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Determination of Physicochemical Parameters and the Correlation with Organotin
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 investigation on the levels of tributyltin(TBT), dibutyltin(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. Tributyltin (TBT), Dibutyltin (DBT), and
(monobutyltin) 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.The
correlation between sites of sampling and concentration are vital information to infer
on the reintroduction of pollutants occurances and fate and toxicity effect to the
environmental.On the other hand,the correlation between organotin concentration and
physicochemical parameters are very much essential in understanding the chemistry
of organotin on rates of pollution ,degradation of organotin compounds particularlt
butyltin species ,remediation methods to be applied (eg.bioremediation of pollutants)
and environmental mitigation measures and purposes.
.
2
Acknowledgements
I would certainly not have been able to finish my thesis without the supervision of
my supervisor, committee members, assistance from friends, and encouragement from
my family .I would like to explicit my deepest gratefulness to my advisor, Dr. Ferdi-
us@Ferdaus Mohamat Yusuff, for her excellent guidance, compassionate, persever-
ance, and providing me with an vivid atmosphere for doing research and who let me
experience the research of organotin analysis of core sediments in the field and prac-
tical issues beyond the textbooks, patiently improved my writing by rectifying them
and financially and monetarily supported my research.
I would also like to thank Dr. Lutfi Wan Johari, Prof. Kannan Narayanan, and Prof.
Mohamad Pauzi Zakaria for administrating my study for the past numerous occasions
and helping me to cultivate my circumstantial in analytical chemistry, geochemistry,
and ecology. Special thanks goes to Mr.Kit Wui Sien, who was enthusiastic to contrib-
ute in my sample collection committee with helping me much in transportation and
mobility purposes.
I would take this chance to thank Mr.Azwan Mejan, who as a wholesome friend,
was continually alacritous to help and give his best recommendations. It would have
been a lonely laboratory without him. Countless thanks to Mr Samsudin, Mr Gaffar,
Miss Thana, Mr Zairi, Miss Lela and other workers in the laboratory of the Environ-
mental Studies Faculty for helping me putting into place all the materials, apparatus,
reagents and necessary laboratory conditions for my study. My research would not
have been achievable without their assistance.
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 accompanying me and boosting me with their best wishes
and always there encouraging me up and stood by me throughout the good times and
bad.
3
Table of Contents
Abstract..................................................................................................................................1
Acknowledgements................................................................................................................2
Table of Contents...................................................................................................................3
List of Figures........................................................................................................................1
List of Tables.........................................................................................................................1
1 Introduction......................................................................................................................2
Background and Context...................................................................................................2
Scope and Objectives.........................................................................................................5
2 Literature Review.............................................................................................................6
Organotin...........................................................................................................................6
2.2.1 Organotin Species.................................................................................................6
2.1.3 Organotin toxicity.................................................................................................7
2.1.4 Transport and Pathway of Organotin Compound and negative effect on Aquatic
Ecosystem...........................................................................................................................8
2.0 Physicochemical Parameters.......................................................................................9
2.1.1 pH........................................................................................................................9
2.1.2 Total Organic Carbon (TOC)...............................................................................9
2.1.3 Sediment Particle Size.......................................................................................10
3.0 Materials and Methods...................................................................................................11
3.1 Method of Selection Criterion of Sampling Site.......................................................11
3.2 Instruments, Apparatus and Glassware, Materials and Reagents Used.....................12
3.2.1 Instruments.........................................................................................................12
3.2.2 Materials and reagent..........................................................................................13
3.2.3 Apparatus and glassware.....................................................................................14
3.3 Sample Collection, Preservation and Storage............................................................14
3.4 Sample Analysis.........................................................................................................15
3.4.1 Gas Chromatography /Mass Spectrometry Analysis..........................................15
3.4.2 Total Organic Carbon Analysis...........................................................................15
3.4.3 pH Analysis.........................................................................................................15
3.4.4 Sediment Particle Size Distribution Analysis.....................................................16
3.5 Data Collection and Statistical Analysis....................................................................16
3.5.1 Data Collection...................................................................................................16
3.5.2 Statistical Analysis..............................................................................................16
4.0 Results and discussion...................................................................................................18
4
4.1 Physicochemical Parameters and Depth....................................................................18
