THE PROFILE OF HYDROCARBONS AND HEAVY METALS IN SURFACE

SEDIMENTS FROM BATANG AI HYDROELECTRIC DAM, LUBOK ANTU,

SARAWAK

Nur Aein Binti Razali (24522)

Bachelor of Science with Honours

(Resource Chemistry)

2012

Faculty of Resource Science and Technology

THE PROFILE OF HYDROCARBONS AND HEAVY METALS IN

SURFACE SEDIMENTS FROM BATANG AI HYDROELECTRIC DAM,

LUBOK ANTU, SARAWAK

NUR AEIN BT RAZALI

This project submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science with Honours

(Resource Chemistry)

FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY

UNIVERSITY MALAYSIA SARAWAK

2012

ii

DECLARATION

I hereby declare that no portion of this work referred to in dissertation has been submitted in

support of an application for another degree or qualification to this university or any other

institution of higher learning.

_________________________

(Nur Aein Bt Razali)

Student Number : 24522

Department of Chemistry

Faculty of Resource Science and Technology

University Malaysia Sarawak

iii

ACKNOWLEDGEMENT

Alhamdullilah and most gratitude to Allah S.W.T. for the strength and courage to gave

me throughput completing this project to success.

I would like to thank the following individuals for helping me to complete my final

year project. Special thank to Prof Zaini Assim for his guidance, advice and support

throughout the period of my research. I also would like to thank to the laboratory assistants for

their cooperation, technical support, advice and help during this project.

I would like to thank, Master student Suhaila Gusni, my friends Ammar Ubaidullah

Rozali, Farahnasya Nabilah Huda Zahari, Widia Natasya Ariffin, Nur Atika Che Rusli and

Nur Hidayah Bt Kamarol Zaman, for their help, support and encouragement.

Lastly, I would like to express my gratitude to my parents Razali Yusof and Fauziah

Samdin, and family members for their love, moral and financial support that had given me the

strength to the completion my final year project.

