Transcript
Deze eindverhandeling was een examen; de tijdens de verdediging vastgestelde fouten werden niet gecorrigeerd. Gebruik als referentie in publicaties is toegelaten na gunstig advies van de KHBO-promotor, vermeld op het titelblad. This thesis was an examination; the mistakes established during the presentation were not corrected. The use as a reference in publications is allowed after recommendation of the KHBOpromoter, mentioned on the front page.
Acknowledgements Presenting this thesis, I would like to thank everybody who contributed to realising it. First of all I’d like to thank the university Tampereen Ammattikorkeakoulu for offering an instructive and pleasant practical training. Many thanks to the international coordinators Ing Dorine Gevaert and Ms Mirja Onduso who made the exchange to Finland possible. I’m heartily thankful to internal supervisor Lic Dirk Gunst and external supervisor Lic Tech Marjukka Dyer for the feedback and assistance completing this thesis. Special thanks to Ms Gwendoline Rogge for feedback on the spelling and grammar, and to Ms Seija Haapamäki for the practical assistance during the project. Last but not least, I would like to thank my parents who gave me the opportunity to accomplish these studies.
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Samenvatting In dit eindwerk wordt het verschil in toxiciteit tussen een natuurlijke en een artificiële haarverf bepaald op Eisenia fetida. Daarnaast wordt er ook een bepaling gedaan van verschillende metalen aanwezig in de beide types haarverf. De bepaling van het verschil in toxiciteit wordt uitgevoerd aan de hand van een acute blootstelling van de testspecies aan een bodem waarin de haarverf aanwezig is. Er wordt getracht de LC 50 van beide haarverven te bepalen over een periode van twee weken. Door een verkeerde interpretatie van de resultaten van de rangetest, werd het onmogelijk aan de hand van de resultaten van de definitieve test de LC 50 te bepalen. Toch werd er een wijziging in gewichtstoename van de species vastgesteld, zowel tussen beide types haarverven als tegenover de blanco stalen. Een opmerkelijk resultaat was een vermindering in gewichtstoename van de species blootgesteld aan lage concentraties artificiële haarverf (onder 0,023 mg/kg droge bodem), terwijl er een verhoging van de gewichtstoename was bij hogere concentraties artificiële haarverf. Species blootgesteld aan de natuurlijke haarverf vertoonden een verhoogde gewichtstoename. De bepaling van de concentraties van de metalen ijzer, koper, zink, cadmium, chroom, nikkel en lood in beide haarverven wordt uitgevoerd met behulp van atomaire absorptie spectrometrie. Er kon geen eenduidig besluit getrokken worden uit de resultaten van de analyse, aangezien alle concentraties onder bepaalbaarheidslimiet lagen. De bepaling van de concentraties van de metalen in de artificiële haarverf werd vermoedelijk ook gestoord door matrixinterferenties.
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Summary In this thesis, the difference in toxicity between a natural and a synthetic hair dye is determined on Eisenia fetida. Besides this, a determination of certain metals present in both types of hair dye is done. The difference in toxicity is determined by an acute exposure of the test species to a soil contaminated with the hair dye. The aim is to determine the LC 50 of both hair dyes in a period of two weeks. Because of a wrong interpretation of the results of the range-finding test, the results of the final test were not suitable to determine the LC 50. Yet a change in the increase in weight of the species was determined, both between the two hair dyes as between the hair dyes and the blanks. A remarkable result was a decrease in the increase in weight for species exposed to low concentrations of synthetic hair dye (below 0,023 mg/kg dry soil), even though there is a bigger increase in weight of the test species exposed to higher concentrations of synthetic hair dye compared to the blanks. Species exposed to the natural hair dye had a bigger increase in weight compared to the species in the blanks. The determination of the concentrations of the metals iron, cupper, zinc, cadmium, nickel, chromium and lead in both hair dyes is done with atomic absorption spectrometry. No univocal conclusions could be drawn from the results of the analysis, because all concentrations were below determination limits. Furthermore, the determination of the metals in the synthetic hair dye was probably disturbed by matrix interference.
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Table of contents Acknowledgements................................................................................................................ 2 Samenvatting ......................................................................................................................... 3 Summary ............................................................................................................................... 4 Table of contents.................................................................................................................... 5 List of pictures ....................................................................................................................... 7 List of tables .......................................................................................................................... 7 List of graphs ......................................................................................................................... 7 List of equations .................................................................................................................... 7 Introduction ........................................................................................................................... 9 Theoretical Part.................................................................................................................... 10 1 Hair dyes .......................................................................................................... 10 1.1 Hair colour ....................................................................................................... 10 1.2 Natural colouring agent .................................................................................... 10 1.3 Synthetic colouring agent ................................................................................. 11 1.3.1 Temporary hair dye .......................................................................................... 11 1.3.2 Semi permanent hair dye .................................................................................. 11 1.3.3 Demi permanent hair dye.................................................................................. 11 1.3.4 Permanent hair dye ........................................................................................... 11 2 Atomic absorption spectroscopy ....................................................................... 13 2.1 General set-up of AAS...................................................................................... 13 2.2 Source of radiation............................................................................................ 14 2.3 Nebulizer and burner ........................................................................................ 15 2.4 The monochromator.......................................................................................... 16 2.5 The detector: the photomultiplier tube .............................................................. 17 2.6 Adjustments before analysing ........................................................................... 17 3 Eisenia fetida in ecotoxicology ......................................................................... 18 3.1 Eisenia fetida.................................................................................................... 18 3.1.1 Taxonomic hierarchy ........................................................................................ 18 3.1.2 General characteristics...................................................................................... 18 3.2 Principles of ecotoxicology............................................................................... 20 3.2.1 Fate of pollutants in individuals and ecosystems ............................................... 20 3.2.2 Major routes of uptake...................................................................................... 21 3.2.3 Fate of pollutants in soils .................................................................................. 22 3.2.3.1 Organic pollutants ............................................................................................ 22 3.2.3.2 Metals............................................................................................................... 23 3.3 The importance of Eisenia fetida in ecotoxicology............................................ 24 Practical part........................................................................................................................ 25 4 Introduction ...................................................................................................... 25 5 Chemical Analysis with AAS ........................................................................... 26 5.1 Principle ........................................................................................................... 26 5.2 Procedure ......................................................................................................... 27 5.3 Results.............................................................................................................. 28 5.4 Conclusions ...................................................................................................... 29 6 Earthworm acute toxicity test............................................................................ 30 6.1 Principle ........................................................................................................... 30 6.2 Description of the test ....................................................................................... 30