4.1.1 Depth and Organotin Concentration...................................................................18
Figure 6.Organotin Compounds Concentration against Depth of Kong-kong 3.........20
4.1.2 pH and depth.......................................................................................................20
4.1.3 Total Organic Carbon (TOC) and depth.............................................................22
4.1.4 Particle Size Distribution and depth...................................................................24
4.2 Physicochemical parameters and sites.......................................................................26
2.1.4 4.2.1 Organotin compound concentration and sites...........................................26
4.2.2 Particle Size Distribution and Sites....................................................................27
4.2.3 Total Organic Carbon (TOC) and sites...............................................................28
4.2.4 Mean pH and sites..............................................................................................29
5.0 Conclusion.....................................................................................................................31
Reference.............................................................................................................................32
Appendix 1...........................................................................................................................36
Appendix 2...........................................................................................................................37
Appendix 1...........................................................................................................................43
Appendix 2...........................................................................................................................44
5
List of Figures
Figure 1. Important Organotin Compounds................................................................................6
Figure 2 .Pathway of Organotin in the Aquatic Ecosystem........................................................8
Figure 3.Map of Sampling Sites and its Coordinates...............................................................11
Figure 4.Organotin Compound Concentration against Depth in Kong-kong 1........................19
Figure 5.Organotin Compound Concentration against Depth in Kong-kong 2........................19
Figure 6.Organotin Compounds Concentration against Depth of Kong-kong 3......................20
Figure 7.Mean pH against Depth for Kong-kong 1..................................................................20
Figure 8.Mean pH against Depth for Kong-kong 2..................................................................21
Figure 9.Mean pH against Depth for Kong-kong 3..................................................................21
Figure 10.Total Organic Carbon against Depth for Kong-kong 1............................................22
Figure 11.Total Organic Carbon against Depth for Kong-kong 2............................................23
Figure 12.Total Organic Carbon against Depth for Kong-kong 3............................................23
Figure 13.Particle Size Distribution against Depth for Kong-kong 1......................................24
Figure 14.Particle Size Distribution against Depth for Kong-kong 2......................................25
Figure 15.Particle Size Distribution against Depth for Kong-kong 3......................................25
Figure 16. Organotin Compound Concentration against Sites.................................................26
Figure 17.Particle Size Distribution against Sites....................................................................27
Figure 18.Total Organic Carbon against Sites..........................................................................28
Figure 19.Mean pH against Sites..............................................................................................29
1
List of Tables
Table 1.Historical Concentration of Organotin Compounds in Malaysia................................12
Table 2 .Instruments used in Analysis......................................................................................13
Table 3.Materials and reagent used in Analysis........................................................................14
Table 4.Apparatus and Glassware used in Analysis.................................................................14
1
1 Introduction
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 sub-
stituent, and X is a halogen: -OH,-SH, or –OR.
Currently , there are existance of over eighthundred organotin compounds which
are synthetic or arise naturally in the environment . The butyltins species (BTs) of
organotin derivatives, such as tributyltin (TBT) and its degradation products namely
the dibutyltin, DBT and monobutyltin, MBT, and the phenyltins species (PhTs) such
as, triphenyltin (TPhT) and its degradation products such as the monophenyltin,
MPhT and diphenyltin, DPhT, are fairly distributed in the marine environment.
Taking into consideration of the extensive distribution and intense toxicity effect of
these compounds on marine organisms, their monitoring and fate analysis in the
environment are significant (M. Hoch et al., 2001).
Incursion and accumulation of considerable amounts of these compounds in the
bottom sediments of the water bodies was the result of several years of using
organotin-based antifouling paints, where in particular tributyltin for inhibiting the
growth of marine organisms on ships’ hulls, and triphenyltin-based pesticides. Estuary
regions in the coastal marine area, and harbour and shipyard basins were
exceptionally affected.
.
2
Lately, there has been a considerable reduction in new loads of OTs into the
marine environment as a result of to the endorsements of the International Convention
on the Control of Harmful Anti-Fouling Systems on Ships (IMO, London, 2001),
framed under the patronages of the International Marine Organisation (IMO), coming
into force.Since 1st January 2003, the use of organotin-based anti-fouling paints on
newly built or painted ships (shorter than 25 metres) has been forbidden (IMO,
London, 2001). However, habours and shipyards are still considered the parts
susceptible to these pollutants because of intensive ship congestion and activities for
instance removal of old paint from hulls and ship scrapping.
Organotin compounds and mainly other highly toxic pollutant have high capacity to
be preserved in the base sediment which acts as the reservoir ; they impose a possible
ecotoxicological risk for live organisms long after the anthropogenic compounds have
dispersed into the environment. OTs compound adsorb onto very small sediment
fractions (particle size 50.063 mm). The portions of these are easily available to
benthic organisms, in particular decomposers and detritus feeders (J. Bolalek et
al.,1999).
