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UNIVERSITI PUTRA MALAYSIAAN ASSESSMENT OF METAL DISTRIBUTION AND METAL SOLUBLEFRACTIONS IN THE EDIBLE MOLLUSCS FROM MALAYSIAFRANKLIN BERANDAH ANAK EDWARD THOMASFS 2009 16

AN ASSESSMENT OF METAL DISTRIBUTION AND METAL SOLUBLEFRACTIONS IN THE EDIBLE MOLLUSCS FROM MALAYSIAByFRANKLIN BERANDAH ANAK EDWARD THOMASThesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, infulfillment of the Requirements for the degree of Master of ScienceJuly 2009

Abstract of the thesis presented to the Senate of Universiti Putra Malaysia infulfillment of the requirements for the degree of Master of ScienceAN ASSESSMENT OF METAL DISTRIBUTION AND METAL SOLUBLEFRACTIONS IN THE EDIBLE MOLLUSCS FROM MALAYSIABYFRANKLIN BERANDAH ANAK EDWARD THOMASJuly 2009Chairman: Associate professor Yap Chee Kong, PhD.Faculty: Faculty of ScienceThe present study focused on the heavy metal concentrations in the different parts of 12species of Malaysian molluscs, six species of bivalves and gastropods, respectively. Theaim of the present study was to provide information on the concentrations of theessential metals: Cu, Fe and Zn and the non-essential metals: Cd, Ni and Pb in the edibletissues of molluscs with particular reference to the food safety and ecotoxicologicalpoints of views. For bivalves, Cu, Fe and Zn concentrations in the edible parts ranged at1.80-79.8 µg/g dw, 42.9-4895 µg/g dw and 28.3-379 µg/g dw, respectively. While forCd, Pb and Ni, they ranged at 0.253-22.4 µg/g dw, 0.558-46.5 µg/g dw and 0.656-23.6µg/g dw, respectively. As for gastropods, Cu, Fe and Zn concentrations in the edibleparts ranged at 1.97-686 µg/g dw, 51.2-2921 µg/g dw, 22.8-337 µg/g dw, respectively.While for Cd, Pb and Ni, they ranged at 0.159-32.9 µg/g dw, 1.20-43.0 µg/g dw and0.222-27.9 µg/g dw, respectively.The study on the soluble and insoluble heavy metal fractions revealed that an abundanceof soluble metals like Cd, Pb and Ni were consistently found in some tissues of theii

molluscs such as in the foot, mantle and muscle and they could potentially be transferredthrough the food web (predators).The total metal concentrations in the different parts were compared with the foodpermissible limits set by six organizations around the world. However, to overcome theoverestimation of food safety based on the total metal concentrations in the differentedible tissues, determination of the metal soluble fractions in the tissues were furtherinvestigated in this study. The soluble concentrations were compared with thepermissible limits set by the Environmental health Criteria (1998, 2001) and theFAO/WHO (1984). From the present findings, it was found that consumption of largeamounts of Per. viridis, G. expansa and most of the gastropods could pose metal toxic totheir consumer. The elevated of Cu and Zn concentrations in most of the edible parts ofthe gastropods suggested that the consumption of large amounts of most gastropodswere not advisable. As for Cd levels, it was found that the byssus of Per. viridis and D.faba; and the digestive gland and mantle of Chi. capucinus were not safe for continuousconsumption (for example: more than a week) since the levels would exceed thepermissible limit. Besides, the continuous consumption of the byssus of Per. viridis andD. faba would also potentially cause Pb toxicity.The information on the metal distributions in the different parts obtained by using thecluster analysis is important to facilitate the biomonitoring of the marine environment,which based on the use of different tissues in the species of molluscs of Malaysia.iii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagaimemenuhi keperluan untuk ijazah Master SainsPENILAIAN TERHADAP TABURAN LOGAM BERAT DAN PECAHANLOGAM BERAT TERLARUT DALAM MOLLUSK BOLEH-MAKANDARIPADA MALAYSIAOlehFRANKLIN BERANDAH ANAK EDWARD THOMASJuly 2009Pengerusi : Prof. Madya Dr. Yap Chee Kong, Ph.D.Fakulti: Fakulti SainsKajian ini menfokuskan terhadap taburan logam berat di dalam pelbagai bahagian 12spesies mollusk di Malaysia, masing-masing enam spesis bivalve dan gastropod. Tujuanutama kajian ini ialah menyediakan maklumat tentang kepekatan Cd, Cu, Fe, Ni Pb danZn di dalam tisu-tisu boleh makan dengan perbincangannya tertumpu khas kepadakeselamatan pemakanannya selain dari segi ekotoksikologi. Untuk bivalve, julatkepekatan Cu, Fe dan Zn di dalam tisu-tisu boleh makan masing-masing adalah 1.8079.8 µg/g dw, 42.9-4895 µg/g dw dan 28.3-379 µg/g dw. Manakala untuk Cd, Pb danNi, kepekatan logam berat tersebut masing-masing berjulat antara 0.253-22.4 µg/g dw,0.558-46.5 µg/g dw dan 0.656-23.6 µg/g dw. Untuk gastropod pula, julat kepekatan Cu,Fe dan Zn di dalam tisu-tisu boleh makan masing-masing adalah 1.97-686 µg/g dw,51.2-2921 µg/g dw dan 22.8-337 µg/g dw. Manakala untuk Cd, Pb dan Ni, kepekatanlogam berat tersebut masing-masing berjulat antara 0.159-32.9 µg/g dw, 1.20-43.0 µg/gdw dan 0.222-27.9 µg/g dw.iv

