Determination of Physicochemical Parameters and the Correlation with Organ-

otin Compounds Concentration in Core Sediment

from Kong-Kong Laut, Johore ,Malaysia.

SELVAGAHNESH PRASAD

2013

Department of Environmental Sciences

Faculty of Environmental Studies

Universiti Putra Malaysia

Abstract

A quantitative study on the levels of tributyl- (TBT), dibutyl- (DBT), and

monobutyltin (MBT) species in core sediment samples was carried out. The sampling

area is located at the Kong-kong Laut estuary in the south east of Johore state,

Peninsula Malaysia, Malaysia. TBT, DBT, and MBT have been analysed in core

sediment samples by Gas Chromatography Mass Spectrometry. The trend of

concentration was comparatively studied with physicochemical parameters namely

pH, Total Organic Carbon (TOC) and Sediment Particle Size Distribution for the

presence of possible correlation.

2

Acknowledgements

I would never have been able to finish my dissertation without the guidance of my

supervisor, committee members, help from friends, and support from my family .I

would like to express my deepest gratitude to my advisor, Dr. Ferdius@Ferdaus Mo-

hamat Yusuff, for her excellent guidance, caring, patience, and providing me with an

excellent atmosphere for doing research and who let me experience the research of

organotin analysis of core sediments in the field and practical issues beyond the text-

books, patiently corrected my writing and financially supported my research.

I would also like to thank Dr. Lutfi Wan Johari, Prof. Kannan Narayanan, and Prof.

Mohamad Pauzi Zakaria for guiding my research for the past several occasions and

helping me to develop my background in analytical chemistry, biochemistry, and eco-

logy. Special thanks goes to Mr.Kit Wui Sien, who was willing to participate in my

sample collection committee with helping me much in transportation and mobility

purposes.

I would like to thank Mr.Azwan Mejan, who as a good friend, was always willing

to help and give his best suggestions. It would have been a lonely lab without him.

Many thanks to Mr Samsudin, Mr Gaffar, Miss Thana, Mr Zairi, Miss Lela and other

workers in the laboratory of the Environmental Studies Faculty for helping me putting

into place all the materials, apparatus, reagents and necessary laboratory conditions

for my research . My research would not have been possible without their help.

Finally, I would also like to thank my parents ,my dad, Mr Marimuthu Ayavoo and

my mom, Miss Tannavali Ramasamy, and my elder sister ,Miss Yogalekshumi Mar-

imuthu. They were always supporting me and encouraging me with their best wishes

and always there cheering me up and stood by me through the good times and bad.

3

Table of Contents

Abstract......................................................................................................................1

Acknowledgements...................................................................................................2

Table of Contents.......................................................................................................3

List of Figures............................................................................................................4

List of Tables.............................................................................................................5

1 Introduction...........................................................................................................6

1.1 Background and Context...............................................................................6

1.2 Scope and Objectives.....................................................................................8

2 Literature Review.................................................................................................9

3 Materials and Methods........................................Error! Bookmark not defined.

3.1 First Section using Heading 2......................Error! Bookmark not defined.

3.1.1 First Subsection using Heading 3............Error! Bookmark not defined.

3.1.1.1 First Subsubsection using Heading 4..............Error! Bookmark not

defined.

3.1.2 Second Subsection...................................Error! Bookmark not defined.

3.2 Second Section.............................................Error! Bookmark not defined.

4 Results and Discussion........................................Error! Bookmark not defined.

4.1 Subheading 1................................................Error! Bookmark not defined.

5 Conclusion...........................................................Error! Bookmark not defined.

5.1 Summary......................................................Error! Bookmark not defined.

5.2 Future Work.................................................Error! Bookmark not defined.

References...............................................................................................................20

Appendix 1..............................................................................................................21

Appendix 2..............................................................................................................22

4

List of Figures

To update this after revisions, right-click in the table and choose Update Field for the

entire table.

Figure 3.1 This is figure 1 in chapter 3. The caption for the figure is always below the

figure......................................................................Error! Bookmark not defined.

Figure 4.1. Caption for figure 2.....................................Error! Bookmark not defined.

5

List of Tables

To update this after revisions, right-click in the table and choose Update Field for the

entire table.

Table 3.1 This is table 1 in chapter 3. The caption for the table is always above the

table........................................................................Error! Bookmark not defined.

Table 4.1. Example for table 2.......................................Error! Bookmark not defined.