The temperature,salinity,qualitative and quantitative composition of organic
matter ,and particle size distribution are namely the physicochemical
parameters ,which alters in timely manner and causes the release of specific forms of
organotin from sediments (D. Schwesig et al.,2005). As a result of the release,they are
widely bioavailable, hence , harmful and damaging to the aquatic orgamnisms living
in the water bodies.
Bottom sediments or core sediment vary in reference to compositions of chemical,
size of particle, source, rate of sedimentation and geographical dispersion and
distribution (J. Pempkowiak et al.,1997).Many synthetic and anthropogenic
sunstances content in core sediments depends on their size of particle,sediment
surface,magnetic properties and density which are cummulatively the physical
properties and chemical properties (i.e., ion exchange capacity, adsorptive properties,
organic matter content and content of inorganic compounds, such as carbonates and
3
oxides as well as on the environmental conditions for instance , pH, salinity and water
temperature) (M.Hoch et al.,2004).
Continental or surface-dweling sediments, particularly from river mouths and
ports, typically comprise of substantial proportions of mineral clays and organic
matter (D. Schwesig et al.,2005). The environmental conditions of river mouths and
ports for instance the depleted salinity is condusive for the buildup of organic
compounds comprising organotin to occur. Moreover, such sites are exceptionally
subjected to the contribution of new fractions of pollutants. The build up of these
sediment become the sink for hydrophobic pollutants. Nevertheless, under certain
environmental acclimatizes they may also undergo transformation as the source of
reintroduced contamination.
4
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).
5
2 Literature Review
Organotin
Compounds of organotins(OTs) are a cluster of organometallics that have a distinct struc-
ture designated by the subsequent molecular formula of RnSnX(4-n), in which Sn represent the
atom of tin , R represents the alkyl group, such as methyl, butyl , octyl or phenyl and X which
corresponds to O-, OH-, Cl-, F,SH- etc., and n ranges from approximately 1 to 4.
2.2.1 Organotin Species
The amount and type of organic substituents bonded to the tin atom determine the
properties and applications of specific organotin compounds. Group X has almost no
effect on an organotin compounds properties, while the alkyl chain length has a signi-
ficant effect on a compound’s toxicity (Thoonen et al., 2004). Based on literature data,
in the case of diorganotin compounds, R2SnX2, it is the organic groups R that determ-
ine the activity potential, while group X controls the delivery of active ions
R2Sn2+.Table 1 presents information about selected organotin compounds.
6
Figure 1. Important Organotin Compounds.
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 facilitate
their acceptance by well-hydrated cells. The lipophilic and hydrophilic properties of organot-
ins 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 -
7
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
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 2 .Pathway of Organotin in the Aquatic Ecosystem.
The negative effect of organotin compounds on the aquatic ecosystem is the reason behind at-
tempts to detect and determine the level of these compounds in various parts of the environment.At
present, it is assumed that the trisubstituted tin compounds pose a significant threat to proper function-
ing 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 compounds of aquatic origin have begun to enter land organ-
isms, causing negative effects in many of those from the highest level in the trophic chain (Wasik,
2012).
8
2.0 Physicochemical Parameters
Sediment physical properties such as the sediment surface proper, size of particles, ,density
and magnetic properties of unconsolidated marine sediments are vital variables of understand-
ing change in geological event of deposition environment effect of mechanical and chemical
diagenesis with burial depth after deposition.(Hamilton and Bachman.,1982).The concentra-
tion of Organotin compound depends on chemical properties for example the exchange of
capacity of ion, organic matter substance, adsorptive properties and content of inorganic com-
pounds, as well as on the environmental circumstances and conditions (i.e., salinity, pH, and
water temperature) (Cima et al., 1996; El Hassani et al., 2005; Gray et al.,1987; Omae, 2003).
2.1.1 pH
pH= -log [H+]
The pH is the most imperative parameter influencing the sorption capacity of adsorption
medium (Sravani et. al., 2012). With the decreasing of pH (up to pH4)(M.Hoch et
al.,2002),adsorption of organotin compounds onto organic matter and clay mineral increases.