Kajian terhadap logam berat terlarut dan tak terlarut mendapati kehadiran logam terlarutyang tinggi Cd, Pb dan Ni secara konsistent di dalam tisu-tisu molluk seperti kaki,mantel dan otot dan berpotensi dipindahkan melalui jaringan makanan (oleh pemangsa).Kepekatan keseluruhan logam berat di dalam pelbagai tisu dibandingkan dengan tahapyang dibenarkan oleh enam organisasi dari seluruh dunia. Walaubagaimanapun, untukmengelakkan anggaran yang kurang tepat yang berdasarkan jumlah keseluruhan logamberat di dalam pelbagai tisu boleh makan tersebut, kajian tentang pecahan terlarut logamdi dalam tisu-tisu itu telah dicadangkan. Kepekatan pecahan terlarut tersebutdibandingkan dengan sukatan yang dibenarkan oleh Environmental health Criteria(1998, 2001) dan FAO/WHO (1984). Melalui perbandingan tersebut, didapatipengambilan Per. viridis, G. expansa dan kebanyakan gastropod mungkin bolehmenyebabkan ketoksikan kepada si pemakannya. Kehadiran Cu dan Zn yang banyak didalam kebanyakan gastropod menunjukkan pengambilannya yang banyak juga tidakdigalakkan. Untuk tahap Cd pula, didapati bisus Per. viridis dan D. faba; dan kelenjarpencernaan dan mantel Chi. capucinus adalah tidak selamat diambil secara berterusan(Contohnya: satu atau lebih minggu) kerana tahapnya akan melebihi tahap yangdicadangkan. Selain itu, pengambilan berterusan bisus Per. viridis dan D. faba mungkinjuga berpotensi menyebabkan ketoksikan Pb.Maklumat tentang taburan logam di dalam pelbagai bahagian tisu yang diperolehimelalui analisis kluster adalah penting untuk panduan dalam mengawal kawasan marin,yang mengaplikasikan pelbagai tisu spesis mollusk di Malaysia.v

ACKNOWLEDGEMENTSFirst of all, I would like to express my gratitude towards my supervisor Assoc. Prof. Dr.Yap Chee Kong. He had given me a lot of ideas, comments, advice, tolerance andpatience while conducting my research. With his proper guidance and motivations, Imanaged to conduct my research smoothly, efficiently and was able to complete thisthesis.I would also like to thank my supervisory committee members, Prof. Dr. Ahmad Ismailand Prof. Dr. Tan Soon Guan for all their support and guidance. Special thanks andappreciation to the Ministry of Science, Technology and Innovation (MOSTI), Malaysiafor awarding me the National Science Fellowship (NSF) Scholarship which covered mystudy and examination fees, and also a monthly allowance for the past 24 months (June2006 - July 2008).Special thanks to my colleagues, Mdm. Norhaidah, Mr. Wan Hee, Mr. Bin Huan, Mr.Mazyar, Mr. Romeo, Mr. Bintal Amin, Mr. Ikram, Mdm. Syafinah and to all thelecturers in the Department of Biology, Faculty of Science, UPM. It was a great andwonderful experience working with them. Thank you so much for all your help andsupport and I really appreciate it.Last but not least, to my beloved wife, Mdm. Chong Sou Ping, thank you for your love,care, help and understanding. To my wonderful family, all the support, care and helpsgiven that droven me all this while. Thank you so much and MAY GOD BLESS YOUALL.vi