6

1 Introduction

1.1 Background and Context

Tin is found in many organic and inorganic compounds. It reacts with chlorine,

sulphur, and oxygen to form inorganic tin compounds, which are found in the earth’s

crust in small amounts (Agency for Toxic Substances and Disease Registry

(ATSDR),2011.). Organotin compounds (OTCs) belong to the group of organometal-

lic compounds (Fent, 1996) with at least one covalent Sn-C bond (Batt, 2006; El Has-

sani et al., 2005; Murata et al., 2008). OTCs can be mono-, di-, tri-, or tetra-substi-

tuted (El Hassani et al., 2005) The general formula of an organotin compound is (El-

Hassani et al., 2005): RmSnX4-m where m = 1, 2, 3, or 4, R is an alkyl or aryl substitu-

ent, and X is a halogen: -OH,-SH, or –OR.

At present, there are over 800 organotin compounds that are man-made and/or

occur in the environment [1]. The butyl species (butyltins, BTs) of organotin

derivatives, such as tributyltin (TBT) and its degradation products (i.e. dibutyltin,

DBT and monobutyltin, MBT), and the phenyl species (phenyltins, PhTs) such as,

triphenyltin (TPhT) and its degradation products (i.e. monophenyltin, MPhT and

diphenyltin, DPhT) are quite broadly spread in the marine environment. Considering

the wide distribution and strongly toxic effect of these compounds on marine

organisms, their monitoring and pathway analysis in the environment are critical (M.

Hoch et al., 2001).

Many years of using organotin-based antifouling paints, in particular TBT for

preventing the growth of marine organisms on ships’ hulls, and triphenyltin-based

pesticides have resulted in the penetration and accumulation of significant amounts of

these compounds in the bottom sediments of water bodies. River mouth areas in the

coastal marine zone, and harbour and shipyard basins were particularly affected.

Recently, there has been a significant decrease in new loads of organotin

compounds into the marine environment as a result of to the recommendations of the

International Convention on the Control of Harmful Anti-Fouling Systems on Ships

(IMO, London, 2001), formulated under the auspices of the International Marine

Organisation (IMO), coming into force.Since 1 January 2003, the use of organotin-

7

based anti-fouling paints on newly built or painted ships (shorter than 25 metres) has

been forbidden (IMO, London, 2001). Nevertheless, shipyards and harbours are still

considered the areas vulnerable to these pollutants because of intensive ship traffic

and activities such as removal of old paint from hulls and ship scrapping.The total ban

on TBT-based paints will start in 2008, and it will also include older vessels,i.e., those

produced before 2003.

Bottom sediments are a reservoir of toxic pollutants including organotin

compounds;they constitute a potential ecotoxicological risk for live organisms long

after the anthropogenic compounds have entered the environment. Organotin

compounds adsorb onto very small sediment fractions (particle size 50.063 mm).

These fractions are easily available to benthic organisms, in particular detritus feeders

(J. Bolalek et al.,1999).

Due to the changing physicochemical conditions in water and sediment, for

example, salinity, temperature, and quantitative and qualitative composition of

organic matter, the release of specific forms of organotins from the sediment may take

place (D. Schwesig et al.,2005). After being released, they become bioavailable and

therefore harmful to the marine organisms inhabiting the water column as well.

Bottom sediments or core sediment differ in reference to chemical composition,

particle size, origin, sedimentation rate and geographical distribution (J. Pempkowiak

et al.,1997). The content of many anthropogenic substances in bottom sediments

depends on their physical (i.e., size of particles, sediment surface proper, density and

magnetic properties) and chemical properties (i.e., ion exchange capacity, adsorptive

properties, organic matter content and content of inorganic compounds, e.g.

carbonates, oxides) as well as on the environmental conditions (i.e., pH, salinity and

water temperature) (M.Hoch et al.,2004).

Terrestrial sediments, especially from ports and river mouths, usually contain

considerable amounts of organic matter or mineral clays (D. Schwesig et al.,2005). In

ports and river mouths other environmental conditions conducive to the accumulation

of organic compounds including organotins occur, for example, low salinity.

8

Moreover, such locations are particularly exposed to the input of new portions of

contaminants. The forming sediments become a kind of trap or sink for hydrophobic

contaminants. However, under certain environmental conditions they may also turn

into a source of renewed contamination.

1.2 Scope and Objectives

1. The general objective of this study is to investigate the concentration of organotin

concentration in core sediment at straights of Johore, particularly Kong-kong Laut and

the physico-chemical parameters of the core sediment.