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6.2.1 Containers ........................................................................................................ 30 6.2.2 Soil................................................................................................................... 31 6.2.3 Selection and preparation of test animals .......................................................... 31 6.2.4 Mixing the test substance into the soil............................................................... 32 6.3 Procedure ......................................................................................................... 32 6.3.1 Determination of the maximum water holding capacity .................................... 32 6.3.2 Determination of soil pH .................................................................................. 33 6.3.3 Test groups and controls ................................................................................... 33 6.3.4 Test conditions ................................................................................................. 34 6.3.5 Range test......................................................................................................... 34 6.4 Results of the range-finding test........................................................................ 35 6.4.1 Natural hair dye ................................................................................................ 35 6.4.1.1 Results.............................................................................................................. 35 6.4.1.2 Conclusions ...................................................................................................... 36 6.4.2 Synthetic hair dye ............................................................................................. 37 6.4.2.1 Results.............................................................................................................. 37 6.4.2.2 Conclusions ...................................................................................................... 38 6.5 Results of the final test...................................................................................... 39 6.5.1 Natural hair dye ................................................................................................ 39 6.5.1.1 Results.............................................................................................................. 39 6.5.1.2 Conclusions ...................................................................................................... 40 6.5.2 Synthetic hair dye ............................................................................................. 41 6.5.2.1 Results.............................................................................................................. 41 6.5.2.2 Conclusions ...................................................................................................... 42 Final conclusions ................................................................................................................. 43 List of references ................................................................................................................. 46
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List of pictures Picture 1.1: lawsone or 1,4-naphtoquinone........................................................................... 10 Picture 1.2: resorcinol or 1,3 dihydroxybenzene................................................................... 12 Picture 2.1: general set-up of AAS ....................................................................................... 13 Picture 2.2: hollow cathode lamp ......................................................................................... 14 Picture 2.3: nebulizer and burner.......................................................................................... 15 Picture 2.4: monochromator in Czerny-Turner mounting ..................................................... 16 Picture 2.5: photomultiplier tube .......................................................................................... 17 Picture 3.1: Eisenia fetida .................................................................................................... 19 Picture 3.2: fate of pollutants ............................................................................................... 20 Picture 3.3: fate of pollutants in soil ..................................................................................... 22 Picture 5.1: AAS.................................................................................................................. 26 Picture 6.1: test containers ................................................................................................... 30 Picture 6.2: preparation of the earthworms ........................................................................... 33
List of tables Table 3.1: taxonomic hierarchy of Eisnia fetida.................................................................... 18 Table 5.1: parameters of AAS .............................................................................................. 27 Table 5.2: results AAS ......................................................................................................... 28 Table 5.3: concentrations metals in both hair dyes................................................................ 29 Table 6.1: properties soil...................................................................................................... 31 Table 6.2: natural hair dye: results range test........................................................................ 35 Table 6.3: synthetic hair dye: results range test .................................................................... 37 Table 6.4: results blanks final test ........................................................................................ 39 Table 6.5: natural hair dye: results final test ......................................................................... 39 Table 6.6: natural hair dye replicas: results range test........................................................... 39 Table 6.7: results blanks final test ........................................................................................ 41 Table 6.8: synthetic hair dye: results final test ...................................................................... 41 Table 6.9: synthetic hair dye replicas: results final test ......................................................... 41
List of graphs Graph 6.1: natural hair dye: increase in weight range test..................................................... 35 Graph 6.2: natural hair dye: mortality range test................................................................... 36 Graph 6.3: synthetic hair dye: increase in weight range test.................................................. 37 Graph 6.4: natural hair dye: increase in weight final test ...................................................... 40 Graph 6.5: synthetic hair dye: increase in weight final test ................................................... 42
List of equations Equation 3.1: BCF ............................................................................................................... 23 Equation 6.1: WHC.............................................................................................................. 32
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Alphabetic list of used symbols and abbreviations AAS
Atomic Absorption Spectrometry
BCF
Bioconcentration Factor
HCL
Hollow Cathode Lamp
ISO
International Organization for Standardization
KOW
Octanol-water partition coefficient
LC
Lethal Concentration
NOAEL
No Observable Adverse-effects Level
NOEC
No Observed Effect Concentration
OECD
Organisation for Economic Co-operation and Development
UV
Ultra Violet
WHC
Water Holding Capacity
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Introduction This thesis is part of a project with the aim of renewing the course ‘Ecotoxicology Laboratory Exercises (E1306)’ (20) in the program Environmental Engineering at Tampereen Ammattikorkeakoulu. In the course, one of the experiments is an acute toxicity test of dimethoate on Eisenia fetida. To keep the course updated to current events in the field, research is done that attempts to show that it is possible to observe a difference in acute toxicity between a natural hair dye and a synthetic hair dye with an 2 week acute toxicity test on Eisenia fetida, with specific reference to the present metals in the hair dyes. If the results are positive, the original test on dimethoate will be replaced. Besides the acute toxicity test on Eisenia fetida, a chemical analysis of the metals iron, cupper, zinc, cadmium, nickel, chromium and lead is done with atomic absorption spectrometry. This analysis is done to provide background information for the experiment and as a possible source for explaining a difference in acute toxicity. To observe the difference in acute toxicity, an estimation of the LC 50 of both hair dyes is attempted. Note that the aim of this thesis is not to make a perfect chemical screening or a detailed toxicity screening of both hair dyes. Summarised, this thesis tries to give a decisive answer about: − is it possible to observe a difference in acute toxicity between the natural and the synthetic hair dye on Eisenia fetida with a 2 week acute toxicity test; − is there a difference in the present metals and their concentration in both hair dyes; − if the present metals and concentrations are the same, is the acute toxicity influenced by the different organic matrix of both hair dyes; − if there is a difference in the present metals and their concentrations, is it possible to observe a difference in acute toxicity relating to it; − is it possible to introduce this experiment in the course ‘Ecotoxicology Laboratory Exercises (E1306)’? This thesis starts with background information about hair dyes, Eisenia fetida and AAS, followed by a description of the used methods and procedures. Then an overview of the results of both experiments is given and the thesis finishes with the final conclusions. Remark: ( paraphrased) literature is referred to with superscripted numbers in the title, subtitle or text, showing the corresponding place in the list of references.
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Theoretical Part 1
Hair dyes
1.1
Hair colour (4,11)
The colour of hair is determined by the pigment molecules, which are protein granules. The protein granules consist of two types of melanin protein, eumelanin and phaeomelanin. Eumelanin is responsible for the colours black to brown, and phaeomelanin causes colours from red to yellow. Next to the melanin granules, also trichosiderin is present, which contains iron and is red. Absence pigment molecules cause white to grey hair. The pigment molecules are stored in the cortex of the hair, underneath the scaly cuticle layer. The type of melanine and the size of the protein granule determine the colour of the hair. The density and the diffusion of the granules determine how dark or light the hair is. 1.2
Natural colouring agent (8,13)
Of all natural colouring agents, Henna is most well known. Henna or Hina (Lawsonia inermis, syn. L. alba) is a flowering plant and is the only species in the genus Lawsonia in the family Lythraceae. It is originating from tropical and subtropical regions of Africa, southern Asia, and northern Australasia. The plant produces an orange-red dye molecule, called lawsone. Lawsone is mostly concentrated in the leaves of the plant, and is in the highest levels in the petioles (the small stalks attaching the leaf blade to the stem) of the leaf. The skin will not be stained if the leaves are whole and unbroken. It is only when lawsone molecules are released from the henna leaf that the skin will be stained. The chemical name of lawsone is naphthoquinone, or more precisely 1,4-naphthoquinone or 4a,8a-Dihydronaphthalene-1,4-dione, which is a principal chemical structure of many natural compounds, such as the K vitamins.
