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
Department
Radioecology
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:
Task leading department:
Dr. Emma Nehrenheim
Mälardalen University,
Dr. Nóra Kováts
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
Tanszék
Radiökológia
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:
A kidolgozás helyszíne:
Dr. Emma Nehrenheim
Mälardalen University,
Dr. Kováts Nóra
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 DDTvel 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
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.
polychlorinated
dibenzo-p-dioxins
Annex
(PCDD),
C
(Unintentional
polychlorinated
production)
contains
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 3500 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 biometabolism, 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
•
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
•
1 beaker with a concentration of 0.05 g DDT/kg dry weight soil
•
1 beaker with a concentration of 0.5 g DDT/kg dry weight soil
•
1 beaker with a concentration of 1.0 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).
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
Total Nitrogen (N)
Phosphorus (P)
Potassium (K)
Magnesium (Mg)
Sulphur (S)
Calcium (Ca)
Concentration (g/m3 )
112
56
120
256
63
2175
Element
Iron (Fe)
Manganese (Mn)
Boron (B)
Copper (Cu)
Zinc (Zn)
Molybden (Mo)
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.
Concentration (g/m3 )
0.7
1.3
0.2
1.0
0.3
1.6
4.1.2. Measured parameters
Table 4.2.Dry weight
Weight of the hour glass
Weight of the soil and the hour glass
Weight of the soil
Weight of the soil after drying (with dish)
Amount of water volatilized from the samples
Dry weight
Mean dry weight
Hour glass 1.
(g)
Hour glass 2.
(g)
Hour glass 3.
(g)
45.492
55.509
10.017
49.062
6.447
3.569
46.200
56.401
10.201
49.811
6.589
3.611
3.616
54.872
65.089
10.216
58.539
6.549
3.667
Hour glass 1.
(g)
Hour glass 2.
(g)
Hour glass 3.
(g)
45.876
60.90
15.024
48.535
2.659
46.202
61.184
14.981
48.896
2.694
2.687
45.628
60.370
14.741
48.337
2.708
Table 4.3.Water holding capacity
Weight of the Petri dish
Weight of the soil and the Petri dish
Weight of the soil
Weight of the samples after drying
Weight of the soil
Actual dry weight
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. 1 beaker with a concentration of 0.05 g DDT/kg dry weight soil (0.027 g DDT)
3. 1 beaker with a concentration of 0.5 g DDT/kg dry weight soil (0.277 g DDT)
4. 1 beaker with a concentration of 1.0 g DDT/kg dry weight soil (0.555 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.
8.3
56.6
31.6
33.3
33.3
6.8
14.9
15.6
18.86
9.4
85
26.1
48.75
57.2
28.5
1044.36
498.7
591.95
741.43
802.5
837.36
291.7
384.95
534.43
595.5
-
545.66
452.41
302.93
241.86
-
65.16
54.03
36.17
28.88
Mortality
Mean % mortality for all
replicates
Standard deviation of
mean % mortality
Variation coefficient of
mean % mortality
Growth inhibition
Mean length for all
replicates
Mean length increment
for all replicates
Mean growth inhibition
(in µ) for all replicates
Mean % growth inhibition
in test sediment
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.
5
100
100
60
100
7.64
-
-
20.81
-
153
-
-
35
-
1097.5
-
-
409
-
887.5
-
-
199
-
-
-
-
688.5
-
-
-
-
77.6
-
Mortality
Mean % mortality for all
replicates
Standard deviation of
mean % mortality
Variation coefficient of
mean % mortality
Growth inhibition
Mean length for all
replicates
Mean length increment
for all replicates
Mean growth inhibition
(in µ) for all replicates
Mean % growth inhibition
in test sediment
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 whitegrey 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
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Day 0
200
250
200
210
200
200
200
200
200
210
Number of dead ostracods per
replicate
Mean % mortality per replicate
Mean % mortality for all
replicates
Reference sediment
Day 6
Replicate
3
4
1100
1100
1050
1000
1050
800
1150
1050
1100
1100
1150
850
1000
1050
1050
1050
1000
800
1100
1
1150
1100
850
1000
1050
1100
1050
1100
1200
-
2
1150
1100
400
1100
1100
1100
1050
1000
-
1
2
1
10
20
10
6.8
Variation coefficient of mean %
mortality
85
Mean length increment for all
replicates
6
600
550
850
1050
1100
1150
1200
1150
1150
1000
0
1
0
0
10
0
990
1072.2
977.7
865.2
770.7
8.3
Standard deviation of mean %
mortality
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
5
1100
1150
1100
1100
850
1150
1100
1000
1100
-
207
1066.6
1087.5
1072.2
1044.36
859.6
880.5
865.2
783
837.36
Mean growth inhibition (in µ) for
all replicates
_
Mean % growth inhibition in test
sediment
_
Table 8.2.: Sample taken from the bioreactor on the 26th of April, 2010 (the 1ST day of the
experiment) Test sediment I.