Objectives

1. To determine the physico-chemical parameters in core sediment samples from

Kong-kong Laut,Johore.

2. To identify the relationship of physico-chemical parameters with organotin com-

pounds concentration in core sediments.

3.To compare the concentration of organotin compounds concentration with historical

datas(before the ban of Organotin and after).

9

2 Literature Review

2.1 Organotin

Organotin compounds are a group of organometallics that have a structure described by the

following molecular formula: RnSnX(4-n), where Sn is the tin atom, R is an alkyl group, such

as methyl (Me,CH3-), butyl (Bu,C4H9–), octyl (Oc, C8H17–) or phenyl, e.g. (Ph, C6H5) and X

corresponds to O-, OH-, Cl-, F-, SH- etc., and n ranges from 1 to 4.

2.1.1 Organotin Species

The amount and type of organic substituents bonded to the tin atom determine the proper-

ties and applications of specific organotin compounds. Group X has almost no effect on an

organotin compounds properties, while the alkyl chain length has a significant effect on a

compound’s toxicity (Thoonen et al., 2004). Based on literature data, in the case of diorgan-

otin compounds, R2SnX2, it is the organic groups R that determine the activity potential, while

group X controls the delivery of active ions R2Sn2+.Table 1 presents information about selec-

ted organotin compounds.

Table 1.

10

2.1.2 Organotin Properties and Characteristics

Organotins bind strongly to solid phase particles, e.g., in sediments (forming oxides and

organic compounds), which results in a risk of contaminating the aquatic environment. OTCs

can also easily bond to proteins (Veltman et al., 2006) and display high affinity for cell mem-

branes (Cima et al., 1996). In order to present a full assessment of the impact of organotin

compounds on the aquatic ecosystem, it is necessary to determine thedegree of accumulation

and harmful effects of these compounds on various organisms in the food chain (Antizar-

Ladislao, 2008;Kannan et al., 1999).Organotin compounds have good lipophilic properties,

which facilitate their penetration through cell membranes.

On the other hand, these compounds also have good hydrophilic properties, which facilit -

ate their acceptance by well-hydrated cells. The lipophilic and hydrophilic properties of or-

ganotins affect these compounds toxicity which in turn may result in cell damage or cell death

(Cima et al., 1996; El Hassani et al., 2005; Gray et al.,1987; Omae, 2003).

2.1.3 Organotin toxicity.

Although the toxicity of organotin compounds in enzymatic systems has not been thor-

oughly investigated (Kimbrough,1976), it is known that due to their solubility, OTCs can eas-

ily penetrate into tissues and the nervous system (Bowen, 1988). Based on the results of nu-

merous studies, tributyltin (TBT), which is characterized by embryotoxicity and genotoxicity

(El Hassani et al., 2005; Jha et al., 2000; Marin et al., 2000), and triphenyltin (TPhT) have

been recognized as the most toxic organotins.

The low toxicity compounds are the derivatives of tri-n-octyltin (Antizar-Ladislao, 2008;

Heroult et al., 2008; Hoch, 2001; Omae, 2003; Ramalho et al., 2010; Riepe et al., 1997). The

harmful effect of TBT was observed in samples of plants and microorganisms; it also showed

a negative effect on higher organisms inhabiting the marine environment (Kannan et al.,

1998; Ramalho et al., 2010; Słaba et al., 2010; Zhenget al., 2005). It was established, inter

alia, that endocrine disordersn occur even at low TBT concentrations (Yang et al., 2010). Be -

cause of the toxic properties of OTCs, mainly with regard to butyltin compounds, the occur-

rence of these chemicals in the environment is becoming more alarming. For this reason, in

11

many scientific circles investigations on the toxicity and ecotoxicological effects of tin com-

pounds have been undertaken (Arambarri et al., 2003; Batt, 2006). The results of these studies

are of widespread interest in society due to the significant increase in pro-environmental

awareness.

2.1.4 Transport and Pathway of Organotin Compound and negative effect on Aquatic

Ecosystem.

Figure 1 illustrates the transport pathways of organotin compounds in the environment.