Picture 1.1: lawsone or 1,4-naphtoquinone Lawsone has an affinity for bonding with proteins, so it binds with keratin, the main protein in hair. Therefore, Henna is considered a permanent hair dye, even though it can be washed out with mineral oil. It cannot be washed out with normal shampoos or rinses.
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1.3
Synthetic colouring agent (4, 9, 10)
1.3.1
Temporary hair dye
Temporary hair dyes coat on the surface of the hair. The dye remains absorbed close to the follicle. A hair follicle is the part of the skin where hair is grown by old cells being packed together. Unless the hair is damaged, the dye will be removed with a single shampooing. 1.3.2
Semi permanent hair dye
Semi permanent hair dyes are designed so that they deposit colour on the hair shaft. The process takes place without lightening the hair. This type of hair dye has smaller molecules than the temporary tinting formulas. Because of this, the dye is able to penetrate the hair shaft. It doesn’t use a developer, but heat can be used to stimulate the penetration. This type of hair dye lasts longer than temporary hair colour, and unless the hair is damaged, it stays intact from 8 up to 14 shampoos. 1.3.3
Demi permanent hair dye
This type of dye uses a mild, creamy developer of a lower volume (1-3 % H2O2) than permanent hair colour. Some demi permanent hair dyes may contain ammonia substitutes to help the penetration. The ammonia substitutes, also referred to as MEA’s, can even lift the natural hair colour, but only slightly. The dye penetrates the hair shaft a bit, leaves the hair shiny and covers or blends some grey hairs. Unless the hair is damaged, demi permanent hair dyes can last from 2 up to 3 months. 1.3.4
Permanent hair dye
Permanent hair dye consists of 2 parts: − peroxide solution; − ammonia solution of intermediates and couplers. The peroxide (developer) solution is in essence a water- or lotion-based solution of 6 % H2O2. The purposes of this solution are: − facilitating the formation of tints within the hair fibre; − lightening the hair by action of the peroxide. The peroxide becomes alkaline when the solution of intermediates and couplers, which contains the alkalising ingredient, is combined with the developer. Because of this, the peroxide is able to diffuse through the hair fibre and to enter the cortex, where melanin is located. The lightening takes place when the melanin is broken up by the alkaline peroxide and replaced with new colours.
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The hair is caused to swell by the ammonia solution (less than 1 % concentration). It also causes the cuticle scales to separate slightly. The primary intermediates give colour on oxidation by the developer. Most common primary intermediates are: − ortho or para diaminobenzenes; − aminohydroxybenzenes; − dihydroxybenzenes. Colour couplers, or performed hair dyes, don’t just oxidise by reaction with the peroxide, but they react with the oxidised primary intermediates. This process gives a wider variety in colours. The most common couplers are: − − − −
phenols; meta disubstituted phenylenediamines; meta disubstituted phenyleneaminophenols; resorcinol derivates.
Picture 1.2: resorcinol or 1,3 dihydroxybenzene
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2
Atomic absorption spectroscopy (1, 3, 7, 15)
AAS is a technique in which the absorption of electromagnetic radiation by atoms is measured. Solutions are atomised in a flame, causing free atoms to be formed. The free atoms are able to absorb radiation of a specific wavelength. The method is very selective. When the spectrophotometer is set up to measure one specific element, other elements won’t absorb any radiation, so they won’t be measured. The spectrometer must be adjusted for every element separately. The intention is that all atoms in the solution of the element being measured are atomised in the flame. Furthermore, the measuring conditions must remain constant during all measurements of standard solutions and samples. Acetylene is always used as burning gas, and the oxidising gas is either air or nitrous oxide. The acetylene-air flame is a low temperature flame, and is therefore used for elements that oxidise quite easily. The acetylene-nitrous oxide flame is a high temperature flame, and is therefore used for elements that don’t oxidise very easily. 2.1
General set-up of AAS
Picture 2.1: general set-up of AAS
Light from the hollow cathode lamp is sent to the flame. The indirect nebulizer is made up of a capillary tube, the nebulizer, where the oxidising gas passes the capillary tube with high velocity, and the spray chamber, with an input of burning gas and an output of the big drops. The non-absorbed light will enter the monochromator through the entrance slit, where the desired wavelength is isolated. Leaving the monochromator through the exit slit, the light reaches the photomultiplier tube. In the photomultiplier tube, the light is converted into an electric amplified signal. The electric signal is sent to a readout device, and a reading of the amplified signal can here be taken. An isolated wavelength is selected so that it is intense enough, and so that there is as less interference as possible, even when working with low concentrations.
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2.2
Source of radiation
Picture 2.2: hollow cathode lamp
The emission curve of an atom has almost the same width as the absorption curve of that atom. Therefore, for every element being determined, its own emission spectrum must be radiated on the atom. As a source for radiation in AAS, the use of a hollow cathode lamp (HCL) is most common. The lamp is made up of a hollow cathode and a rod-shaped anode. The outside of the cathode is covered with glass isolation, causing the cathode to be negative only on the inside. At the inside of the lamp, inert gas is present under low pressure, mostly Ne or Ar. Neon is used for elements that emit light in the visible range, because neon itself doesn’t emit any light in that range. Argon is used as a filling gas for elements that emit light in the UV range. When a potential difference of about 300 volt is applied, with a current intensity between 1 and 50 mA, the inert gas starts to ionise, and the inert gas ions start to bombard the hollow cathode. Because of this, a cloud of atoms of element X is created within the hollow cathode. New inert gas ions collide with the cloud of atoms, by which the atoms of element X are excited. When returning to the ground state, the emission spectrum of element X is emitted. The elements of element X in the flame can absorb this light. To avoid interferences, the pressure of the inert gas must be low enough, and the current intensity must remain below a certain maximum as well.
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Nebulizer and burner
2.3
Picture 2.3: nebulizer and burner
The oxidising gas rushes past the capillary tube, causing an underpressure. Because of the underpressure, the solution is drawn up and atomised. The design of the nebulizer is to limit the size of the atomized sample or droplets introduced to the flame to a very small size. Droplets larger than this are stopped by baffles or spoilers and end up flowing to waste. The parameters that are important to the size of the droplets are: − − − −
the velocity of the oxidising gas; the density of the solution; the viscosity of the solution; the volume flow rates of both the oxidising gas and the solution.
The efficiency of the atomisation can be increased considerably by the use of baffles and spoilers. They cause a spray of even smaller droplets, and too big droplets end up in flowing to waste. Depending on the efficiency that is wanted, the baffle or spoiler can be adjusted, moving further or closer to the nebulizer. Obtaining a spray of very small droplets is very important, considering that in an acetylene-air flame the flow rate is about 200 cm/sec. If the light beam is 2 mm in diameter, the atoms will only be in the light beam for 0,001 sec. So a good atomisation is required and the input of the sample should be long enough.
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2.4
The monochromator
Picture 2.4: monochromator in Czerny-Turner mounting The function of the monochromator is to isolate light emitted from the primary radiation source and to isolate the most intense resonance line from non-absorbing lines close to it. These lines may come from the cathode metal, the filler gas of the hollow cathode lamp, molecular emission and other background continua originating from the flame. The Czerny-Turner mounting is the one most commonly used. A polychromatic light beam enters the entrance slit, which can be few millimetres in height and between 10 and 100 micrometers in width. The entrance split is located in the focus of the collimator lens, which converts the divergent rays into parallel rays. When the parallel rays reflect on the grid monochromator, dispersion takes place. The parallel rays resulting from the dispersion each contain one wavelength of the radiation, and are again focussed by a second collimator lens. A beam of monochromatic light leaves the monochromator through the exit slit.
