, and Fejes Lászlóné

Utasi Anett, for their assistance and help with the Erasmus Internship. Last bot not least I

would like to thank my mother for her continuous support and good words in the hardest

times, and all my family and friends especially Gabriela Krasteva, and Johannes Radicke and

for being there for me when I needed help, and a good word, and Mikael Åsberg for his

support during the long hours of work.

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