Length
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Number of dead ostracods per
replicate
Mean % mortality per replicate
Mean % mortality for all
replicates
Day 0
200
250
200
210
200
200
200
200
200
210
Test sediment I.
Day 6
Replicate
3
4
300
400
350
450
500
-
1
450
600
550
400
-
2
400
400
300
350
400
300
-
6
4
8
60
40
80
5
850
900
450
600
450
600
-
6
800
850
600
600
800
-
7
4
5
70
40
50
450
641.6
730
243
434.6
523
56.6
Standard deviation of mean %
mortality
14.9
Variation coefficient of mean %
mortality
26.1
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
207
487.5
358.3
325
498.7
280.5
151.3
118
Mean length increment for all
replicates
291.7
Mean growth inhibition (in µ) for
all replicates
545.66
Mean % growth inhibition in test
sediment
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
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Number of dead ostracods per
replicate
Mean % mortality per replicate
Mean % mortality for all
replicates
Day 0
200
250
200
210
200
200
200
200
200
210
Test sediment II.
Day 6
Replicate
3
4
900
450
600
600
600
450
850
650
400
600
900
600
400
500
1
400
450
600
550
400
450
550
550
-
2
650
850
600
600
600
600
600
650
-
2
2
6
20
20
60
5
400
550
600
600
850
550
-
6
600
650
400
600
400
-
0
4
5
0
40
50
555
591.6
530
348
384.6
323
31.6
Standard deviation of mean %
mortality
15.6
Variation coefficient of mean %
mortality
48.75
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
207
493.8
643.8
737.5
591.95
286.8
436.8
530.5
Mean length increment for all
replicates
384.95
Mean growth inhibition (in µ) for
all replicates
452.41
Mean % growth inhibition in test
sediment
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
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Number of dead ostracods per
replicate
Mean % mortality per replicate
Mean % mortality for all
replicates
Day 0
200
250
200
210
200
200
200
200
200
210
Test sediment III.
Day 6
Replicate
3
4
800
600
800
600
850
900
800
900
800
400
800
600
800
850
800
-
1
600
600
600
800
600
-
2
600
550
800
800
850
600
800
-
5
3
1
50
30
10
5
900
600
850
800
850
850
850
850
800
-
6
850
900
600
850
-
4
1
6
40
10
60
666.6
816.6
800
609.6
593
33.3
Standard deviation of mean %
mortality
18.86
Variation coefficient of mean %
mortality
57.2
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
207
640
714.3
811.1
741.43
433
507.3
604.1
459.6
Mean length increment for all
replicates
534.43
Mean growth inhibition (in µ) for
all replicates
302.93
Mean % growth inhibition in test
sediment
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
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Number of dead ostracods per
replicate
Mean % mortality per replicate
Mean % mortality for all
replicates
Day 0
200
250
200
210
200
200
200
200
200
210
Test sediment IV.
Day 6
Replicate
3
4
850
800
800
850
850
800
900
800
800
850
850
850
850
-
1
900
850
1150
850
850
850
850
-
2
600
750
850
900
600
600
800
-
3
3
2
30
30
20
5
800
1200
500
600
900
850
1200
-
6
850
850
800
850
800
650
-
5
3
4
50
30
40
820
864.3
658.3
613
657.3
451.3
33.3
Standard deviation of mean %
mortality
9.4
Variation coefficient of mean %
mortality
28.5
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
207
900
728.6
843.75
802.5
693
521.6
636.7
Mean length increment for all
replicates
595.5
Mean growth inhibition (in µ) for
all replicates
241.86
Mean % growth inhibition in test
sediment
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
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Day 0
Reference sediment
Day 6
Replicate
3
4
1000
1200
1100
1150
1150
1150
1100
1100
1150
900
1100
1200
800
800
1100
850
1150
900
800
1200
1
1200
900
1150
1100
1200
1200
1200
1200
-
2
1150
750
1150
1150
1150
1200
1150
1150
1200
-
Number of dead ostracods per
replicate
2
1
0
Mean % mortality per replicate
20
10
0
200
200
250
200
200
200
200
250
200
200
Mean % mortality for all
replicates
7.64
Variation coefficient of mean %
mortality
153
Mean length increment per
replicate
Mean length increment for all
replicates
6
1100
1150
1100
1100
1150
1200
1100
850
1100
1150
0
0
0
0
0
0
1045
1135
1100
925
890
5
Standard deviation of mean %
mortality
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
5
1100
1200
1250
1100
1200
1050
1100
1100
1150
1100
210
1144
1116.6
1045
1097.5
934
906.6
835
835
887.5
Mean growth inhibition (in µ) for
all replicates
_
Mean % growth inhibition in test
sediment
_
Table 8.7.: Sample taken from the bioreactor on the 3rd of May 2010 (7th day of the
experiment) Test sediment III.