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The detector: the photomultiplier tube
2.5
A photomultiplier tube is an extremely sensitive detector in ultraviolet, visible and near infrared. It’s basically a photoelectric cell with internal amplification, constructed from a vacuum glass tube, holding a photocathode, several dynodes and an anode. The electrons being produced as a consequence of the photoelectric effect in the cathode are directed to a dynode which has a potential difference with the cathode of 90 V. The collision of the photoelectrons with the dynode causes a bigger amount of secondary electrons to be set free from the dynode. These secondary electrons are attracted to the second dynode, where the same process takes place. By these multiplications, the signal reaching the anode doesn’t have to be amplified anymore before it’s sent to the readout device.
Picture 2.5: photomultiplier tube
Adjustments before analysing
2.6
Optimizing the conditions in which the analysis takes place will increase the sensitivity of the flame-AAS drastically. Some important parameters are: − − − − − −
the height of the flame; the composition and flow rate of the flame; the current of the lamp; the flow rate of the sample; the width of the slit of the monochromator; the length of the burner.
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Eisenia fetida in ecotoxicology
3.1
Eisenia fetida
3.1.1
Taxonomic hierarchy (19) Rank Kingdom Subkingdom Phylum Class Subclass Superorder Order Suborder Superfamily Family Subfamily Genus Species
Name Animalia Eumetazoa Annelida Oligochaeta Diplotesticulata Megadrili Opisthopora Lumbricina Lumbricoidea Lumbricidae Lumbricinae Eisenia fetida
Table 3.1: taxonomic hierarchy of Eisnia fetida 3.1.2
General characteristics (5, 6, 17)
Eisenia fetida, or redworm, is a species of earthworm that has adapted to an environment of decaying organic matter. It is rarely found in common soils, but instead it is found in soils where other species cannot survive, like rotting vegetation, compost or manure. Nutrients are derived by E. fetida from the fungi and bacteria that grow upon these materials. They fragment organic matter and strongly help to recycle the nutrients it contains. Its specific name is based on the pungent liquid it exudes when being handled roughly, as sort of a chemical defence mechanism. Eisenia fetida is closely related to the species Eisenia andrei, also referred to as Eisenia fetida andrei. A simple way of making a distinction between them is the lighter colour of Eisenia fetida. Molecular analysis has shown that they truly are two different species, and breeding experiments have also shown that they do not produce hybrids. When the species is introduced to a new soil, it will dramatically change the soil structure, and because of that they will damage ecosystems. Soils of sandy loam, pine barrens, and possibly other soils are typical areas for this. This introduction can, for example, be caused by soil or trees transported from one place to another. Eisenia fetida is commercially available, as they are used for vermiculture. This technique depends on the ability of Eisnia fetida to turn compost into organic matter, which results in vermicompost. They are also sold as bait.
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Eisenia fetida is an Epigeic worm. Epigeic worms live on the surface of the soil or in the top 15 cm of the topsoil under the litter layer. The average incubation period for Eisenia fetida is between 32 and 73 days. 8 to 10 weeks are needed for E. fetida to sexually mature and begin the production of cocoons. Once it starts breeding and laying cocoons, it can lay 2 to 3 cocoons per week for 6 months to 12 months. All of this is dependant on environmental factors such as moisture, temperature, available food, etc.
Picture 3.1: Eisenia fetida Eisenia fetida provides several beneficial functions to the soil they live in. An overview of the most important functions: a)
Stimulate microbial activity
Although E. fetida obtains its nutrition from micro organisms, a lot more micro organisms are present in its faeces or casts than in the organic matter it consumes. As organic matter passes through its entrails, it is broken up into smaller pieces and inoculated with micro organisms. Increased microbial activity assists the cycling of nutrients from organic matter and it aids their conversion into forms easily taken up by plants. b)
Mix and aggregate soil
Wastes are excreted in the form of casts, as they consume organic matter and minerals. The casts are a type of soil aggregate. E. fetida can move big amounts of soil from a deeper layer of soil to the surface and can also carry organic matter down into lower strata. A large proportion of soil passes through the entrails of the worms, and they can turn over the top 15 cm of soil in 10 to 20 years.
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c)
Increase infiltration
E. fetida improves porosity as it moves through the soil. It makes permanent small mostly horizontal tunnels in the top layer of the soil. These small tunnels, or burrows, can persevere long after the inhabitant has died, and can be a major channel for soil drainage, especially under heavy rainfall. At the same time, the channels cause minimisation surface water erosion. d)
Improve the water-holding capacity
By fragmenting organic matter and increasing soil porosity and aggregation, E. fetida can considerably increase the water-holding capacity of soils. 3.2
Principles of ecotoxicology (2, 21)
3.2.1
Fate of pollutants in individuals and ecosystems
In a simple model that represents the fate of a xenobiotic in an individual, the movements, interactions and biotransformations that occur after exposure are shown.
Picture 3.2: fate of pollutants
Irrespective of the type of xenobioticum, the specific differences in these processes lead to a corresponding different toxicity for different organisms (selective toxicity). In the model, 5 types of sites are shown: − − − − −
sites of uptake; sites of metabolism; sites of action; sites of storage; sites of excretion.
The arrows show the movements of the xenobiotics between the different sites.
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Once a pollutant has entered the organism, there are 4 sites it can reach: I
Sites of action
Molecular interaction leads to the appearance of toxic expressions in the whole organism. With molecular interaction, the interaction of the toxic from with a macromolecule (e.g. protein, DNA) or structure (e.g. membrane) within the organism is meant. The chemical acts on the organism II
Sites of metabolism
Enzymes transform the xenobiotics by means of biotransformation. Usually, biotransformation causes detoxication, but sometimes biotransformation can cause activation. The organism acts on the chemical III
Sites of storage
From a toxicological point of view, the xenobiotic stays in a neutral state in this site. It’s not acting upon the organism, neither is it being acted upon. IV
Sites of excretion
Excretion can include the original pollutant, and/or products of biotransformation (metabolites or conjugates). After terrestrial animals have been exposed to lipophilic xenobiotics, excretion includes mostly products of biotransformation, and few original compounds. 3.2.2
Major routes of uptake
Terrestrial invertebrates can take up lipophilic pollutants by their nutrition or across the skin or cuticle. The mobility of the organism is important in determining the speed of taking up the pollutant across cuticle or skin. Mobile species will come into contact with more pollutant present in the soil because of their mobility. This raises the question how available compounds which are bound to soil, clay and organic matter are. While compounds that are dissolved in soil water are widely available for uptake by the species, it is not well understood how the compounds that are bound to the soil, clay or organic matter are available for uptake. The respiratory system is another way by which pollutants may be absorbed. The respiratory system of Eisenia fetida,is diffusion through the moist outer skin Pollutants which are in the gaseous state are suitable for this way of absorption. When the pollutants are particles or droplets which may be left behind in the respiratory tract, a more complex situation occurs.