Referring to the adsorptivity of clay minerals, for instance, TBT is the most effectively adsorp
at pH=6 for montmoryllonite, and at pH=7 for kaolinite. At such pH of these values , the in-
tensity of the cations of organotin compounds is the highest. This happens when the pH is
closed to the pKa – for TBT pKa=6.3 and for TPhT pKa=5.2 (C.G Arnold,1998). The formed
cations can effortlessly adsorbed onto the negatively charged superficial of minerals
(K.Medrzycka et al., 2006),while the consequential bonds have electrostatic character. Sedi-
ments rich in organic matter have high adsorptivity which is most effective at pH 6–7, when a
cation is the dominant TBT form. Then the formation of complex bonds between TBT+ ion
and depronated ligands of organic matter are formed (K.Medrzycka et al., 2006).
2.1.2 Total Organic Carbon (TOC)
Organic carbon is the most substantial factor that controls the concentration of organotins
in sediments (M.Hoch et al.,2002).The partition coefficient of sediment and water or known
as kd for organotin undertakes very low values in the case of pure and low organic carbon
content minerals,e.g. kd for pure kaolinite is 51L kg-1,while the supplement of 5% of organic
matter result in the abrupt kd escalation up to 2700L kg-1.(Alonso-Azcarate et al .,2003).
9
2.1.3 Sediment Particle Size
The finest size fractions (<0.063mm) exhibit principally good sorptive properties, for in-
stance silty-clays (M.Hoch et al.,2004).This is for the reason that the physicochemical factors
(high proportion of clay minerals)(J.Pempkowiak et al.,1997).Reference to the surface proper
of sediment particles such as Silty-clay sediments are considered by the surface suitable with
high sorptive properties at the level of number of metres squared per gram, while for gravels,
it does not surpass centimetres squared per gram (J.Pempkowiak et al.,1997).Percentage of
inflatable 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. In addition of organic matter, higher
sorption of TBT can be expected onto kaolinite as different minerals adsorb organic matter
with fluctuating strength. The arrangement in which organic matter adsorbs on minerals is as
respects of : kaolinite>montmorilonitequartz (Schwesig et al.,2004).
10
3.0 Materials and Methods
3.1 Method of Selection Criterion of Sampling Site.
Figure 3.Map of Sampling Sites and its Coordinates.
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]
11
Table 1.Historical Concentration of Organotin Compounds in Malaysia.
3.2 Instruments, Apparatus and Glassware, Materials and Reagents Used
3.2.1 Instruments
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 -
12
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 2 .Instruments used in Analysis.
3.2.2 Materials and reagent
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
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
13
Ethanol Pesticide residue analysis
Diethyl ether Pesticide residue analysis
Sodium hexametaphosphate 250g
Distilled water -
Milli-Q water -
CaCO3 standards (TOC analysis) 5g
Table 3.Materials and reagent used in Analysis.
3.2.3 Apparatus and glassware
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
Sieve (500,250,150,100,70μm) 1 unit (each)
Spatula -
Test tubes -
Zip lock bags -
Vacuum filter set -
Table 4.Apparatus and Glassware used in Analysis.
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.
14
3.4 Sample Analysis
3.4.1 Gas Chromatography /Mass Spectrometry Analysis
Organotin compounds were extracted from the sediment followed the application sugges-
ted by (L.Edbon et al,.1998):Freezed-dried 0.5g of sediment in a centrifugal tube,was added
with 100ml of TPrT (50 mgL-1) as an internal standard. After treatment for about 10 minutes,
2 g of sodium chloride (NaCl), 15mL of toluene containing 0.1% tropolone, and 10mL of 1
mol L-1 of methanolic HCl were added. Then the capped tubes were mixed for sixty minutes,
and 10mL of Milli-Q water was added. The tubes were mechanically shaken well for 10 min
and then centrifuged at 2000 rpm for 4 minutes. Next, toluene part was concentrated to 5mL
for further analysis. NaBEt4 was pertained as a derivatization agent for organotin compounds
in sediments as stated : 5mL of 1 molL-1 acetate buffer of pH 5 was added to the acetone part.
Fifteen millilitres of milli-Q water and 1mL of 5% NaBEt4 were added. The tubes were
shaken mechanically for 10 min for extraction and ethylation step, and then centrifuged. Pas-
teur pipette was used to collect the layer of hexane from the centrifugal tube. Water traces in
the sample were removed by smearing 3 g of anhydrous Na2SO4. Finally, the hexane extract
was evaporated to 100 mL applying stream of nitrogen gas. 1 mL was injected into the gas
chromatography instrument for organotin compound concentration analysis.