Length
(µm)
Test organism
1
2
3
4
5
6
7
8
9
10
Mortality
Day 0
Test sediment III.
Day 6
Replicate
3
4
600
400
450
600
400
500
-
1
600
450
600
600
700
650
-
2
500
450
550
-
Number of dead ostracods per
replicate
4
7
10
Mean % mortality per replicate
40
70
100
200
200
250
200
200
200
200
250
200
200
Mean % mortality for all
replicates
5
450
400
400
400
-
6
450
450
400
450
500
-
4
6
5
40
60
50
492
413
450
282
203
240
60
Standard deviation of mean %
mortality
20.81
Variation coefficient of mean %
mortality
35
Growth inhibition
Mean length of ostracods per
replicate
Mean length for all replicates
Mean length increment per
replicate
Mean length increment for all
replicates
210
600
500
409
390
290
199
Mean growth inhibition (in µ) for
all replicates
688.5
Mean % growth inhibition in test
sediment
77.6
IX. References
[1] Agriinfo.in, My agriculture information bank; 2009; Available at http://agriinfo.in/?
page=topic&superid=5&topicid=174 (Accessed 1 July, 2010)
[2] O. Muter, E. Nehrenheim, M. Odlare, A. Rodriguez, G. Cepurnieks, V. Bartkevics; 2008;
Demilitarization industry sludge: assessment of toxicity and biodegradation potential; School
of sustainable development of society and technology, Mälardalen University, Västerås,
Sweden; s.l.
[3] R. V. Subba-Rao and Martin Alexander; 1984; Bacterial and fungal co-metabolism of 1, 1,
1-trichloro-2, 2-bis (4- chlorophenyl) ethane (DDT) and its breakdown products; Appl.
Environ. Microbiol. 1987 September; 53(9); Available at
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC204048/ (Accessed 3 May, 2010)
[4] AKG, Természettudományok, Elsivatagosodás, éhínség, erdőírtás; 2010; Available at
http://termtud.akg.hu/okt/9/valtozof/6elsivat.htm (Accessed 16 May, 2010)
[5] Rachel Carson, 1999, Silent Spring, Penguin Books, p. 27.
[6] DDT, DDE and DDD, Chemical and physical information; Available at
http://www.atsdr.cdc.gov/toxprofiles/tp35-c4.pdf (Accessed 26 march 2010)
[7] Újabb fordulat a DDT történetében, Wolfner András; January 1996; Természet világa,
Available at http://www.kfki.hu/chemonet/hun/eloado/tan/ddt.html (Accessed 28 February 2010)
[8] Essential soil science, A clear and concise introduction to soil science; M. R. Ashman and