I certify that an Examination Committee has met on 13 July 2009 to conduct the finalexamination of Franklin Berandah Anak Edward Thomas of his degree thesisentitled “AN ASSESSMENT ON THE METAL DISTRIBUTION AND METALSOLUBLE FRACTIONS IN THE EDIBLE MOLLUSCS FROM MALAYSIA” inaccordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and UniversitiPertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommendsthat the student be awarded the Master of Science (Ecotoxicology)Members of the examination Committee were as follows:Lecturer,Faculty of ScienceUniversiti Putra Malaysia(Chairman)Lecturer,Faculty of ScienceUniversiti Putra Malaysia(Internal Examiner)Lecturer,Faculty of ScienceUniversiti Putra Malaysia(Internal Examiner)Lecturer,Faculty of ScienceUniversiti Putra Malaysia(External Examiner)HASANAH MOHD. GHAZALI, PhDProfessor and DeanSchool of Graduate StudiesUniversiti Putra MalaysiaDate :vii

This thesis was submitted to the Senate of Universiti Putra Malaysia and has beenaccepted as fulfillment of the requirement for the degree of Master of Science(Ecotoxicology). The members of the Supervisory Committee were as follows:Lecturer,Professor/Associate Professor/IrFaculty of ScienceUniversiti Putra Malaysia(Chairman)Lecturer,Professor/Associate Professor/IrFaculty of ScienceUniversiti Putra Malaysia(Member)Lecturer,Professor/Associate Professor/IrName of Department or FacultyName of Organisation(Member)HASANAH MOHD. GHAZALI, PhDProfessor and DeanSchool of Graduate StudiesUniversiti Putra MalaysiaDate :viii

DECLARATIONI declare that the thesis is my original work except for quotations and citations whichhave been duly acknowledged. I also declare that it has not been previously, and is notconcurrently, submitted for any other degree at Universiti Putra Malaysia or at any otherinstitution.FRANKLIN BERANDAH ANAK EDWARD THOMASDate :ix

TABLE OF EDGEMENTSAPPROVALDECLARATIONLIST OF TABLESLIST OF FIGURESLIST OF ABBREVIATIONSCHAPTER1INTRODUCTION1.1 Background of study1.2 Objectives of study234Page13LITERATURE REVIEW2.1 Heavy metals Studies in Malaysian Molluscs2.2 Heavy Metals in Molluscs: Different by Species, Habitatand Location2.3 Heavy Metals in Molluscs: Food Safety Concern2.4 Soluble and insoluble fractions of heavy metals inmolluscsMATERIALS AND METHODS3.1 Sampling sites descriptions3.2 The sampling map3.3 Sample preparation3.3.1 Molluscs3.3.2 Sediments3.4 Digestion of molluscs tissues3.5 Estimation of Soluble and Insoluble Heavy Metal in theMolluscs3.6 Speciation of Cd, Cu, Ni, Pb and Zn in Sediments3.7 Metal determination3.8 Quality control and quality assurance3.9 Statistical Analytical ProceduresRESULTS4.1 Heavy metal concentrations in the total tissues ofmolluscs4.1.1 Heavy metal concentrations in the total tissues ofbivalves4.1.2 Distribution of heavy metal concentrations in thetotal tissues of gastropodsx5781012131819202121242526282830