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3.2.3
Fate of pollutants in soils
3.2.3.1
Organic pollutants
Organic compounds become distributed between soil water, soil air and the available surfaces of soil minerals and organic matter, when they enter soils. If the introduced compound is associated with – or in the form of – droplets or particles, some time is needed before individual molecules of the compound will be distributed between these different compartments of the soil. The Kow, vapour pressure and chemical stability of the compound are the most important factors that influence the distribution of it. A distinction between polar and less polar (apolar) compounds can be made. Polar compounds have a low Kow and tend to dissolve in water. Except for ions, they can only be adsorbed to soil colloids to a limited degree. Less polar, or apolar compounds can be strongly adsorbed to surfaces of clay and organic matter. They have a high Kow, resulting in very low concentrations of them in the soil water. Compounds with high vapour pressure can volatize into the soil air or straight from the soil surface into the atmosphere. If they volatize into the soil air, they may be kept within the soil for a while but eventually, they will pass into the atmosphere. A schematic representation of these processes is given in the following picture:
Picture 3.3: fate of pollutants in soil
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3.2.3.2
Metals
The fate of metals when introduced in soil depends on 4 factors: − localisation; − persistence; − bioconcentration and bioaccumulation factors; − bioavailability.
a)
Localisation
When the concentration of a pollutant exceeds a threshold value in a particular environmental compartment it is defined as ‘toxic’. The ultimate compartment is the whole planet, but it can be smaller like individual organisms, or even single cells or cell organelles within a cell. It has been claimed that ‘the solution to pollution is dilution’. Tall chimneys function on the ‘safe dilution’ approach to releases to the environment. The total amount of pollutants being released stays the same, but the concentration decreases in the local area. Note that this solution is only a short term solution, as the spreading of pollutants can cause even bigger problems (e.g. acid rain). To prevent problems with essential biochemical reactions in the cytoplasm, at cellular level organisms may possibly compartmentalise potential toxins in insoluble residues. For example, acting as intercellular sites of storage detoxification, the epithelium of the midgut of many invertebrates contains metal-rich granules. b)
Persistence
Metals don’t break down in nature and they are not biodegradable, but a formation and degradation of specific compounds such as methyl mercury is possible. Metals have a long residence time once they get into the soil or sediments, so it takes a long time before they are eluted to other compartments. c)
Bioconcentration and bioaccumulation factors
The uptake of some inorganic pollutants by organisms is bigger than others. This can be represented in the bioconcentration factor (BCF), which is expressed in the following way:
BCF =
conc. of the chemical in the organism conc. in the surrounding environment
Equation 3.1: BCF For a terrestrial organism, the surrounding environment is usually the soil. If a high bioconcentration factor is shown for a particular chemical by an organism, this may be the result of its biochemistry.
24
One of the factors the level of long term bioaccumulation depends on is the speed of excretion, even though it has never been perfectly proven. Therefore, for example, the bioaccumulation of cadmium is high relative to most other metals as it is assimilated rapidly and excreted slowly. d)
Bioavailability
A particular substance may be more biologically available which might result in a higher bioconcencentration factor than one which is less biologically available. The pH has a evident effect on the solubility of most metals in soils and water. If the pH declines, for example because of acid deposition, some metals may become more soluble than others. This may result in a higher bioavailability. 3.3
The importance of Eisenia fetida in ecotoxicology (17, 18)
The attention for earthworms in ecotoxicology started with some bigger events in the corresponding area, namely the inception, ring-testing and international standardisation of the acute earthworm toxicity test (OECD 1984). This procedure was largely developed by Prof. Clive Edwards (1983) and was planned to be included in the risk assessment context for newly registered chemicals and pesticides. Eventually this turned the earthworm Eisenia fetida into a model organism for measuring the effects of chemicals on terrestrial saprotrophic invertebrates. Saprotrophic organisms obtain their nutrients from non-living organic matter, generally dead and decaying plants or animals, by absorbing soluble organic compounds. “When developing the acute toxicity test, it is unlikely that Prof. Edwards was aware he was seeding a ‘community’ among ecotoxicologists. The fact there have now been three IWEE events, however, bears testament tot this. Evidence of the importance of earthworms in ecotoxicology can be taken from an analysis of published papers. In 2001, 234 ISI listed papers mentioned earthworms in the title, keywords or abstract (search terms = earthworm*), 60 of which (25 %) discussed aspects of ecotoxicology. For springtails (the only other soil invertebrate group for which there is a standardised toxicity test) only 125 papers were published (search terms = collembol* and springtail*), of which 22 (18 %) were ecotoxicological.” (Pedobiologia 47, 2003, p. 588-606).
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Practical part 4
Introduction
A commercially available natural hair dye and a synthetic hair dye were chosen so that they result in the same colour when being applied. A natural hair dye chosen with henna as a base. The synthetic hair dye was a permanent hair dye, because – as described in paragraph 1.2 – henna is a permanent hair dye as well. The hair dyes will be referred to as ‘natural hair dye’ and ‘synthetic hair dye’. Before any analysis was done, a procedure to make the stock solutions of both hair dyes was determined. The 2 parts of the synthetic hair dye (see paragraph 1.3.4) are mixed together and are left to react as described in the instructions of the dye. A stock solution is made by analytically weighing and dissolving the wanted amount of the mixture. The solution is then diluted in a volumetric flask. Because the hair dye is too viscous, analytically weighing is preferred to using pipettes or a burette to make the stock solutions The natural hair dye is obtained in powder form. The remains of the crushed henna leaves complicate dissolving it. As described in the introduction, any procedure in this thesis is supposed to fit in an exercise in a laboratory course. Therefore, procedures are developed so that they don’t require too much excessive work when the experiment is carried out. An obvious choice would be to digest the hair dye using microwave destruction. Unfortunately, the digestion could lead to a change in the toxicity of the hair dye as some molecular structures are destroyed. Several ways were attempted to dissolve the remains of the crushed henna leaves in a simple way, including dissolving in acid, dissolving in organic solvents (alcohols, ethers and acetone) and ultrasonic destruction. As these methods didn’t work out, a simple procedure using filtration was chosen. Some practical research showed that maximum about 10 % of the henna powder dissolves in water. Therefore, a surplus (10 times as much as needed) of the natural hair dye weighed analytically, and mixed in a sufficient amount of water. The mixture is then filtered through a Büchner funnel, using analytically weighed quantitative filter paper. The residuum + filter paper is then dried to constant mass. The filtrate is then diluted in a volumetric flask. The difference in mass between the original filter paper and the residuum + filter paper allows the concentration of the hair dye in the solution to be calculated. All dilutions of the hair dyes were made with deionised water. Qualitative filter paper of Schleicher & Schuell was used for filtration. Weighing the hair dyes was done with an analytical balance of Sartorius Competence Series.
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5
Chemical Analysis with AAS
5.1
Principle
AAS uses an indirect technique. The measured values are compared with the values of samples with a known amount of the element that is to be determined. In this experiment, the external standard method is used. External standards consist of a series of solutions with increasing and exactly know concentrations of the element that is to be determined. During the analysis, the series is measured and the results are converted into a calibration curve by the software of the AAS device. The specific extinctions of the unknown samples are converted to concentrations by the software using the calibration curve. All analysis were done with a flame AAS of the Solaar M Series of Thermo Elemental.