G. Puri, Blackwell publishing, 2002, Blackwell Science Ltd., United Kingdom, p. 163.
[9] The Pied Piper, DDT, Stuart M. Bennett, 2001, Available at http://www.thepiedpiper.co.uk/th13(l).htm (Accessed 19 May 2010)
[10] M. R. Ashman and G. Puri; 2002, Essential Soil Science-A clear and concise
introduction to soil science, Blackwell Science Ltd., p. 164
[11] Marion R. Wells, James B. Phillips, George G. Murphy; June 1974; Treatment with
Aldrin and Dieldrin; Springer New York, Bulletin of Environmental Contamination and
Toxicology, Volume 11, Number 6, June, 1974; Available at
http://www.springerlink.com/content/n8p5t71125kv1tg5/ (Accessed 30 March, 2010)
[12] Stockholm Convention on persistent organic pollutants (POPs), What are POPs ?; 2008;
Available at http://chm.pops.int/Convention/ThePOPs/tabid/673/language/en-US/Default.aspx
(Accessed 29 March 2010)
[13] EPA United States Environmental Protection Agency, Technology Innovation Program,
Contaminated Site Clean Up Information, Persistent Organic Pollutants, POPs, Health Effects
of POPs; 2007; Available at http://www.cluin.org/contaminantfocus/default.focus/sec/Persistent_Organic_Pollutants_(POPs)/cat/Health_Effects
(Accessed 29 March 2010)
[14] Substance Profiles, Dichlorodiphenyltrichloroethane, DDT, Report on carcinogens, 11th
edition; 1985; Available at http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s064ddt.pdf (Accessed
20 March 2010)
[15] Toxicological Profile for DDT, DDE, DDD, US. Department of Health and Human
Services, Public Health Service, Agency for Toxic Substances and Disease Registry; 2002;
Available at http://www.atsdr.cdc.gov/toxprofiles/tp35.pdf (Accessed 20 March 2010)
[16] WWF Lexikon, DDT; 2006, Available at http://www.wwf.hu/index.php?
p=tudomany&sub=lexikon&betu=D (Accessed 14 March 2010)
[17] Agency for toxic substances and disease registry, Division of Toxicology, ToxFaQs DDT
DDE and DDD; Sept 2002; Available at http://www.atsdr.cdc.gov/tfacts35.pdf (Accessed 26
March 2010)
[18] Richard D. Porter and Stanley N. Wiemeyer; 1969; Dieldrin and DDT: Effects on
Sparrow Hawk Eggshells and Reproduction; Science 11 July 1969:
Vol. 165. no. 3889, pp. 199 – 200, Available at
http://www.sciencemag.org/cgi/content/abstract/165/3889/199 (Accessed 19 May 2010)
[19 ] DDT @ 3D chem.com, Banned Insecticide; 2007, Available at
http://www.3dchem.com/molecules.asp?ID=90 (Accessed 14 March 2010)
[20] DDT Factsheet, Pesticide Action Network, UK, June 1998, Pesticides News, No. 40, pp
18-20, Available at http://www.pan-uk.org/pestnews/Actives/ddt.htm (Accessed 18 May 2010)
[21] VIMS research reveals DDT persists in Antarctic ice, 2008, Available at
http://www.asoc.org/Portals/0/DDT%20persists%20in%20Antarctic%20Ice.pdf (Accessed 18 May,
2010)
[22] Persistent Bioaccumulative and Toxic (PBT) Chemical Program, US. EPA; 2010;
Available at http://www.epa.gov/pbt/pubs/ddt.htm (Accessed 21 March 2010)
[23] M. Kirchner, T. Faus-Kessler, G. Jakobi, W. Levy, B. Henkelmann, S. Bernhöft, J.
Kotalik, A. Zsolnay, R. Bassan, C. Belis, N. Kräuchi, W. Moche, P. Simončič, M. Uhl, P.
Weiss and K. W. Schramm; July 2009; Vertical distribution of organochlorine pesticides in
humus along Alpine altitudinal profiles in relation to ambiental parameters; Environmental
Pollution, Volume 157, Issue 12, December 2009, pp 3238-3247, Available at
http://www.ncbi.nlm.nih.gov/pubmed/19596160 (Accessed 16 March 2010)
[24] F. Sjoeib, E. Anwar, M. S. Tungguldihardjo; 1994; Behavior of DDT and DDE in
Indonesian tropical environments, Journal of Environmental Science and Health, Part B, Volume
29, Issue 1 1994, pp 17 – 24, Available at
http://www.informaworld.com/smpp/content~content=a905516138&db=all (Accessed 28 April
2010)
[25 ] Status report on UNEP’s and other related activities on persistent organic pollutants
(POPs), 1998, Available at http://www.chem.unep.ch/pops/indxhtms/POPSTATE.html (Accessed