iv

TABLE OF CONTENT

CONTENT Page

DECLARATION ii

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES ix

ABSTRACT x

ABSTRAK x

CHAPTER ONE: INTRODUCTION

1.1 General Introduction 1

1.2 Objectives of the Project 2

CHAPTER TWO: LITERATURE REVIEWS

2.1 Hydrocarbons in Aquatic Sediment 3

2.2 Heavy metals in Aquatic Sediment 5

2.3 Importance of Core Sediment in Environmental Studies 6

CHAPTER THREE: MATERIALS AND METHODS

3.1 Sampling Sites 8

3.2 Hydrocarbon in Sediment

3.2.1 Extraction of Geolipid 9

3.2.2 Column Chromatography Fractionation of Geolipid 9

3.2.3 Gas Chromatography-Flame Ionization Detector (GC-FID)

Analysis

10

3.2.4 Data Analysis

3.2.4.1 Qualitative Analysis 11

3.2.4.2 Quantitative Analysis 12

3.3 Heavy Metal Analysis

3.3.1 Sample Preparation 13

v

3.3.2 Atomic Absorption Spectrophotometer (AAS) Analysis 13

CHAPTER FOUR : RESULTS AND DISCUSSION

4.1 Response Factors (RFs) for Hydrocarbons

4.1.1 Aliphatic Hydrocarbon 16

4.1.2 Polycyclic Aromatic Hydrocarbons (PAHs) 18

4.2 Aliphatic Hydrocarbon in Sediments of Batang Ai Hydroelectric

Dam

4.2.1 Distribution of Aliphatic Hydrocarbon 19

4.2.2 Biomarker Indices of Aliphatic Hydrocarbons in Sediments

4.2.2.1 Carbon Preference Index (CPI) 23

4.2.2.2 Pristane/Phytane (Pr/Phy) Ratio 23

4.2.2.3 The Ratio of Isoprenoid/n-Alkane 24

4.3 Aromatic Hydrocarbon in Sediments of Batang Ai Hydroelectric

Dam

4.3.1 Distribution of PAHs 25

4.3.2 Biomarker Indices of PAHs in Surface Sediments

4.3.2.1 Fluoroanthene/Pyrene (Fluo/Pyr) 30

4.3.2.2 Phenanthrene/Antharacene (Phe/Ant) 30

4.3.2.3 Benzo (a) anthracene /Chrysene (B(a)A/Chry) 30

4.4 Distribution of Hydrocarbons from Other Places

4.4. 1 Aliphatic Hydrocarbons 32

4.4.2 Aromatic Hydrocarbons 34

4.5 Heavy Metals in the Surface Sediments from Batang Ai

Hydroelectric Dam

4.5.1 Calibration of Standard Heavy Metals on AAS 35

4.5.2 Distribution of Heavy Metals in the Surface Sediments from

Batang Ai Hydroelectric Dam

40

4.5.3 Spatial Distribution of Heavy Metals in the Surface Sediments

from Batang Ai Hydroelectric Dam

4.5.3.1 Copper, Manganase, Zinc and Chromium 42

vi

4.5.3.2 Argentum, Nickel and Tin 45

4.5.4 Correlation Coefficient of Heavy Metals in the Surface

Sediments from Batang Ai Hydroelectric Dam

45

4.5.5 Sediment quality criteria and Environmental Status of Sediment

from Batang Ai Hydroelectric Dam based on Heavy Metals

Content

46

CHAPTER FIVE: CONCLUSION AND RECOMMENDATION

5.1 Conclusion 48

5.2 Recommendation 49

REFERENCES 50

vii

LIST OF TABLES

Table

Page

Table 3.1 : The eluting solvent used during geolipid fractionation

10

Table 3.2 : Concentration of heavy metal standards in calibration

analysis on AAS

14

Table 4.1 : RFs value for aliphatic hydrocarbon standard

17

Table 4.2 : RFs value for polycyclic aromatic hydrocarbon (PAH)

standard

18

Table 4.3 : Distribution of aliphatic hydrocarbon in the surface

sediments from Batang Ai hydroelectric Dam from

different sampling stations

20

Table 4.4 : Biomarker indices for aliphatic hydrocarbon in surface

sediments at Batang Ai Hydroelectric Dam

25

Table 4.5 : Distribution of PAHs in the surface sediments from Batang

Ai hydroelectric Dam from different sampling stations.

29

Table 4.6 : Biomarker indices for PAHs in surface sediments at

Batang Ai Hydroelectric Dam

31

Table 4.7 : Concentration of aliphatic hydrocarbons in sediments from

other locations

33

Table 4.8 : Concentration of PAHs in sediments from other areas

34

Table 4.9 : Calibration curve equations of heavy metals analyzed

on AAS

38

Table 4.10 : The value of LOD, LOQ and sensitivity of heavy metals

analyzed on AAS

39

Table 4.11 : Distribution of heavy metal in the surface sediments from

Batang Ai Hydroelectric Dam

41

viii

Table 4.12 : Pearson correlation (PC) coefficient matrix between heavy

metals in surface sediments of Batang Ai Hydroelectric

Dam

46

Table 4.13 : Concentration of heavy metals in surface sediment from

Batang Ai hydroelectric dam in comparison with USEPA

guideline classification values for sediment metal

concentration (µg/g).

47

ix

LIST OF FIGURES

Figures

Page

Figures 3.1 : Location of sampling sites at Batang Ai hydroelectric Dam

at Lubok Antu District, Sri Aman, Sarawak.