Picture 5.1: AAS
27
Procedure
5.2
Following standard solutions for the calibration curve were prepared to fit to the optimum range (in ppm): − − − − − − −
Zn: 0.40, 0.80, 1.20, 1.60, 2.00. Cr: 1.00, 2.00, 4.00, 6.00, 8.00; Ni: 2.00, 4.00, 6.00, 8.00, 10.00, 12.00; Cu: 1.00, 2.00, 4.00, 6.00, 8.00; Fe: 2.00, 4.00, 6.00, 8.00, 10.00, 12.00; Pb: 5.00, 10.00, 15.00, 20.00, 25.00; Cd: 0.50, 1.00, 1.50, 2.00, 2.50;
Following solutions of the hair dyes were prepared: − natural hair dye: 1028 ppm − synthetic hair dye: 1134 ppm Following parameters are used in the analysis: Wavelength
Background correction
Flame type
Burner height
Nebulizer uptake
Fuel flow
Zn
213.9 nm
D2
Air-C2H2
7.0 mm
4s
1.2 l/min
Cr
357.9 nm
Off
N2O-C2H2
8.0 mm
4s
4.2 l/min
Ni
232.0 nm
D2
Air-C2H2
7.0 mm
4s
0.9 l/min
Cu
324.8 nm
D2
Air-C2H2
7.0 mm
4s
1.1 l/min
Fe
248.3 nm
D2
Air-C2H2
7.0 mm
4s
0.9 l/min
Pb
217.0 nm
D2
Air-C2H2
7.0 mm
4s
1.1 l/min
Cd
228.8 nm
D2
Air-C2H2
7.0 mm
4s
1.2 l/min
Table 5.1: parameters of AAS
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5.3
Results Zn Standards Concentration (ppm ) 0,000 0,400 0,800 1,200 1,600 2,000 Natural hair dye Synthetic hair dye Ni Standards Concentration (ppm ) 0,000 2,000 4,000 6,000 8,000 10,000 12,000 Natural hair dye Synthetic hair dye
Signal 0,003 0,035 0,067 0,100 0,130 0,160 0,013 0,000
Signal 0,000 0,038 0,076 0,113 0,150 0,185 0,218 0,002 0,001
Fe Standards Concentration (ppm ) 0,000 2,000 4,000 6,000 8,000 10,000 Natural hair dye Synthetic hair dye
Signal 0,000 0,038 0,077 0,114 0,150 0,187 0,006 0,004
Cd Standards Concentration (ppm ) 0,000 0,500 1,000 1,500 2,000 2,500 Natural hair dye Synthetic hair dye
Signal 0,011 0,064 0,121 0,176 0,235 0,289 0,011 0,009
Table 5.2: results AAS
Cr Standards Concentration (ppm ) 0,000 1,000 2,000 4,000 6,000 8,000 Natural hair dye Synthetic hair dye
Signal 0,000 0,016 0,031 0,062 0,092 0,119 0,000 0,000
Cu Standards Concentration (ppm ) 0,000 1,000 2,000 4,000 6,000 8,000 Natural hair dye Synthetic hair dye
Signal 0,000 0,028 0,058 0,122 0,178 0,245 -0,001 -0,004
Pb Standards Concentration (ppm ) 0,000 5,000 10,000 15,000 20,000 25,000 Natural hair dye Synthetic hair dye
Signal 0,000 0,061 0,122 0,185 0,246 0,305 0,004 0,001
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Conclusions
5.4
All concentrations of all the metals analysed were below the lowest concentration of the standard series. Due to a lack of time, no second analysis could be done with higher concentrations of hair dyes. From the results of the analysis we can conclude (in mg/g hair dye):
Zn Cr Ni Cu Fe Pb Cd
Natural hair dye < 0,389 < 0,973 < 1,946 < 0,973 < 1,946 < 4,864 < 0,486
Synthetic hair dye < 0,353 < 0,882 < 1,764 < 0,882 < 1,764 < 4,409 < 0,441
Table 5.3: concentrations metals in both hair dyes The analysis of the synthetic hair dye was possibly disturbed by the matrix interference. In literature (1) is mentioned that the analyses could be disturbed by the presence of paraffins. It is very likely that paraffins are present as additives in the synthetic hair dye. Another issue is that the matrices of the hair dye solutions are completely different from the matrices of the standard solutions. Therefore, a standard addition method might be more suitable for this analysis.
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6
Earthworm acute toxicity test (11, 12, 14, 16, 20)
6.1
Principle
Adult worms are exposed to a range of concentrations of the hair dye. The hair dye is mixed into the soil. The range of the test concentrations is chosen to cover those likely to cause both sub-lethal and lethal effects over a period of 2 weeks. Mortality and growth effects are determined after 2 weeks of exposure. The mortality and growth effects of the worms exposed to the test substance are compared to those of the controls in order to determine the LC 50. A regression model is used to estimate the concentration that would cause a 50 % mortality. The test concentrations are chosen so that the LC 50 then comes from interpolation rather than extrapolation. 6.2
Description of the test
6.2.1
Containers
Test containers of about one to two litres capacity and made of chemically inert material are used. The containers have a cross-sectional area of approximately 200 cm² so that a moist substrate depth of 5-6 cm is achieved when 500 to 600 g dry mass of substrate is added. The container cover permits gaseous exchange between the substrate and the atmosphere and access to light. If the amount of test substrate used is substantially more than 500 to 600 g per test container the number of worms increases proportionately
Picture 6.1: test containers
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6.2.2
Soil
The dry constituents of the soil are mixed thoroughly in a well ventilated area. Before starting the test, the dry artificial soil is moistened by adding the solution of the test substance (and if necessary additional deionised water) to obtain approximately half of the final water content, being 40 % to 60 % of the maximum water holding capacity (corresponding to 50 ± 10 % moisture dry mass). This will produce a substrate that has no standing or free water when it is compressed in the hand. Soil moisture content is determined at the beginning and at the end of the test and soil pH is determined as well. These determinations are carried out in a sample of control soil and a sample of each test concentration. The moisture content is monitored throughout the test by weighing the containers daily. For the experiment, a commercially available soil is used. The soil has following properties: Brand Magnesium-containing lime
Biolem ® Puutarhan mustamulta 6 kg/m³
Compost conductivity N P K Grain size
90 l/m³ 40 mS/m 100 ppm 80 ppm 400 ppm < 35 mm
Table 6.1: properties soil
As the soil contains enough nutrients for the worms, no additional food is added over the 2 weeks of the experiment. 6.2.3
Selection and preparation of test animals
The species used in the test is Eisenia fetida. Adult worms between two months and one year old and with a clitellum are essential to start the test. The worms are selected from a synchronised culture with relatively homogeneous age structure. Individuals in a test group may not differ age by more than 4 weeks. The selected worms are acclimatised for at least 24 hours with the type of artificial soil substrate to be used for the test. During this period the worms should be fed on the same food to be used in the test, but as the type of soil doesn’t require any additional food, no extra food is added here as well. Groups of 10 worms are weighed individually and randomly. The groups are assigned to the test containers at the start of the test. The worms are washed prior to weighing and the excess water is removed by placing the worms on filter paper for a short time. The wet mass of individual worms should be between 200 and 600 mg.