20 July, 2010)
[26 ] Shen H., Henkelmann B., Levy W., Zsolnay A., Weiss P., Jakobi G., Kirchner M., Moche W.,
Braun K., Schramm K. W.; 2009; Altitudinal and chiral signature of persistent organochlorine
pesticides in air, soil, and spruce needles (Picea abies) of the Alps; Environ. Sci. Technol. 2009
Apr 1; 43(7):2450-5; Available at http://www.ncbi.nlm.nih.gov/pubmed/19452900 (Accessed 20
July, 2010)
[27] M. R. Ashman and G. Puri; 2002, Essential Soil Science-A clear and concise
introduction to soil science, Blackwell Science Ltd., p. 168
[28] Stockholm Convention on persistent organic pollutants (POPs), About the convention,
2008, Available at http://chm.pops.int/Convention/tabid/54/language/en-US/Default.aspx#convtext
(Accessed 29 March 2010)
[29] Stockholm Convention on persistent organic pollutants (POPs), What are POPs ?, 2008,
Available at http://chm.pops.int/Convention/ThePOPs/tabid/673/language/en-US/Default.aspx
(Accessed 29 March 2010)
[30] Per Jonsson, Jan Eckhéll, Per Larsson, August 2000, PCB and DDT in Laminated
Sediments from Offshore and Archipelago Areas of the NW Baltic Sea, AMBIO: A Journal of
the Human Environment, pp 268-276, Available at http://www.bioone.org/doi/abs/10.1579/00447447-29.4.268 (Accessed 20 March 2010)
[31] Talajremediáció tervezése és kivitelezése, Gruiz Katalin, Available at
http://enfo.agt.bme.hu/drupal/sites/default/files/Remedi%C3%A1ci%C3%B3%20tervez
%C3%A9se1.pdf (Accessed 20 March 2010)
[32] United Nations, Compendium of soil clean up technologies and soil remediation
companies, 2nd edition; 2000; United Nations, New York, Geneva and Trieste
[33] Montserrat Pérez, Francesc Torrades, Xavier Doménech and José Peral; June 2002;
Fenton and photo-Fenton oxidation of textile effluents; Water Res. PubMed.Com, 2002 Jun;
36(11):2703-10.; Available at http://www.ncbi.nlm.nih.gov/pubmed/12146857 (Accessed 04
April 2010)
[34] Triton® X-100 Nonionic Surfactant, The miracle surfactant with many applications, SPI
Supplies; 2010, Available at http://www.2spi.com/catalog/supp/triton-x-100.html (Accessed 20
March 2010)
[35] Triton X-100, octylphenolpoly (ethyleneglycolether) x, especially purified for membrane
research; Roche Diagnostics GmbH Roche Applied Science, Germany; Sept 2005, Available
at http://www.roche-applied-science.com/pack-insert/1332481a.pdf (Accessed 20 March 2010)
[36] Marve Hyman, R. Ryan Dupont; 2001, Ground water and soil remediation-Process
design and cost estimating of proven technologies, American Society of Civil
Engineers(ASCE) Press, p. 193
[37] Ricardo D. Villa, Alam G. Trovó, and Raquel F. Pupo Nogueira, September 2009, Soil
remediation using a coupled process: soil washing with surfactant followed by photo-Fenton
oxidation; Journal of Hazardous Materials, Volume 174, Issues 1-3, 15 February 2010, pp 770775; Available at http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGF-4XBR4TJ5&_user=10&_coverDate=02%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_doca
nchor=&view=c&_searchStrId=1398336627&_rerunOrigin=google&_acct=C000050221&_version=1
&_urlVersion=0&_userid=10&md5=58fb5bba8ea99ee79d616f85e3071e08 (Accessed 26 March
2010)
[38] Science daily, Science reference, Biodegradation; 2010; Available at
http://www.sciencedaily.com/articles/b/biodegradation.htm (Accessed 27 April 2010)
[39] M. Vidali; 2001; Bioremediation, an overview; Pure Appl. Chem., Vol. 73, No. 7, pp.
1163–1172, 2001 IUPAC; Available at
http://www.iupac.org/publications/pac/2001/pdf/7307x1163.pdf (Accessed 02 April 2010)
[40] Marve Hyman, R. Ryan Dupont; 2001; Ground water and soil remediation-Process
design and cost estimating of proven technologies, American Society of Civil Engineers
(ASCE) Press, p. 203
[41] M. R. Ashman and G. Puri; 2002, Essential Soil Science-A clear and concise
introduction to soil science, Blackwell Science Ltd., pp 69-70.