The negative effect of organotin compounds on the aquatic ecosystem is the reason behind

attempts to detect and determine the level of these compounds in various parts of the environ-

ment.At present, it is assumed that the trisubstituted tin compounds pose a significant threat to

proper functioning of the environment, while the decomposition products, or the di- and

mono-substituted derivatives, are less harmful (Belfroid et al., 2000; Guruge et al., 1996,

1997; Harino et al., 1997; Wasik,2012). In recent years, it was noted that organotin com-

pounds of aquatic origin have begun to enter land organisms, causing negative effects in

many of those from the highest level in the trophic chain (Wasik, 2012).

12

2.2 Physicochemical Parameters

Sediment physical properties(i.e., size of particles, sediment surface proper, density and

magnetic properties) of unconsolidated marine sediments are important variables of under-

standing geological event of deposition environment effect of mechanical and chemical dia-

genesis with burial depth after deposition.(Hamilton and Bachman.,1982).The concentration

of Organotin compound depends on chemical properties (i.e., ion exchange capacity, adsorpt-

ive properties, organic matter content and content of inorganic compounds, e.g. carbonates,

oxides) as well as on the environmental conditions (i.e., pH, salinity and water temperature)

(Cima et al., 1996; El Hassani et al., 2005; Gray et al.,1987; Omae, 2003).

2.2.1 pH

pH= -log [H+]

The most important parameter influencing the sorption capacity is the pH of adsorption

medium (Sravani et. al., 2012). Adsorption of organotin compounds onto clay minerals and

organic matter increases with the decreasing pH (up to pH4)(M.Hoch et al.,2002). In the

case of clay minerals, the adsorption of, for example, tributyltin is the most effective at pH=6

for montmoryllonite, and at pH=7 for kaolinite. At such pH values the concentration of organ-

otin cations is the highest. This happens when the pH is closed to pKa (the acidy constant) –

for TBT pKa=6.3 and for TPhT pKa=5.2 (C.G Arnold,1998). Cation forms can be easily ad-

sorbed onto negatively charged surface of minerals (K.Medrzycka et al., 2006),while the res-

ulting bonds have electrostatic character. Adsorption onto sediments rich in organic matter is

the most effective at pH 6–7, when a cation is the dominant TBT form. Then the complex

bonds between TBT + and depronated organic matter ligands are formed (K.Medrzycka et al.,

2006).

2.2.2 Total Organic Carbon (TOC)

Organic carbon is the most significant factor that controls the content of organotins in sedi-

ments (M.Hoch et al.,2002).The sediment – water partition coefficient (kd) for organotin as-

sumes very low values in the case of pure and low organic carbon content minerals,e.g. k d for

pure kaolinite is 51L kg-1,whilw the addition of 5% of organic matter result in the abrupt kd

increase up to 2700L kg-1.(Alonso-Azcarate et al .,2003).

13

2.2.3 Sediment Particle Size

The finest size fractions (<0.063mm) display particularly good sorptive properties, i.e.

silty-clays (M.Hoch et al.,2004).This is due to physical (large surface proper) and chemical

factors (high content of clay minerals)(J.Pempkowiak et al.,1997).Regarding surface proper

of sediment particles such as Silty-clay sediments are characterised by the surface proper with

high sorptive properties at the level of number of metres squared per gram, while for gravels,

it does not exceed centimetres squared per gram (J.Pempkowiak et al.,1997).Percentage of

expandable clay minerals in sediments are characterised by clay minerals which show a high

sorption capacity to organic contaminations of cationic or polar character (M.Hoch et

al.,2002). It depends on their surface area and reactivity (negatively charged surface). Sorp-

tion coefficient (Kd) for TBT (at pH=6) and surface proper values for the mineral particles can

be arranged in the following sequence: montmorillonite (Kd=89 L kg_1; surface area ca 32m2

g_1)> kaolinite (Kd=51 L kg_1; surface area=10m2 g_1)> quartz (Kd=25L kg_1; surface

area=0.31m2 g_1)(M.hoch et al.,2004). Particularly strong sorption of TBT in pure montmoril-

lonite is caused by its big surface area. (Hoch and Schwesig.,2004) observed the increase in

Kd value from 25 to 67 L kg_1 for pure quartz sand after adding 10% of montmoryllonite, and

to Kd=94L kg_1 after adding 20% of montmorillonite. When adding organic matter, higher

sorption of TBT can be expected onto kaolinite because different minerals adsorb organic

matter with varying strength. The sequence in which organic matter adsorbs onto minerals is

as follows: kaolinite>montmorillonitequartz (Schwesig et al.,2004).