8

Figures 4.1 : Gas chromatogram of aliphatic hydrocarbon standards

obtained from GC-FID analysis

15

Figures 4.2 : Gas chromatogram of aromatic hydrocarbon standards

obtained from GC-FID analysis

15

Figures 4.3 : Gas chromatogram of aliphatic fraction from (a) ST1, (b)

ST2, (c ) ST3 and (d) ST4

21

Figures 4.4 : Gas chromatogram of aliphatic fraction from (a) ST5, (b)

ST6 and (c) ST7

22

Figures 4.5 : Gas chromatogram of aromatic fraction from (a) ST1, (b)

ST2 and (c) ST3

26

Figures 4.6 : Gas chromatogram of aromatic fraction from (a) ST4, (b)

ST5 and (c) ST6

27

Figures 4.7 : Gas chromatogram of aromatic fraction from ST7

28

Figures 4.8 : Calibration graph of (a) Ni, (b) Cu, (c) Mn and (d) Pb

analyzed on AAS

35

Figures 4.9 : Calibration graph of (a) Cd, (b) Pb, (c) Zn and (d) Cr

analyzed on AAS

36

Figures 4.10 : Calibration graph of (a) Sn, (b) Ag, (c) As and (d) Bi

analyzed on AAS

37

Figures 4.11 : Spatial distribution of (a) Cu, (b) Mn, (c) Zn and (d) Cr in

surface sediment at Batang Ai hydroelectric Dam.

43

Figures 4.12 : Spatial distribution of (a) Ag, (b) Ni and (c) Sn in surface

sediment at Batang Ai hydroelectric Dam.

44

x

Profiles of Hydrocarbons and Heavy Metals in Surface Sediments

from Batang Ai Hydroelectric Dam, Lubok Antu, Sarawak

Nur Aein Binti Razali

Department of Chemistry

Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

A study was carried out to determine distribution of hydrocarbons and heavy metals in fresh water surface

sediments from Batang Ai hydroelectric Dam, Lubok Antu, Sarawak. The surficial sediments from seven

sampling sites in the vicinity of Batang Ai hydroelectric Dam were analyzed for hydrocarbons (aliphatic and

aromatic) using gas chromatography flame ionization detector (GC-FID) and also heavy metal using atomic

absorption spectrophotometer (AAS). The total concentration aliphatic hydrocarbons varied from 804 – 1014.39

µg/g, while total concentration of PAH varied from 1248 - 7827 ng/g dry weights. Eleven heavy metals analyzed

are Ni, Cu, Mn, Pb, Cd, Zn, Cr, Sn, Ag, As and Bi. Sediment from sampling site ST2 which is near to the

outflow area were highly deposited with heavy metals compared to other sampling sites at Batang Ai

hydroelectric Dam. The concentration level of chromium (Cr) and copper (Cu) from site ST2 can be classified as

heavily polluted.

Key Words: Heavy metals, hydrocarbons, surface sediments, atomic absorption spectrophotometer (AAS), gas

chromatography-flame ionization detector (GC-FID)

ABSTRAK

Satu kajian telah dijalankan untuk menentukan taburan hidrokarbon dan logam berat dalam enapan permukaan

air tawar daripada empangan hidroelektrik Batang Ai, Lubok, Antu, Sarawak. Enapan permukaan daripada

tujuh lokasi pensampelan dalam lingkungan empangan hidroelektrik Batang Ai telah dianalisis untuk

hidrokarbon (alifatik dan aromatic) menggunakan kromatografi gas/pengesan pengionan nyalaan (KG-PPN)

dan juga logam berat menggunakan spektrofotometer serapan atom (SSA). Jumlah kepekatan hidrokarbon

alifatik adalah dalam julat 804 – 1014.39 µg/g, manakala jumlah kepekatan hidrokarbon aromatik adalah dalam

julat 1248 – 7827 ng/g berat kering. Sebanyak 11 logam berat yang telah dianalisis iaitu Ni, Cu, Mn, Pb, Cd,

Zn, Cr, Sn, Ag, As dan Bi. Enapan dari stesen pensempelan ST2 yang berdekatan dengan aliran keluar air

empangan menupukkan logam berat yang tinggi berbanding dengan stesen pensempelan lain di empangan

hidroelektrik Batang Ai. Kepekatan kromium (Cr) dan tembaga (Cu) dalam enapan dari stesen ST2 boleh

diklasifikasikan sebagai sangat tercemar.