[42] Soil bacteria, Soil biology basics, Profitable and sustainable primary industries, 2010,
NSW DPI, Available at
http://www.dpi.nsw.gov.au/__data/assets/pdf_file/0017/41642/Soil_bacteria.pdf (Accessed 29 April
2010)
[43] Soil health, biological, chemical, physical; 2008, Available at
http://www.soilhealth.com/bacteria/ (Accessed 27 April 2010)
[44] United Nations Environment Programme, Division of Technology, Industry and
Economics, Phytotechnologies; 200?, Available at
http://www.unep.or.jp/ietc/publications/freshwater/fms7/16.asp (Accessed 25 July 2010)
[45] S. K. Sharma, K. V. Sadasivam and J. M. Dave; August 1986, DDT degradation by
bacteria from activated sludge, Environment International, Vol. 13, pp 183-190, 1987;
Available at http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V7X-48YXCRTDV&_user=10&_coverDate=12%2F31%2F1987&_rdoc=1&_fmt=high&_orig=search&_sort=d&_do
canchor=&view=c&_searchStrId=1407322554&_rerunOrigin=google&_acct=C000050221&_version
=1&_urlVersion=0&_userid=10&md5=22305ef1791440c6786757de031111f3 (Accessed 30 March
2010)
[46] Marve Hyman, R. Ryan Dupont; 2001, Ground water and soil remediation-Process
design and cost estimating of proven technologies, American Society of Civil
Engineers(ASCE) Press, p. 205.
[47] Marve Hyman, R. Ryan Dupont; 2001, Ground water and soil remediation-Process
design and cost estimating of proven technologies, American Society of Civil
Engineers(ASCE) Press, pp 207-210.
[48] A. Hussain, S. H. Tirmazi, U. Maqbool, M. Asi, F. A. Chaughtai; 1994, Studies of the
effects of temperatures and solar radiation on volatilization, mineralization and binding of
14
C-DDT in soil under laboratory conditions, Journal of Environmental Science and Health, Part
B, Volume 29, Issue 1 1994 , pp 141 – 151, Available at
http://www.informaworld.com/smpp/content~db=all~content=a905516150 (Accessed 28 April
2010)
[49] Marve Hyman, R. Ryan Dupont; 2001, Ground water and soil remediation-Process
design and cost estimating of proven technologies, American Society of Civil
Engineers(ASCE) Press, p. 209.
[50] W. D. Guenzi and W. E. Beard, 1976, DDT Degradation in flooded soil as related to
temperature, J. Environ. Qual. 5:391-394, Available at
http://jeq.scijournals.org/cgi/content/abstract/5/4/391 (Accessed 18 May, 2010)
[51] M. M. Andrea, R. Y. Tomita, L. C. Luchini and M. R. Musumeci; 1994, Laboratory
studies on volatilization and mineralization of 14c-p,p'-DDT in soil, release of bound residues
and dissipation from solid surfaces; Journal of Environmental Science and Health, Part B, Volume
29, Issue 1. 1994, pp 133 – 139, Available at
http://www.informaworld.com/smpp/content~content=a905516149&db=all (Accessed 30 April
2010)
[52] Dr Cliff Synder; Efficient fertilizer use manual, PH, Mozaic; Available at
http://www.back-to-basics.net/efu/pdfs/pH.pdf (Accessed 28 April 2010)
[53] European Commission, Environment, Soil; June 2010, Available at
http://ec.europa.eu/environment/soil/index_en.htm (Accessed 20 July 2010)
[54] Commission of the European Communities, Communication from the Commission to the
council, the European Parliament, the Economic and Social Committee and the Committee of
the Regions, Towards a Thematic Strategy for Soil Protection; 2002, Available at http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:2002:0179:FIN:EN:PDF (Accessed 20 July
2010)
[55] European Soil Policy Thematic Strategy on Soil Protection, Soil Contamination; 2003, Available
at http://viso.jrc.ec.europa.eu/contaminated_lands/presentations/EUSoilPolicy.pdf (Accessed 20 July
2010)
[56] Soudek P., Petrová Š., Benešová D., Kotyza J., Vaněk T.; 2008; Phytoremediation and
possibilities of increasing it’s effectivity; Chemické listy, 102 [5]: 346-352; Available at
http://www.ueb.cas.cz/en/content/fytoremediation-and-possibilities-increasing-its-effectivity
(Accessed 01 April 2010)
[57] B. Suresh, P. D. Sherkhane, S. Kale, S. Eapen, G. A. Ravishankar; 2005; Uptake and
degradation of DDT by hairy root cultures of Cichorium Intybus and Brassica Juncea;
Chemosphere Volume 61, Issue 9, December 2005, pp 1288-1292; Available at
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V74-4G4MMDK5&_user=10&_coverDate=12%2F31%2F2005&_rdoc=1&_fmt=high&_orig=search&_sort=d&_doca
nchor=&view=c&_searchStrId=1398345725&_rerunOrigin=google&_acct=C000050221&_version=1
&_urlVersion=0&_userid=10&md5=2268578cabdfc2ef68b2f4a4e0c500c2 (Accessed 10 April
2010)
[58] What is phytoremediation, Available at http://arabidopsis.info/students/dom/mainpage.html
(Accessed 22 March 2010)
[59] Anthony Randazzo; 2001; Restoration and reclamation review, Phytoremediation:
prospects and limitations; Available at
http://horticulture.cfans.umn.edu/vd/h5015/99fpapers/Randover.htm (Accessed 24 March 2010)
[60] Jerald L. Schnoor, Louis A. Light, Steven C. Mc Cutcheon, N. Lee Wolfe and Laura H.