3.4.2 Total Organic Carbon Analysis
Total organic carbon or TOC was measured in percentage at Faculty of Agrobio ,UPM oper-
ating a Total Organic Carbon Analyzer (Shimadzu TOC-VCSH), in which was well-appointed
with a Solid Sample Module(Shimadzu SSM-5000A) by following the manufacturer’s
method (Shimadzu, 2001). The difference in combustion and oxidation and the carbonate
acidification reaction (analysis of inorganic carbon) was used to calculate and compute TOC.
3.4.3 pH Analysis.
The pH reading was determined by applying (METHOD 9045D, USEPA). Weighted 20 g
of dried sediment was placed into 50-mL beaker, then mixed with 20 mL Milli-Q water,
covered, and the suspension is stirred vigorously for 5 minutes. Additional dilutions are per-
missible if occupied with hygroscopic sediments and soil or other tough matrices. Then, the
sediment suspension was let to stand for about an hour to allow most of the suspended clay to
settle out from the suspension or filtered or centrifuged off from the aqueous phase for pH
measurement. Next, the pH meter electrodes are adjusted using the clamps of the electrode
holder in order to, upon lowering the electrodes into the beaker, the glass electrode will be
immersed just deep enough into the clear supernatant solution to ascertain a good electrical
15
contact through the fibre-capillary hole or ground-glass joint. The electrodes are inserted into
the sample solution in this manner. For permutation of electrodes, immerse just below the sus-
pension. The pH reading is determined.
3.4.4 Sediment Particle Size Distribution Analysis.
The Hydrometer method as recommended by the USDA Soil Survey and Canadian Soil
Survey Committee was used in determining the particle size distribution in the core sediment.
Qualitatively, this method regulates the quantities of 3 distinct sizes of primary sediment
particles, in which is determined via their settling rates in the aqueous solutions using well
calibrated standard hydrometer. Proportion of sediments are characterized by class sizes with
the range from 2000 - 50 um; Silt ranging from50-2.0 um and clay < 2.0 um. The principal Of
particle settling rates are based primarily on sedimentation confined by the Stroke’s Law and
measured using a hydrometer. In this investigation, the standard ASTM 152H-Type hydro-
meter is established on a standard temperature of 20 oC and a particle density of 2.65gcm-3
and units are articulated as grams of soil per litre. For specific samples the method may need
the pre-treatment removal of soluble salts, carbonates, organic matter and iron oxides with
successive dispersion using sodium hexametaphosphate (Day 1965). Determining the hydro-
meter reading of a blank solution will cater the correction for temperature and solution viscos-
ity. The method employs a detection limit of 2.0% sand, silt and clay (dry basis) and is gener-
ally 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 representation
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.
Correlation analysis is the most widely used and reported statistical analysis method
.It is vital in determining a relationship exist between two distinct variables(Goldrath
N et al.,1988). For instance, depth and physicochemical parameters (eg. total organic
carbon, pH, particle size distribution and orgatotin compounds concentration) and also
depth within sites and sites by concentration of organotin compounds in this investig-
16
ation.The analysis of variance or commonly abbreviated as ANOVA is a statistical
tool used to perceive variances between means of experimental group. ANOVA is
justified in experimental designs with a dependent variable which is a continuous
parametric numerical outcome measure, and multiple experimental groups within one
or more independent or categorical factor. In ANOVA terminology, independent vari-
ables are called factors, and groups within each factor are referred to as levels(Wack-
erly DD et al.,2002).ANOVA is actually a very convincing technique and there are
many accounts which will not be mentioned in this writing, the interested reader can
pursue these in Armitage and Berry (chapters 7 and 8, 1994).