14

3.0 Materials and Methods

3.1 Method of Selection Criterion of Sampling Site.

Map 3.1 Sampling Site Map.

The sampling site of Kong-kong Laut ,Pasir Gudang,Johor which is situated to the south-

east of southern peninsula state of Johore, Malaysia was chosen as the sampling site for the

study due to its geographically highest in concentration of organotin compound in whole Pen-

insula Malaysia(Harino et al .,2008). Coastal and estuaries of Johor Straits and its surrounding

vacinity had the highest concentration among other sites in Peninsula Malaysia. (Harino et

al .,2009).Table 2 below shows the concentration of organitin compounds in Peninsula Malay-

sia.[Red Circle: highest concentration of Organotin]

.

Source: (Harino et al .,2008)

15

3.2 Instruments, Apparatus and Glassware, Materials and Reagents Used

3.2.1 Instruments

The list of instruments used during analysis is shown in Table 3.1.

List of instruments used during

analysis Instruments

Model

Core sampler (1metre) Self-made silicone core sampler

Gas chromatography/mass

spectrometry

Hewlett-Packard Model 6890 GC

with Quadra pole mass spectrometry

(HP5973MSD)

152H Hydrometer -

Thermometer -

Stop watch Casio 98D

Incubator vertical rotary shaker -

pH meter ORION 2 STAR

Centrifuge Rotofix 32 (Hettich Zentrifugen)

Electronic balance Shimadzu AY 220

Dessicator -

Oven

TOC Analyser Shimadzu TOC VCN-S

Table 3.1

3.2.2 Materials and reagent

The list of materials and reagents used is shown in Table 3.2.

Materials and reagent Materials and

Reagents

Grade

Monobutyltin trichloride Reagent grade

Dibutyltin dichloride Reagent grade

Tributyltin chloride Reagent grade

Monophenyltin trichloride Reagent grade

Diphenyltin dichloride Reagent grade

Triphenyltin chloride Reagent grade

MBT trichloride-d5 Reagent grade

DBT dichloride-d10 Reagent grade

16

TBT chloride-d27 Reagent grade

Tetrabytyltin (TeBT)-d20 Reagent grade

Sodium Tetraethylborate >98%

Sodium acetate Reagent grade

Hydrochloric acid Metal analysis

Ethyl acetate Pesticide residue analysis

Sodium sulphate anhydrous Pesticide residue analysis

Copper(grain,20-60 mesh) Reagent grade

Potassium Hydroxide Reagent grade

Sep-Pak plus Florisil Content (50mg)

n-Hexane Pesticide residue analysis

Acetone Pesticide residue analysis

Methanol Pesticide residue analysis

Ethanol Pesticide residue analysis

Diethyl ether Pesticide residue analysis

Sodium hexametaphosphate 250g

Distilled water -

Milli-Q water -

CaCO3 standards (TOC analysis) 5g

Table 3.2

3.2.3 Apparatus and glassware

The list of apparatus and glassware used during analysis is shown in Table 3.3.

List of apparatus and glassware

used during analysis

Volume Apparatus and Glassware

Beakers 500mL x 5

Measuring cylinder 1000mL x 8

Conical flasks 100mL x 5

Volumetric flasks 100mL, 500mL, 1000mL

Pipettes 1mL, 10mL

Centrifuge tubes 50mL

Mortar and pestle 1 unit

Sedimentation cylinder 1000mL x 2

17

Sieve (500,250,150,100,70μm) 1 unit (each)

Spatula -

Test tubes -

Zip lock bags -

Vacuum filter set -

Table 3.3

3.3 Sample Collection, Preservation and Storage.

After the sample is collected using a core sampler, the sediment sample is refrigerated at -

20oC and free from light from the time of collection until extraction. A 3-day maximum ex-

tract storage time is recommended. The sediment are kept at -20oC until analysis procedure

applied.