Carreia; July 1995; Phytoremediation of organic and nutrient contaminants; Environ. Sci.
Technol., 1995, 29 (7), pp 318A–323A; Available at
http://pubs.acs.org/doi/abs/10.1021/es00007a002 (Accessed 22 March 2010)
[61] T. Scheper and D. T. Tsao, 2003, Advances in biochemical engineering biotechnology,
Phytoremediation [e-book]; Springer Verlag-Berlin Heidelberg, New York; p. 4; Available
athttp://books.google.no/books?
id=fJGHEfyWy_0C&pg=PA51&lpg=PA51&dq=Advances+in+biochemical+engineering,
+biotechnology,
+Phytoremediation&source=bl&ots=Toet41bBrS&sig=4WjU_oKrTWRquJnhSQf69czgf0I&hl=hu&ei
=P7pNTKaFNcqbOMeIlJYD&sa=X&oi=book_result&ct=result&resnum=4&ved=0CDIQ6AEwAw#
v=onepage&q=Advances%20in%20biochemical%20engineering%2C%20biotechnology%2C
%20Phytoremediation&f=false (Accessed 22 March 2010)
[62] New Mexico Climate Center, Chapter 9., Soil water; 2007; Available at
http://weather.nmsu.edu/teaching_material/soil252/Chap9.htm (Accessed 26 April 2010)
[63] Agvise laboratories, Water Holding capacity, 2009, Available at
http://www.agvise.com/tech_art/whc.php (Accessed 26 April 2010)
[64] Initiation of anti-dumping investigations concerning import of cyclohexanone originating
in or exported from Taiwan, European Union and United States of America
(USA).No.14/17/2003-DGAD, Government of India Ministry of Commerce & Industry
(Department of Commerce), October 2003, Available at
http://commerce.nic.in/adint_cyclohexanone.htm (Accessed 26 April 2010)
[65] Gujarat State Fertilizers and Chemicals Ltd., Cyclohexanone, 2008, Available at
http://www.gsfclimited.com/cyclohexanone.asp?mnuid=3&fid=3 (Accessed 26 April 2010)
[66] J. W. Arkis and , G. W. Flint; 1952; Solvents for DDT; Research Department, Standard
Oil Co. (Indiana), Whiting; Ind. Advances in Chemistry, Vol. 7, Chapter 6, pp 52–55;
Available at http://pubs.acs.org/doi/abs/10.1021/ba-1951-0007.ch006 (Accessed 16 March 2010)
[67] Ulla C. Brinch, Flemming Ekelund and Carsten S. Jacobsen; 2002; Method for Spiking
Soil Samples with Organic Compounds; Applied and Environmental Microbiology, April
2002, pp 1808-1816, Vol. 68, No. 4; Available at http://aem.asm.org/cgi/content/short/68/4/1808
(Accessed 20 March 2010)
[68] Grant L. Northcott and Kevin C. Jones; 2000; Spiking hydrophobic organic compounds
into soil and sediment: a review and critique of adopted procedures; Environmental
toxicology and chemistry; Volume 19, Issue 10, pp 2418-2430; Available
athttp://www3.interscience.wiley.com/journal/122672486/abstract?CRETRY=1&SRETRY=0
(Accessed 20 March 2010)
[69] Göran Dave and Eva Nilsson, 1999, Sediment toxicity and contaminants in the Kattegat
and Skagerrak, Aquatic Ecosystem Health & Management, Volume 2, Issue 4 1999 , pp 347 –
360, Available at http://www.informaworld.com/smpp/content~db=all~content=a905225699
(Accessed 17 March 2010)
[70] Veronica Ribé, 2006, Determination of the toxicity of Cu and Zn to the freshwater
sediment-dwelling ostracod Heterocypris incongruens, Department of Biology and Chemical
Engineering, Mälardalen University, Sweden; s.l.