Kata kunci: Logam berat, hidrokarbon, enapan permukaan, spektrofotometer serapan atom (SSA), kromatografi

gas/pengesan pengion nyalaan (KG-PPN)

1

CHAPTER ONE

INTRODUCTION

1.1 General Introduction

The important sources of water in Malaysia are from the lakes and reservoirs. Lakes

and reservoirs function as storage basins for municipal and industrial water supply, agriculture

and hydropower. Water quality studies mainly focuses on water quality analysis, pollution

source identification and water quality improvement techniques (Sharip and Zakaria, 2008).

Batang Ai hydroelectric Dam serves Kuching and its surroundings with uninterrupted water

supply. Rapid land development activities upstream could contribute substantially to sediment

formation at lake bottoms. Creation of hydropower generation causes stratification problem.

Management measures on lake and reservoir requires understanding of key process that thrive

the ecosystem (Sharip and Zakaria, 2008).

Hydrocarbon generated by biological or diagnetic processes naturally at low content in

sediments and are a part of the naturally hydrocarbon baseline of ecosystem (Gao and Chen,

2008). According to Long et al. (1998), the presence of high concentration of certain

hydrocarbon has an adverse effect and may cause toxicity on aquatic ecosystem. Hydrocarbon

can become dangerous if they enter the food chain since several of the compound such as

PAHs is carcinogenic (Perelo, 2009). In order to measure the level of the total concentration of

hydrocarbon, the total aliphatic hydrocarbon (TAH) and polycyclic aromatic hydrocarbon

(PAH) were analyze after a single extraction.

2

Heavy metals are widespread pollutants of great environmental concern as they are non

degradable, toxic and persistent with serious on aquatic ecology. Metal will be easily trapped

in the sediments it is because when sediment composed of fine sand and silt the sediment

become in stable condition. In general, industrialization and human activities are the main

contributors to heavy metals discharges into ecosystem. These metal subsequently enter into

the food chain directly or in directly and could give affect on human health (Hamzah et al.,

2001).

1.2 Objectives of the Project

The objectives of this project are :

a. to determine the profile of hydrocarbons and heavy metals in surface sediments from the

Batang Ai hydroelectric Dam,

b. to assess the spatial distribution of hydrocarbons and heavy metals in fresh water

sediments from Batang Ai hydroelectric Dam,

c. to evaluate the environmental status of sedimentary environment of Batang Ai

hydroelectric Dam based on the level of hydrocarbons and heavy metals in sediment.

3

CHAPTER TWO

LITERATURE REVIEW

2.1 Hydrocarbons in Aquatic Sediment

Hydrocarbon is naturally occurring compounds and one of the important components

of sedimentary organic matter (Gao and Chen, 2008). According to Fadli (2010), the major

classes of hydrocarbons are n-alkanes, cycloalkanes, alkenes and aromatic compound. The

hydrocarbons synthesized by organisms occur normally in the biosphere. Among the biogenic

hydrocarbons, n-alkanes are the predominant group and they have been identified in many

species of plants and animals. Both terrestrial and marine organisms synthesize n-alkanes

where chains with odd carbon numbers predominate. N-alkanes of terrestrial origin are mainly

associated with higher plants, presenting chains with odd carbon numbers above n-C23. Marine

phytoplankton synthesize n-alkanes (lower than n-C23) with odd carbon numbers (Nishigima

et al., 2001).

The aliphatic hydrocarbon consist of both fully saturated normal alkane (paraffin) from

C2 to C60 with a smooth distribution between odd and even number of alkanes including

isoprenoid hydrocarbon. The aliphatic hydrocarbons comprise n-alkanes, branched alkanes,

isoprenoids and cyclic compounds, including geochemical biomarkers, such as hopanes and

steranes. Their analysis can be used to fingerprint spilled oils and provides additional

information on the source of hydrocarbon contamination and the extent of degradation of the

oil spill.

4

Polycyclic aromatic hydrocarbons (PAH) are rarely found as products of biosynthesis.