3.4 Sample Analysis

3.4.1 Gas Chromatography /Mass Spectrometry Analysis

The extraction of organotin compounds from sediments was performed as follows [24]: to

0.5 g of freeze-dried sediment in a centrifugal tube, 100 mL of TPrT (50 mgL-1) was added as

an internal standard. After 10 min, 2 g of NaCl, 12mL of toluene containing 0.1% tropolone,

and 10mL of 1 mol L-1 of HCl methanol were added. The tubes were capped and mixed for 60

min afterwards, and 10mL of Milli Q water was added. The tubes were shaken for 10 min and

thencentrifuged at 2000 rpm for 3 min. The toluene part was concentrated to 5mL for further

analysis. NaBEt4 was applied as a derivatization agent for organotin compounds in sediments

as follows: 5mL of 1 molL-1 acetate buffer of pH 5 was added to the acetone part. Fifteen mil-

lilitres of milli Q water and 1mL of 5% NaBEt4 were added. The tubes were shaken for 10

min for ethylation and extraction, then centrifuged. The hexane layer was collected from the

centrifugal tube using a Pasteur pipette. Water traces were removed from the sample by ap-

plying 2 g of anhydrous Na2SO4. Finally, the hexane extract was evaporated to 100 mL using

a stream of nitrogen gas. 1 mL was injected into the GC for OTCs analysis.

18

3.4.2 Total Organic Carbon Analysis

Total organic carbon (TOC) was measured at Faculty of Agrobio ,UPM using a Total Or-

ganic Carbon Analyzer (Shimadzu TOC-VCSH), which was equipped with a Solid Sample

Module(Shimadzu SSM-5000A) following the manufacturer’s method (Shimadzu, 2001). The

TOC value was calculated by the difference of the results of the combustion–oxidation reac-

tion (total carbon analysis) and carbonate acidification reaction (inorganic carbon analysis).

3.4.3 pH Analysis.

To 20 g of soil in a 50-mL beaker, added 20 mL of reagent water, covered, and the suspen-

sion is stirred for 5 min. Additional dilutions are allowed if working with hygroscopic soils

and salts or other problematic matrices. Let the soil suspension stand for about 1 hr to allow

most of the suspended clay to settle out from the suspension or filter or centrifuge off the

aqueous phase for pH measurement. Adjust the electrodes in the clamps of the electrode

holder so that, upon lowering the electrodes into the beaker, the glass electrode will be im-

mersed just deep enough into the clear supernatant solution to establish a good electrical con-

tact through the ground-glass joint or the fiber-capillary hole. Insert the electrodes into the

sample solution in this manner. For combination electrodes, immerse just below the suspen-

sion. The pH reading is determined. (METHOD 9045D, USEPA).

3.4.4 Sediment Particle Size Distribution Analysis.

This method quantitatively determines the physical proportions of three sizes of primary

soil particles as determined by their settling rates in a aqueous solution using a hydrometer.

Proportions are represented by stated class sizes: sand ranging from 2000 - 50 um; Silt ran-

ging from50-2.0 um and clay < 2.0 um and those stated by the USDA Soil Survey and Cana-

dian Soil Survey Committee. Settling rates of primary particles are based on the principle of

sedimentation as described by Stokes’ Law and measured using a hydrometer. The use of the

ASTM 152H-Type hydrometer is based on a standard temperature of 20 oC and a particle

density of 2.65gcm-3 and units are expressed as grams of soil per liter. For specific samples

the method may require the pretreatment removal of soluble salts, organic matter, carbonates

and iron oxides with subsequent dispersion using sodium hexametaphosphate (Day

19

1965).Corrections for temperature and for solution viscosity is made by taking a hydrometer

reading of a blank solution. The method has a detection limit of 2.0% sand, silt and clay (dry

basis) and is generally reproducible to within ± 8%.

3.5 Data Collection and Statistical Analysis

3.5.1 Data Collection

All analytical data are collected, tabulated and generated into suitable graphical representa-

tion using Microsoft Excel 2013.Data are rearranged, classified according to intervals and

suitable bin values and stored with backup of hardcopy.

3.5.2 Statistical Analysis.

The hypothesis that the means of two groups are equal can be assessed by an appropriate t-

test, or possibly by some distribution-free analogue such as a Mann Whitney test. Analysis of

variance, often abbreviated to ANOVA, is the technique that is employed when there are

more than two groups to compare. Of course, just as with a t-test where there are paired and

non-paired versions available for data with different structures, there are several versions of

ANOVA.ANOVA is actually a very powerful technique and there are many versions which

will not be mentioned here: the interested reader can pursue these in Armitage and Berry

(chapters 7 and 8, 1994).

20

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21

Appendix 1

You may have one or more appendices containing detail, bulky or reference material

that is relevant though supplementary to the main text: perhaps additional specifica-

tions, tables or diagrams that would distract the reader if placed in the main part of the

dissertation. Make sure that you place appropriate cross-references in the main text to

direct the reader to the relevant appendices.

22

Appendix 2

23