564.2 Heavy metal concentrations in the different parts ofmolluscs4.2.1 Heavy metal concentrations in the different partsof bivalve4.2.2 Heavy metal concentrations in the different partsof gastropods4.2.3 Biota-sediment accumulation factors of selectedmolluscs324.3 Soluble and insoluble heavy metals in the different partsof molluscs4.3.1 Soluble and insoluble heavy metals in the differentparts of bivalves4.3.2 Soluble and insoluble heavy metals in the differentparts of gastropods513237425159DISCUSSION5.1 Heavy metal concentrations in the different parts ofmolluscs: Ecotoxicological point of views5.1.1 Heavy metal concentrations in the different parts ofmolluscs (Cluster analysis)5.1.2 Biota-sediment accumulation factors (BSAF) ofselected molluscs5.2 Soluble heavy metal fractions in edible tissues ofmolluscs and their potential transfer through the foodweb5.3 Heavy metal concentrations in the different parts ofmolluscs: Food safety point of views5.3.1 Evaluation of Cu, Cd, Zn and Pb from the foodsafety standpoint in the molluscs’ edible tissues basedon the soluble fractions5.4 Summaries and ATA OF STUDENTLIST OF PUBLICATIONS108121117119xi667176798388

LIST OF TABLESTable2.10Some previous studies of heavy metal concentrations (μg/g) inMalaysian marine molluscsPage63.10The descriptions of the sampling locations and information on themolluscs and sediments collected143.11Analytical results for the reference material and the certified value foreach metal (µg/g dry weight)264.10aEssential heavy metal concentrations (mean µg/g dw) in the differentparts of bivalves collected from coastal areas of Malaysia334.10bNon-essential heavy metal concentrations (mean µg/g dw) in thedifferent parts of bivalves collected from coastal areas of Malaysia354.11aEssential heavy metal concentrations (mean µg/g dw) in the differentparts of gastropods collected from coastal areas of Malaysia384.11bNon-essential heavy metal concentrations (mean µg/g dw) in thedifferent parts of gastropods collected from coastal areas of Malaysia.404.12Basic statistics (Minimum and maximum) for bivalve data (all theheavy metal concentrations in μg/g dw)424.13Basic statistics (Minimum and maximum) for gastropods data (all theheavy metal concentrations in μg/g dw)434.14Basic statistics (ranges) for sediment data (all the heavy metalconcentrations in μg/g dw)444.15Pearson correlation between the heavy metal concentrations (based on 46Cd, Cu, Ni, Pb and Zn) in the different parts of bivalves (Kpg. PasirPuteh) and those concentrations in the sediment (Sequential ExtractionTechnique-SET).4.16Biota-sediment (non-resistant fractions of SET) accumulation factors(BSAF) based on the different parts of bivalves4.17Pearson correlation between the heavy metal concentrations (Cd, Cu,49Ni, Pb and Zn) in the different parts of gastropods and thoseconcentrations in the sediment (Sequential Extraction Technique-SET)Biota-sediment (non-resistant fractions of SET) accumulation factors50(BSAF) based on the different parts of gastropods4.18xii47

5.10Soluble and insoluble heavy metals fractions in bivalves and theirrelationships with the total concentrations of the different parts815.11Soluble and insoluble heavy metals fractions in gastropods and theirrelationships with the total concentrations of the different parts825.12Summary of comparison of some edible parts of bivalves with thepermissible levels of the food safety guideline set by five differentcountries (µg/g dw)845.13Summary of comparison of some edible parts of gastropods with thepermissible levels of the food safety guideline set by six differentcountries (µg/g dw)855.14Evaluation of food safety of bioavailable metal intake by human based 90on the Cu soluble fraction of bivalves in comparison with therecommended tolerable intakes5.15Evaluation of food safety of bioavailable metal intake by human based 92on the Cd soluble fraction of bivalves in comparison with theprovisional tolerable weekly intake5.16Evaluation of food safety of bioavailable metal intake by human based 94on the Zn soluble fraction of bivalves in comparison with therecommended tolerable intakes5.17Evaluation of food safety of bioavailable metal intake by human based 96on the Pb soluble fraction of bivalves in comparison with theprovisional tolerable weekly intake5.18Evaluation of food safety of bioavailable metal intake by human based 98on the Cu soluble fraction of gastropods in comparison with therecommended tolerable intake5.19Evaluation of food safety of bioavailable metal intake by human based 99on the Cd soluble fraction of gastropods in comparison with theprovisional tolerable weekly intake5.20Evaluation of food safety of bioavailable metal intake by human based 100on the Zn soluble fraction of gastropods in comparison with therecommended tolerable intake5.21Evaluation of food safety of bioavailable metal intake by human based 101on the Pb soluble fraction of gastropods in comparison with theprovisional tolerable weekly intakexiii