Aromatic hydrocarbons contain one or more aromatic rings which are connected as fused ring

(naphthalene) or line ring (biphenyl) and normally consist of unsubsituted or parent aromatic

structure and like structure with multiple alkyl substitution. These hydrocarbons present a

higher toxicity for organisms. The PAH that are formed in processes of incomplete

combustion of gasoline, diesel oil and other refined petroleum products, generate particles

composed of PAH with high molecular weight, transported to the ocean by the atmosphere

and rivers . The PAH are associated with particulate and dissolved material and tend to be

deposited in the sediments. The presence of PAH in sediments can be utilized as an indication

of oil pollution (Nishigima et al., 2001).

Aliphatic and polycyclic aromatic hydrocarbons are sedimentary contaminants due to

their tendency to accumulate in sediments. Sedimentary aliphatic hydrocarbon has both

biogenic and anthropogenic sources (Peng et al., 2008). Biogenic sources are generated by

biological processes. Biological sources include terrestrial plant, phytoplankton, animals,

bacteria, microalgae and macroalgae (Nishigima et al., 2001). Anthropogenic source generated

by human such as industrials and domestic wates, emissions from the transportation, storage,

processing and combustion of fossil fuels (Doskey, 2000).

Human activities are significantly influenced the composition and distribution of

hydrocarbon and the substances coming from various sources including biogenic, diagenetic,

petrogenic and pyrogenic (Gao and Chen, 2008).

5

2.2 Heavy Metals in Aquatic Sediments

Metals and metal compounds are natural constituents of all ecosystems, moving

between atmosphere, hydrosphere, lithosphere, and biosphere. Their distribution in the

environment is a result of natural processes (volcanoes, erosion, spring water, bacterial

activity) and anthropogenic activities (fossil fuel combustion, industrial and agricultural

processes) (Florea and Busselberg, 2005).

Heavy metal are released into the environment via airborne, contaminants, rural land

use activities, sewage sludge, mine waste, industrial waste, wastewater, pesticides and

fertilizer application (Hamzah et al., 2001). Metal may be presented in the estuarine system as

dissolve species, as free ion or forming organic complex with acid (Spencer and MacLeod,

2002).

The high concentrations of heavy metals are derived from anthropogenic input from

industrial activites around the estuary (Harikumar and Nasir, 2010). Some heavy metal are

potentially harmful (Cd, Hg, Pb) but some are essentially important to human health (Fe, Ca,

Mg). However high concentration of heavy metals could affect human health (Hamzah et al.,

2001). Exposure to heavy metals is potentially harmful especially for those metal-compounds,

which do not have any physiological role in the metabolism of cells. The ingestion of metals

via food or water could modify the metabolism of other essential elements such as Zn, Cu, Fe

and Se) (Florea and Busselberg, 2005).

6

An adverse impact on aquatic ecosystem will occur by the heavy metals which are

mostly detachable from sediments and therefore contribute to hazardous waste. Thus, to

determine heavy metal portioning, selective chemical teaching techniques will be used. This

technique has been widely used to reconstruct the history of metal pollution (Hosono et al.,

2010).

Heavy metals from non degradable materials often accumulate causing biological

effect (Kar et al., 2008). A better understanding of the contaminant data to assists in the

interpretation and in making decision will be achieved by knowing the spatial and temporal

variance in the concentration of heavy metals in the aquatic environment. Therefore,

monitoring these metals is important for safety of the environment and human health in

particular.

2.3 Importance of Core Sediment in Environmental Studies

Bottom sediments consist of particles that have been transported by water, air, or

glaciers from the sites of origin in a terrestrial environment and have been deposited on the

floor of a river, lake, or ocean. Bottom sediment will also contain materials precipitate from

chemical and biological processes. Natural processes responsible for the formation of bottom

sediments can be altered by the anthropogenic activities. Sediments will accumulate due to the

effect from changes in the climate linked to the changes in the ocean (Kamaruzzaman et al. ,

2011). These accumulate in sediments via several pathways, including disposal of liquid

effluents, terrestrial runoff and leachate carrying chemicals originating from numerous urban,

industrial and agricultural activities as well as atmospheric deposition (Kassim, 2010)

7

Sediment core analysis is very useful because it is commonly used to characterize

contamination in deeper sediments, documentation of historical changes in vertical

distribution of contaminants, reducing oxygen exposure needed for sample analysis and

correlate organism exposure to specific sediment layer (Jeng, 2007). The geochemical

characteristics of the sediments can be used to determine the weathering trend and the source

of pollution (Harikumar and Nasir, 2010).