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6.2.4
Mixing the test substance into the soil
A solution of the test substance in deionised water is prepared in a quantity sufficient for all replicates of one concentration. The solution is prepared immediately before starting the test. Furthermore, it is convenient, though not necessary, to prepare an amount of solution required to reach the final moisture content (40 to 60 % of maximum water holding capacity). The solutions may be prepared in a smaller amount, if the additional water to reach the final moisture content is added during the mixing process. The solution is mixed thoroughly with the soil substrate before introducing it into a test container. In addition to determining the exact concentration of the test substance in the dry soil, a determination of the water in the dry soil is done by analytically weighing a defined quantity of dry soil and drying it to constant mass. With the difference in mass, the moist content of the dry soil can be determined. The moist content of the dry soil is then count in the calculations of the concentration of the test substance in the dry soil. 6.3
Procedure
6.3.1
Determination of the maximum water holding capacity
A defined quantity of the test soil substrate is collected in a tube of about 30 cm in length and with an inner diameter of about 0.5 cm. The bottom of the tube is covered with a piece of filter paper, filled with water and then placed on a rack in a water bath. The tube is gradually submerged until the water level is higher than the top of the soil. It is then left in the water for about three hours. Since not all water absorbed by the soil capillaries can be kept, the soil sample is allowed to drain for a period of two hours in a covered container, so that it is prevented from drying. The sample is then weighed and dried to constant mass at 105 °C. The water holding capacity (WHC) is then calculated as follows:
WHC =
S −T − D × 100 D
Equation 6.1: WHC Where: WHC = water holding capacity in % of dry mass S = water-saturated substrate + mass of tube + mass of filter paper T = tare (mass of tube + mass of filter paper) D = dry mass of substrate
33
6.3.2
Determination of soil pH
A quantity of soil is dried at room temperature for at least 12 hours. A suspension of at least 5 gram of soil is then made up in five times its volume of either a 1 M solution of analytical grade potassium chloride (KCl) or a 0.01 M solution of analytical grade calcium chloride (CaCl2). The suspension is then shaken thoroughly for five minutes and left to rest for at least 2 hours but not for longer than 24 hours. The pH of the water phase is then measured using a pH-meter that has been calibrated before each measurement using an appropriate series of buffer solutions. To meaure the pH, a Meterlab PHM210 standard pH meter was used, which was calibrated with Merck CertiPUR buffer solutions of pH 4 and 7. 6.3.3
Test groups and controls
A loading of 10 earthworms in 500 – 600 g dry mass of artificial soil (i.e. 50-60 g of soil per worm) is used. The 10 worms are prepared for each control and treatment container. The worms are washed with deionised water, wiped and then placed on absorbent paper for a short period to allow excess water to drain.
Picture 6.2: preparation of the earthworms To avoid systematic errors in distributing the worms to the test containers, the homogeneity of the test population is determined by individually weighing 20 worms sampled randomly from the population of which the test worms are to be taken. Having ensured homogeneity, batches of 10 worms are then selected, weighed individually and assigned to the test containers. After adding the test worms, the weight of each test container is measured as an initial weight that can be used as a basis to monitor soil moisture content during the test. The test containers are then covered and placed in the test chamber. Appropriate controls are prepared according to the relevant procedures described, except that the test substance is not added. Weighing the earthworms was done with an analytical balance of Sartorius Competence Series was.
34
6.3.4
Test conditions
The test temperature is 20 ± 2 °C. The test is carried out under controlled light-dark cycles (preferably 16 hours light and 8 hours dark) with illumination of 400 to 800 lux in the area of the test containers. For the range-finding test (as described in paragraph 6.3.6), timers were used to control the light-dark cycles. For the definitive test, natural sun light was used, as the natural light-dark cycles were about 16 hours light and 8 hours dark. The test temperature was obtained by normal room temperature control. Measurements were taken before starting the test and during the test, showing the minimum and maximum temperatures of the room. All tests were executed in the range 20 ± 2 °C. The test containers are not aerated during the test but the design of the covers allows gaseous exchange whilst limiting evaporation of moisture. The water content of the soil substrate in the test containers is maintained throughout the test by reweighing the test containers daily. Losses are refilled as necessary with deionised water. The water content may not vary by more than 10 % from that at the start of the test. Weighing the test containers was done with a precision balance of Mettler Toledo. To determine the LC 50, 5 treatment concentrations in a geometric series are used. 2 replicas for each treatment and 2 controls are prepared as well. The concentrations should be spaced by a factor not exceeding 1.8. 6.3.5
Range test
To obtain prior knowledge of the toxicity of the test substance, a range finding test is performed with 4 test concentrations of about 0.01, 0.45, 21.5 and 1000 mg/kg (dry mass of soil). Test conditions and procedures remain the same, except that no replicas and no blanks are used in the range-finding test.
35
6.4
Results of the range-finding test
6.4.1
Natural hair dye
6.4.1.1
Results
Table 6.2: natural hair dye: results range test
Natural hair dye: Increase in weight
Increase in weight (%)
30 25 20
2
R = 0,993
15 10 5 0 0
200
400 600 800 Concentration (mg/kg dry soil)
Graph 6.1: natural hair dye: increase in weight range test
1000
1200
36
Natural hair dye: Mortality 60 Mortality (%)
50 40
2
R = 0,980
30 20 10 0 0
200
400
600
800
1000
1200
Concentration (mg/kg dry soil)
Graph 6.2: natural hair dye: mortality range test 6.4.1.2
Conclusions
The results of the range-finding test of the natural hair dye show that the LC 50 can be determined around 1000 mg/kg dry soil. Therefore, a series of 5 concentrations will be set up around this value in the final test. The concentrations will be spaced by a factor not exceeding 1.8. No other unusual behaviour or changes in morphology were recorded during the range test. Looking at the increase in weight, a connection can be determined with the concentration of the natural hair dye. An increase in concentration seems to lead to an increase in weight. As the range test didn’t include blanks and replicas, no further conclusions can be drawn from these results.
37
6.4.2
Synthetic hair dye
6.4.2.1
Results
Table 6.3: synthetic hair dye: results range test
Synthetic hair dye: Increase in weight
Increase in weight (%)
30 25 20
2
R = 0,998
15 10 5 0 -5 0
200
400
600
800
Concentration (mg/kg dry soil)
Graph 6.3: synthetic hair dye: increase in weight range test
1000
1200
38
6.4.2.2
Conclusions
The results of the synthetic hair dye show that the LC 50 cannot be determined within the range used in the range-finding test. The 10 % mortality in every test container is most probably due to other environmental factors or by chance and not caused by presence of the hair dye. As no replicas or blanks were used in the range test, no univocal conclusions can be drawn regarding the cause of this 10 % mortality. No other unusual behaviour or changes in morphology were recorded during the range test. Looking at the increase in weight, again a connection can be determined with the concentration of the synthetic hair dye. An increase in concentration seems to lead to an increase in weight. Though at low concentrations (0.01 mg/kg dry soil), a decrease in weight seems to occur. Because no replicas or blanks were used in the range test, no univocal conclusions can be drawn about the cause of this decrease in weight. Therefore, a series of 5 concentrations will be set up around this value in the final test. The concentrations will be spaced by a factor not exceeding 1.8. Literature (16) proposes a limit test when no mortality is observed at the highest concentration of the range-finding test, to demonstrate that the NOEC (no observed effect concentration) is greater than the limit concentration whilst minimising the number of worms used in the test. As this is not relevant for this project, a limit test will not be performed.