17
4.0 Results and discussion.
4.1 Physicochemical Parameters and Depth
4.1.1 Depth and Organotin Concentration
Concentrations of TBT in the investigated area of Kong-kong 1(Figure 2.) ranged between
790.0 to 7.0 μg/L with corresponding to each depth intervals. It is clear that TBT is the pre-
dominant species of OTC with a ratio to total OT species ranged to 73.82%. The maximum
value of 790.86 ug/L. Overall OT concentration and increasing depth was found to correlate
negatively with r=-0.464(p=0.05) and the significance is strongest for DBT species against
depth. A strong significant correlation with r = 0.745 (p = 0.01) was found between the con-
centration of TBT and DBT reflecting the degradation of TBT as a main source of DBT in the
Kong-kong 1 site. At site Kong-kong 2(Figure 3.), the concentration of both TBT and DBT
correlates positively strong with r=0.804(p=0.01) which emulating degradation of TBT as a
dominant source of DBT in the Kong-kong 2 site. However, total mean concentration of MBT
of site 2 is relatively higher than site 1, and concentration of TBT is relatively is relatively
smaller in site 2 than site 1, this scenario can be contributed by high degradability factor at
site 2 or minimal current reintroduction of organotin compounds at site 2 than site 1 which is
concentrated with anthropogenic activities. Reference to the depth, DBT has negatively strong
correlation against depth, r=-0.685(p=0.05),this might due to certain physicochemical factors
affecting the adsorption of butyltin compounds. At Kong-kong 3(Figure 3.), there is a strong
positive correlation between MBT and depth with r=0.733(p=0.05) which resonates the occur-
rences of MBT settling at the site, which is the furthest site from site 1, which is highly pol -
luted. There is no statistically significant evidence of TBT concentration correlating with
depth at site 3, which suggest the site is far away from the pollutant sources or current reintro-
duction. However, there is a strong negative correlation between DBT and MBT of r=-
0.812(p=0.05) which reflect degradation of DBT as a main source of MBT in the Kong-kong
3 site. Thus, there is statistical evidence that organotin compounds concentration have
significant negative correlation towards depth. Hence, this condition sets a benchmark for
further investigation on other parameters compared to depth.
18
Figure 4.Organ-
otin Compound Concentration against Depth in Kong-kong 1.
Figure 5.Organotin Compound Concentration against Depth in Kong-kong 2.
19
0-3cm 7-9cm 13-15cm
19-21cm
25-27cm
30-33cm
36-39cm
42-45cm
0
200
400
600
800
1000
1200
1400
Organotin Compounds Concentration against Depth of Kong-kong 1
MBT DBT TBTDepth /cm
OT
conc
entr
ation
ug/
L
0-3cm
6-9cm
12-15cm
18-21cm
24-27cm
30-33cm
36-39cm
42-45cm
48-51cm
54-57cm
0
50
100
150
200
250
300
350
400
Organotin Compounds Concentration against Depth of Kong-kong 2
MBT DBT TBT
Depth /cm
OT
conc
entr
ation
ug/
L
Figure 6.Organotin Compounds Concentration against Depth of Kong-kong 3
4.1.2 pH and depth
At site 1(Figure 5), pH of core sediments by depth intervals has a strong positive correla-
tion with r=0.929(p=0.01).Thus, as depth increases, pH also increases almost linearly, in
which the basicity of the sediment increases. This condition may due to the increase of bac-
terial activity with depth which elevates the pH (Wang and Alva, 2000; Weston et al., 2010).
Statistically there is no significant correlation between pH and depth in sample from site
2(Figure 6) and 3(Figure 7).
Figure 7.Mean pH against Depth for Kong-kong 1
20
0-3cm 6-9cm 12-15cm 18-21cm 24-27cm 30-33cm0
20
40
60
80
100
120
140
160
180
Organotin Compounds Concentration against Depth of Kong-kong 3
MBT DBT TBT
Depth /cm
OT
conc
entr
ation
ug/
L
Figure 8.Mean pH against Depth for Kong-kong 2.
Figure 9.Mean pH against Depth for Kong-kong 3.
21
4.1.3 Total Organic Carbon (TOC) and depth.
Statistically no significance difference in correlation between TOC and depth in sample
from site 1(Figure 8).However, there is a strong negative correlation between TOC and depth
of site 2(Figure 9) with r=-0.733(p=0.05).The reduction in TOC with increasing depth display
that site 2 is relatively further to industrial and anthropogenic influence which affects the
TOC usually with positive correlation. There is a strong negative correlation between TOC
and depth of site 3(Figure 10) with r=-0.868 (p=0.05).The higher reduction ratio of TOC cor-
responding to its depth at site 3, reflects site 3 is even relatively further than site 2 to indus-
trial and anthropogenic influence which affects the TOC usually with positive correlation.
This is true in the case of sampling site selection in which site 1 with busy anthropogenic
activities, site 2 and 3 is further from site 1 relatively.
Figure 10.Total Organic Carbon against Depth for Kong-kong 1.
22
Figure
11.Total Organic Carbon against Depth for Kong-kong 2.
Figure 12.Total Organic Carbon against Depth for Kong-kong 3.
23
4.1.4 Particle Size Distribution and depth
There is no significant correlation between particle size distribution of site 1(Figure11) and
depth. However percentage of gravel and silt correlate with r=-0.850(p=0.01). At site 2(Fig-
ure12), gravel and depth have strong correlation with r=0.687(p=0.05).However sand and silt
do not display any significant correlation towards depth. There is no significant correlation
between particle size distribution of site 3(Figure13) and depth. However percentage of sand
and silt correlate strongly with r=-0.986(p=0.01).To windup, there is no evidence statistically
significant correlating depth to particle size distribution.