Textural properties of lake sediments (e.g., porosity, water content) can serve as tools

for evolving and assessing the possible effects of sediment focusing, slumping and

inhomogeniety in the sediment composition. Sediment focusing is a process whereby water

turbulence moves sedimented material from shallower to deeper zones of a lake (Kassim,

2010).

Many researchers have used sediments to study the behavior of metals. The occurrence

of increase levels of metal especially in the sediments can be good indication of man induced

pollution of high level of heavy metals can often be attribute to anthropogenic influences

rather than natural enrichment of the sediment by geological weathering (Harikumar and

Nasir, 2010).

8

CHAPTER THREE

MATERIALS AND METHODS

3.1 Sampling Sites

The location of sampling area at Batang Ai Hydroelectric Dam is shown in Figure 3.1.

Seven sampling sites were selected at Batang Ai Hydroelectric Reservoir. The surface

sediments were collected using stainless steel grab sampler. The sediment sample for heavy

metals analysis was placed in plastic bag, while the sediment samples for hydrocarbon

analysis was wrapped with aluminum foil. The sediment samples were stored in cooler box

during transportation. Upon arrived in UNIMAS laboratory, the sample was stored in freezer

at -18 0C until further analysis.

Figure 3.1: Location of sampling sites at Batang Ai Hydroelectric Dam at Lubok Antu

District, Sri Aman, Sarawak.

9

3.2 Hydrocarbon in Surface Sediment

3.2.1 Extraction of Geolipid

The extraction and fractionation procedure for hydrocarbons analysis was carried out

according to procedure described by Zakaria et al. (2000). Extraction of geolipids from

sediments was performed using Soxhlet extraction method. Exactly 10 g (2 mm) sediment was

placed in the extraction thimbles (30 mm x 100 mm, Whatman) and extracted with 200 mL

dichloromethane for 8 hours extraction times. Exactly 50 µL of the internal standards

mixture containing (50 ppm of each component), anthracene-d10 and n-ecosene in

dichloromethane was spiked into the sample. Then, the crude extract was reduced to nearly

dryness using rotovap. The residue was diluted with dichloromethane and evaporates to

dryness by blowing with gentle stream of pure N2. The residue in vial is considered as total

extractable lipids (TEL).

3.2.2 Column Chromatography Fractionation of Geolipid

TEL was dissolved in 5 ml n-hexane and then subjected to fractionate on a column

chromatography (1.1 cm x 50 cm) which are packed with 7.5 g activated silica gel (60 mesh).

The eluting solvents used are listed in the Table 3.1.

10

Table 3.1: The eluting solvents used during geolipid fractionation

Fraction Eluting solvent Group of compounds

extracted on silica gel

column chromatography

F1 40 mL hexane Aliphatic hydrocarbons

F2 40 mL mixture of dichloromethane: hexane (1:3,

v/v)

PAHs

The total aliphatic hydrocarbon (TAH), F1 fraction was collected by eluting the

chromatography column with 40 mL of hexane, while the PAH fraction, F2 was collected by

eluting the column with mixtures of 40mL methylene chloride: hexane (1:3, v/v). Each

fraction were then evaporated and transferred to a vial with 1 ml of dichloromethane. The

solvent in each vial was evaporated just to the point of dryness under a gentle stream of

nitrogen.