39
6.5
Results of the final test
6.5.1
Natural hair dye
6.5.1.1
Results
Table 6.4: results blanks final test
Table 6.5: natural hair dye: results final test
Table 6.6: natural hair dye replicas: results range test
40
Natural hair dye: Increase in weight 120,0 2
Increase in weight (%)
R = 0,997 100,0 80,0 2
R = 0,996 60,0
test concentrations replicas
40,0 20,0 0,0 0
500
1000 1500 2000 Concentration (mg/kg dry soil)
2500
3000
Graph 6.4: natural hair dye: increase in weight final test 6.5.1.2
Conclusions
No mortality was observed during the final test, even though the range test predicted this differently. The mortality of the range-finding test is most possibly caused by other environmental factors or by chance. No LC 50 can be determined with the results of the final test. Looking at the increase in weight, a marked correlation with the concentration of the natural hair dye can be determined. This can be explained by the ability of Eisenia fetida to fragment organic matter and derive nutrients from the micro organisms growing on it (as described in 3.1.2). The natural hair dye consists of crushed henna leaves. Most probably Eisenia fetida uses the natural hair dye as food. No other unusual behaviour or changes in morphology were recorded during the final test.
41
6.5.2
Synthetic hair dye
6.5.2.1
Results
Table 6.7: results blanks final test
Table 6.8: synthetic hair dye: results final test
Table 6.9: synthetic hair dye replicas: results final test
42
Synthetic hair dye: Increase in weight 15 2
Increase in weight (%)
R = 0,991 10 5 2
R = 0,992 0 0,000 -5
0,010
0,020
0,030
0,040
0,050
0,060
test concentrations
0,070
replicas
-10 -15 Concentration (mg/kg dry soil)
Graph 6.5: synthetic hair dye: increase in weight final test
6.5.2.2
Conclusions
No mortality was observed during the final test. No LC 50 can be determined with the results from the final test. As the range-test implicated, the increase in weight decreases at low concentrations. The concentration where the increase in weight is the same as in the blanks is calculated at 0,023 mg/kg dry soil. Below this concentration, a lower increase in weight is recorded compared to the blanks. Above this concentration, a higher increase in weight is observed compared to the blanks. This concentration is sometimes referred to as the NOAEL (no observable adverse-effects level). Below a NOAEL the effects a toxicity that initially decreases as concentration decreases, might increase again eventually. In other words, the toxicity of a substance can be higher at low concentrations than at high concentrations, because of a different bio-chemical acting of the organism on the toxin or of the toxin on the organism. No other unusual behaviour or changes in morphology were recorded during the final test.
43
Final conclusions
a)
2 week acute toxicity test − Natural hair dye
No mortality was observed during the final test for the natural hair dye, even though the range test predicted this differently. The mortality of the range-finding test could have been caused by other environmental factors or by chance. The only different parameter between the rangefinding test and the final test were the controlled light-dark cycles. In the range test, the cycles were controlled by timers, which were replaced in the final test by exposure to natural sunlight. It is very unlikely that this parameter could have caused such an influence on the test, because the light-dark cycles of the sun approached the theoretical cycles of 16 hours of light and 8 hours of darkness. Looking at the increase in weight, a distinct correlation with the concentration of the natural hair dye can be determined. This can be explained by the ability of Eisenia fetida to fragment organic matter and derive nutrients from the micro organisms growing on it. The natural hair dye consists of crushed henna leaves. Most probably Eisenia fetida uses the natural hair dye as food. No LC 50 of the natural hair dye could be determined with a 2 week acute toxicity test. − Synthetic hair dye No mortality was observed during the final test. As the range-test implicated, the increase in weight decreases at low concentrations. The concentration where the increase in weight is the same as in the blanks is calculated at 0,023 mg/kg dry soil. Below this concentration, a lower increase in weight is recorded compared to the blanks. Above this concentration, a higher increase in weight is observed compared to the blanks. This concentration is sometimes referred to as the NOAEL (no observable adverse-effects level). Below a NOAEL the effects a toxicity that initially decreases as concentration goes down, might increase again eventually. In other words, the toxicity of a substance can be higher at low concentrations than at high concentrations, because of a different bio-chemical acting of the organism on the toxin or of the toxin on the organism. No LC 50 of the synthetic hair dye could be determined with a 2 week acute toxicity test.
44
b)
Analysis on AAS
All concentrations of all the metals analysed were below the lowest concentration of the standard series. From the results of the analysis we can conclude (in mg/g hair dye):
Zn Cr Ni Cu Fe Pb Cd
Natural hair dye < 0,389 < 0,973 < 1,946 < 0,973 < 1,946 < 4,864 < 0,486
Synthetic hair dye < 0,353 < 0,882 < 1,764 < 0,882 < 1,764 < 4,409 < 0,441
The analysis of the synthetic hair dye was possibly disturbed by matrix interferences. In literature (1) is mentioned that the analyses could be disturbed by the presence of paraffins. It is very likely that paraffins are present as additives in the synthetic hair dye. Another issue is that the matrices of the hair dye solutions are completely different from the matrices of the standard solutions. Therefore, a standard addition method might be more suitable for this analysis.
c)
Discussion
The 2 week toxicity test performed as described in this thesis is not suitable to be integrated in the course ‘Ecotoxicology Laboratory Exercises (E1306)’ (20). Though with some improvements, it might become suitable. First of all, a new range test can be performed for the natural hair dye. The range test should contain at least 1 replica of every test concentration, and at least 2 blanks. This would largely prevent wrong interpretation of results that might be influenced by other parameters than the toxin. For the synthetic hair dye, a new range test can be set up around the NOAEL determined in this thesis. Again the range test should include at least 1 replica of every test concentration, and at least 2 blanks. It might be interesting to investigate if the natural hair dye has an increasing toxicity at low dose concentrations as well. In that way, an acute toxicity test on Eisenia fetida could be set up to determine the difference in toxicity between both hair dyes at low dose concentrations by determining the NOAEL of both hair dyes, and perhaps the LC 50. If no increasing toxicity is found at low concentrations for the natural hair dye, an exercise could be set up in which just the increasing toxicity of the artificial hair dye at low concentrations is determined. A last, more drastic option is to replace the 2 week acute toxicity test by an earthworm reproduction test. Previous research (17) has shown that the presence of metals has a distinct influence on the reproduction. The results of the AAS analysis show that most of the investigated metals are present in the hair dyes, though the concentrations of most metals in the analysed
45
solutions of the hair dyes were below determination limits, but not below detection limits. A new analysis could be performed, this time with higher concentrations of hair dye, and using a standard addition method instead of external standards.
46
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