[71] Patryk Oleszczuk, 2007, Heterocypris incongruens as a tool to estimate sewage sludge
toxicity, Environ. Toxicol. Chem. PubMed.Com, 2008 Apr; 27(4):864-72., Available at
http://www.ncbi.nlm.nih.gov/pubmed/18333677 (Accessed 28 April 2010)
[72] Tonu Meidla, 2007, Ostracods from the Upper Ordovician Borenshult fauna, Sweden;
GFF, Volume 129, Issue 2 June 2007, pp 123 – 132, Available at
http://www.informaworld.com/smpp/content~db=all~content=a913717153 (Accessed 28 March
2010)
[73] J. Athersuch, D. J. Horne and J. E. Whittaker; 1989, Marine and brackish water
ostracods; Synopses of the British Fauna (New Series), No. 43., The Linnean Society of
London, p. 35; Available at
http://www.nhbs.com/sbf_volume_43_marine_and_brackish_water_ostracods_tefno_30400.html
(Accessed 3 May 2010)
[74] Standard operational procedure of Ostradcodtoxkit FTM-Chronic “direct contact” Toxicity
Test for Freshwater Sediments, Micro Bio Test Inc., Gent, Belgium, 2010, p. 2.-3.
[75] On some interstitial fauna in the marine sands on Indian cost; s.l., No 22, Nov 20, 1970,
p. 505; Available at http://www.ias.ac.in/jarch/currsci/39/00000714.pdf (Accessed 01 May 2010)
[76] Bobby L. Glass, 1972, Relation between the degradation of DDT and the iron redox
system in soils; J. Agric. Food Chem., 1972, 20 (2), pp 324–327; Available at
http://pubs.acs.org/doi/abs/10.1021/jf60180a043 (Accessed 18 May, 2010)
[77] Duangporn Kantachote, 2001, The use of microbial inoculants to enhance DDT
degradation in contaminated soil, s.l., Available at
http://digital.library.adelaide.edu.au/dspace/bitstream/2440/21703/1/09phk165.pdf
(Accessed 18 May, 2010)
[78] PAN Pesticides Database, Chemicals, 2010, Available at
http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC33454
(Accessed 18 May, 2010)
[79] Chemicalland 21, Cyclohexanone, 2010, Available at
http://chemicalland21.com/industrialchem/solalc/CYCLOHEXANONE.htm (Accessed 18 May
2010)
[80] PAN Pesticides Database, Chemicals, 2010, Available at
http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC32981 (Accessed 18 May 2010)
[81] Cyclohexanone Material Safety Data Sheet, 2003, Available at
http://www.nanotech.wisc.edu/CNT_LABS/MSDS/Solvents/MSDS%20Cyclohexanone.pdf
(Accessed 18 May 2010)
[82] European Commission, Treaties Office Database; 2009, Available at
http://ec.europa.eu/world/agreements/prepareCreateTreatiesWorkspace/treatiesGeneralData.do?
step=0&redirect=true&treatyId=1141 (Accessed 18 May 2010)
[83] J.M.Aislabie, N.K.Richards and H.L.Boul; 1997, Microbial degradation of DDT and its
residues-a review, New Zealand Journal of Agricultural Research, 1997, vol. 40, pp 269-282,
Available at http://www.royalsociety.org.nz/Site/publish/Journals/nzjar/1997/24.aspx (Accessed 19
May 2010)
[84] Dr. Bo Wahlström, 200?, Managing POPs in Sweden, Swedish National Chemicals
Inspectorate; Available at
http://www.chem.unep.ch/pops/POPs_Inc/proceedings/bangkok/WAHLS1.html (Accessed 21
March 2010)
[85] Persistent Organic Pollutants: A Global Issue, A Global Response, U. S. Environmental
Protection Agency (EPA); 2010, Available at http://www.epa.gov/international/toxics/pop.htm
(Accessed 15 March 2010)
[86] European Commission, Environment, Chemicals, REACH; 2010, Available at
http://ec.europa.eu/environment/chemicals/reach/reach_intro.htm (Accessed 22 July 2010)
[87] Chain of contamination- The food link, WWF; 2006, Available at
http://www.wwf.org.uk/filelibrary/pdf/contamination.pdf (Accessed 16 March 2010)
[88] Applied phytoremediation, David M. Singer, August 2003, Available at
http://pangea.stanford.edu/~dmsinger/Applied_Phytoremediation.pdf (Accessed 25 July 2010)
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.