Figure 13.Particle Size Distribution against Depth for Kong-kong 1.
24
Figure 14.Particle Size Distribution against Depth for Kong-kong 2.
25
Figure 15.Particle Size Distribution against Depth for Kong-kong 3.
4.2 Physicochemical parameters and sites.
2.1.4 4.2.1 Organotin compound concentration and sites.
Figure 16. Organotin Compound Concentration against Sites.
Butyltin species were detected throughout the sediment core in all sites’ intervals. It
has been reported that The Australian sediment quality guidelines for TBT are 5 ug/L
and 70 ug/L for low and high threshold values (S.Diez et al,.2002). The concentra-
tions of TBT in sediment samples from all 3 stations in Kong-kong Laut were found
to be higher than the highest threshold value, suggesting these sediments may pose a
threat to a benthic biota(S.Diez et al,.2002). Moreover, high levels of TBT in Kong-
kong Laut sediments reflects its widespread contamination and could be an indicative
of the continuing usage of TBT based antifouling paints on ship hulls. Via graphical
representation in (Figure 14), qualitatively Kong-kong 1 has high TBT/DBT ratio may
be attributed to recent input of TBT and/or to low degradation of TBT to DBT into
this station. It is known that TBT degradation rates in sediment are slower than in wa-
ter column, particularly in anaerobic conditions. The half-life of TBT in sediments is
in the range of years rather than days or weeks in the water column (F.Cima et al,.2003)
26
Kong-kong 1 Kong-kong 2 Kong-kong 30
200
400
600
800
1000
1200
Organotin Compound Concentration against Sampling Sites
Monobutyltin Dibutyltin Tributyltin
Sampling Site
Conc
entr
ation
ug/
L
.OT compound concentration at site 1 is the highest compared to site 2 and 3 respectively. Overall, the
concentration mean of Dibutyltin is the highest compared to Tributyltin and Monobutyltin ,which is
38.99 ug/L for MBT,85.90ug/L and 61.46ug/L for DBT and TBT respectively, it can be inferred that the
highest concentration of DBT is from the source of degradation of TBT. There is a significant negative
correlation between DBT concentration and site with r=-0.523(p=0.01).Thus, it can be concluded that
further the site from main polluted site (Kong-kong 1/site 1), there is a linear decrease in organotin
concentration.
4.2.2 Particle Size Distribution and Sites.
Figure 17.Particle Size Distribution
against Sites.
The current investigation confirms that sediments in kong-kong laut, Johore are
generally consist of silty-clay (>80%) USDA and UK-ADAS textural
triangle.Statistically there is a correlation significantly between sand and sites, which
is r=-0.437(p=0.05),hence when sand and organotin tin concentration were tested for
correlation, there is a significantly strong correlation between them with
r=0.690(p=0.01). A study to assess the degradation kinetics of butyltin and phenyltin
in sandy soil collected in INRA (Pierroton Experimental Unit, near Bordeaux, France)
noted that the order of persistence in the soil was as follows:
MBT>DBT>MPhT>TBT>DPhT>TPhT. Therefore, it can be deduced that sand has high
adsorptivity towards OT compounds, particularly butyl species. Therefore, TBT, DBT
and MBT accumulated in sediments of the Kong-kong laut was affected by grain size,
significantly, sand even though with the least percentage of its constituent at each site.
27
Kong-kong 1 Kong-kong 2 Kong-kong 30%
10%20%30%40%50%60%70%80%90%
100%
Particle Size Distribution against Site
Gravel Sand SiltSand Silt Clay
4.2.3 Total Organic Carbon (TOC) and sites.
Figure 18.Total Organic Carbon against Sites.