3.2.3 Gas Chromatography- Flame Ionization Detector (GC-FID) Analysis

Computerized capillary gas chromatography analysis Clarus 680 series equipped with

standard flame ionization detector (FID) was used to perform gas chromatographic analysis of

hydrocarbon fractions, F1 and F2. The FID response proportionately to the number of CH2

groups introduced to the flame. Prior the GC-FID analysis, samples was diluted with 100 µL

hexane in 3 mLl capacity vial. Exactly 1 µLl sample was injected in less split mode and

separated on fused silica capillary column (25 m x 0.22 mm internal diameter 0.25 µm film

11

thickness) of DB-5 phase (crosslinked 5% diphenyl and 95% dimethylpolysiloxane). The

temperature in the oven were maintained 500C for 5 minutes and temperature was ramped at

310 0C at a rate 6.5

0C/min. The final temperature was held for 16.5 minutes. Temperature for

injector and detector was set at 280 0C and 320

0C respectively. The H2 compressed air and N2

gas flow was set at 30 mL/min, 400.0 mL/min and 25.0 mL/min respectively.

3.2.4 Data Analysis

3.2.4.1 Qualitative Analysis

The retention time for n-alkane on GC-FID detector were determined by comparing the

retention time with n-alkane in a mixture of standards. However, n-alkane standards only

consist of even number carbon n-alkane. The qualitative analysis for odd number was

determined based on the two adjacent alkanes. As for isoprenoid pristane is calculated by

average retention time of C17 - C18 and phytane is calculated by average retention time of C18-

C19.

Various biomarkers indices can be used in order to determine the origin of organic

matters in the surface sediment samples. The ratios of pristine/phytane, phytane/C18,

pristane/C17 and nC25/nC15 were calculated as biomarker indices for aliphatic hydrocarbon.

12

The carbon preference index (CPI) was calculated according to Bray and Evans (1961)

as equation 3.1:

CPI = 1 C25+C27+C29+C31+C33 + C25+C27+C29+C31+C33 …equation 3.1

2 C26+C28+C30+C32+C34 C24+C26+C28+C30+C32

where, Cn = alkane with n number of carbons

3.2.4.2 Quantitative Analysis

The relative response factor (RFs) for even number carbon n-alkanes in F1 and each of

the PAH compound for F2 determined according to Howsam and Puttmann, (1989) as

equation 3.2:

RFs = (Cstd / Astd) x (Ais x Cis) … equation 3.2

where, Cstd = Concentration standard analyte

Astd = Gas chromatogram for standard analyte

Ais = Gas chromatogram for internal standards

Cis = Concentration of internal standards

RFs for odd carbon of n-alkane were calculated using average of two adjacent even

numbers n-alkane carbon. The RFs are used in the calculation of the concentration for

individual alkane in sediments by using the equation 3.3:

Concentration of analytes = (Cis/ Ais) X Ax X RF …equation 3.3

where, Ax = Chromatogram for analyte x

13

3.3 Heavy Metal Analysis

3.3.1 Sample Preparation

The sediment sample preparation was carried out according to procedure described by

Binning and Baird (2001). The sediment was air dried in petri-dishes, then ground into a

powder. Approximately 0.5 g of each dried sediment sample was placed in a beaker and mixed

with 20 mL Aqua Regia (cHNO3: cHCl; 1:3; v/v) and allowed to stand overnight. The mixture

was heated to near dryness and allowed to cool before 1 mL of a HNO3 solution was added.

The sediment samples was then allowed to stand overnight and then filtered through Whatman

No 41 filter paper. The filtrates was transferred to a 100 mL volumetric flask and made up to

the mark with deionized water.

3.3.2 Atomic Absorption Spectrophotometer (AAS) Analysis

The solution was analyzed for the metals content using AAS model Thermo Scientific

iCE 3000 SERIES using the calibration curve method. The samples were analyzed for Ni, Cu,

Mn, As, Pb, Cd, Bi, Zn, Cr, Sn and Ag. Prior to AAS analysis calibration solution were

analyzed. The concentration of respective heavy metals standard used during calibration

analysis is shown in Table 3.2.