The highest TOC content percentage (39.97%) was recorded for kong-kong 2 ,this
may due to it being near to small residential area and the nearby area composes of
small food industry region,hence total organic carbon may due to influx of organic
material to the site (Walkley.A et al.,1947).TOC percentage for site kong-kong 1 and
kong-kong 3 are 22.70% and 19.86% respectively whereby kong-kong 1 have
substantial amount of anthropogenic activities and site 3 with least anthropogenic
activities .Correlation between TOC and organotin compound concentration is
significant between MBT species and TOC which correlates negatively with r=-
0.383(p=0.01). To relate between MBT,TOC and particle size distribution(sand),in
brief MBT correlates negatively with TOC ,but MBT correlates positively with
particle size distribution (sand),hence it can be inferred that MBT is more strongly
adsorbed to mineral phase (sand) than organic matter (TOC) and to further strengthen
this point,the study area(south Johor) investigated has high constituent of
montmorillonite mineral which makes up the sand majorly (Khairuddin et
al.,1992) ,in which montmorillonite has the highest adsorptivity towards butyltin
species (refer 2.2.3 Sediment Particle Size). In addition, the adsorption properties of
OTs cannot be solely determined or correlated with only considering TOC because
OTs adsorption properties also depends on properties of minerals in sediment (eg.
montmorillonite, kaolinite and quartz) which has different sorption coefficient kd.To
wrap up, the total organic carbon has least affects on the organotin concentration at
28
Kong-kong 1 Kong-kong 2 Kong-kong 305
10152025303540
TOC against Site
Kong-kong laut than the type of mineral content which makes the component of the
particles of the sediments.
4.2.4 Mean pH and sites.
Figure 19.Mean pH against Sites.
The mean pH value of all three site is in the range of ±0.24 and tend to display low
variation between sites. From the statistical correlation test done, all species of OT
concentration have strong negative correlation with pH with MBT having r=-
0.929(p=0.01), r=-0.881(p=0.01), and r=-0.833(p=0.05) for TBT and DBT
respectively. Therefore, in lower pH the concentration of OT is higher than in higher
pH. At low pH where pH<pKa, the organotin compound is present primarily as cation
and the main adsorption process is expected to be a cationic exchange mechanism. At
pH higher than pKa, the neutral organotin species are prevailing and adsorption is
mainly controlled by hydrophobic interactions. In general, the highest adsorption of
TBT is detected between pH 6 and 7, which reveals the area of maximum overlap
between the total negative surface charge and the concentration of TBT cations in
solution (Fent,1996; Weidenhaupt, 1997; Hoch, 2001; Hoch, 2004).Thus, the optimal
pH value at Kong-kong Laut is suitable for adsorption and cationic exchange
mechanism on sediments.Thus ,the monitoring of the concentration of organotin in
29
Kong-kong laut is very vital to protect the marine ecosystem and human health
toxicity effect(Howells et al .,1997).
30
5.0 Conclusion
The recent study exposed that the concentration of organotin compounds in Kong-
kong Laut is still higher than the recommended threshold level and considered highly
polluted. Primarily this is due to increased recreational boating activities, perhaps due
to anthropogenic inputs from marinas activity, which is still moderately active till to-
day. Furthermore, the concentration mean of OTs(tributyltin,dibutyltin dan
monibutyltin) had elevated from 232 ug/L [2006 data], (Harino et al.,2008) to
398ug/L [2013 data] at recent study, hence after seven years there is an increase of
72%,hence if divided equally, it is of 9% increment annually of organotin concentra-
tion in Johore. Therefore, the banned organotin compound containing paints are still
being utilized till now, thus ,it is recommended that the enforcement and environmen-
tal departments should investigate the usage of anti-fouling paints as soon as possible
to avoid future contamination and severe ecotoxicological consequences . Factor of
depth correlates negatively with organotin concentration due to the various factor in-
fluencing the penetration of these compounds into the sediments which acts as the
sink, this might due to adsorptivity which was discussed earlier correlates positively
with influence of type of minerals which it constitute in sediment particle,the sand
portion of the Kong-kong Laut sediment which consist of montmorillonite(mineral
that makes up the sand proportion of sediment particle distribution) ,in which highly
adsorps butyltin compounds than kaolinite and quartz (Khairuddin et al.,1992).In ad-
dition, total organic carbon (TOC) influences the least significance in concentration of
organotin compounds in Kong-kong Laut due to the greater effects of sediment min-
erals which suppresses the effect of TOC. In relation to pH and depth, pH increases
with increase in depth due to increase in bacterial activity (Wang and Alva et
al .,2000) consequently increase in pH causes decrease in organotin concentration
due to adsorptivity of organotin species decreases with increasing pH (Fent,1996;
Weidenhaupt, 1997; Hoch, 2001; Hoch, 2004) . In conclusion,the in depth and further
understanding of the physicochemical parametres effects towards organotin concen-
tration particularly the butyltin species, should be studied for greater understanding of
the nature and fate of organotin compounds.
31
32
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Appendix 1
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Appendix 2
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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 specifications, 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.
46
Appendix 2
y
47