LIST OF FIGURESFigure3.103.11Map of Peninsular MalaysiaThe Six species of bivalves collected from the intertidal area ofMalaysiaPage13153.12The six species of gastropods collected from the intertidal area ofMalaysia163.13Sampling to the southern parts of Peninsular Malaysia.173.14Sampling to the northern parts of Peninsular Malaysia.183.15Sediments are sifted through a 63 µm stainless steel aperture193.16Digestion of samples in a digestion block at 40 C for 1 hour andthen fully digested at 140 C for 2-3 hours203.17Estimation of soluble and insoluble heavy metal fractions in 22molluscs [Modified from Bragigand et al. (2004)]3.18The sample were continuously shaken for 3 hours with 50 ml of1.0 M ammonium acetate (NH4CH3COO).3.19Air-acetylene Perkin-ElmerTM flame atomic absorption 24spectrophotometer model AAnalyst 800 for heavy metals4.10Heavy metal concentrations (µg/g dw) in the total soft tissue of the 29bivalves4.11Heavy metal concentrations (µg/g dw) in the total soft tissue of the 31gastropods4.12The soluble and insoluble fractions of Cu in the different softtissues of the bivalves524.13The soluble and insoluble fractions of Cd in the different softtissues of the bivalves534.14The soluble and insoluble fractions of Zn in the different softtissues of the bivalve554.15The soluble and insoluble fractions of Pb in the different softtissues of the bivalves564.16The soluble and insoluble fractions of Ni in the different soft58xiv23

tissues of the bivalves4.17The soluble and insoluble fractions of Cu in the different softtissues of gastropods604.18The soluble and insoluble fractions of Cd in the different softtissues of gastropods614.19The soluble and insoluble fractions of Zn in the different softtissues of gastropods624.20The soluble and insoluble fractions of Pb in the different softtissues of gastropods634.21The soluble and insoluble fractions of Ni in the different softtissues of gastropods655.10Hierarchical Cluster Analysis of the different parts of bivalvesfrom Malaysia intertidal area based on heavy metal (Cu, Cd, Fe,Ni, Pb and Zn) concentrations [Log10 (x 1)]735.11Hierarchical Cluster Analysis of the different parts of gastropods 74from Malaysia intertidal area based on heavy metal (Cu, Cd, Fe,Ni, Pb and Zn) concentrations [Log10 (x 1)]xv

LIST OF ABBREVIATIONSμmmicrometreμg/gmicrogram per gramµg/Lmicrogram per litreCIcondition indexDDWdouble distilled waterg/cm3gram per centimetres cubicH2NO4sulphuric acidHClhydrochloric acidHNO3nitric acidppmpart per millionrpmrotation per minuteSEstandard errorSNKStudent-Newman-KuelsSTsoft tissuesxvi

CHAPTER 1INTRODUCTION1.1 Background of StudyMarine molluscs are a major internationally traded seafood commodity. They inhibittheir natural habitat, the intertidal area, which are usually close to estuaries. Hence,the chance of their exposure to various type of contaminants and pollutants fromanthropogenic (land-based activities) through the riverine system and the sea-basedsources is very high. Moreover, the tissues of molluscs are well known for theiraccumulation of a wide range of contaminants such as heavy metals (Goldberg et al.,1978). Hence, the information on the safety of molluscs as food is important andcrucial.In the literature, studies on the food safety of molluscs focused on the total softtissues such as the studies conducted by Storelli (2008) in the Adratic Sea(Cephalopod molluscs), Amiard et al. (2008) in France, UK and Hong Kong(Buccinum undatum, Crassostrea gigas, Ostrea edulis, Saccostrea cucullata, Pernaviridis, Marcia hiantina and Chlamys nobilis), Espana (2007) in the Straits ofMagellan (Mytilus chilensis and Perunytitus purpuratus), Fung et al. (2004) in theeast coast of China (Perna viridis and Mytilus edulis), Chiu et al. (2000) in HongKong (Per. viridis). In Malaysia, a detailed study on the food safety of Per. viridisfrom Peninsular Malaysia was conducted by Yap et al. (2004).However, studies on the f

UNIVERSITI PUTRA MALAYSIA . . Name of Department or Faculty . Name of Organisation (Member) . concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. _ FRANKLIN BERANDAH ANAK EDWARD THOMAS . Date : ix. TABLE OF CONTENTS . ABSTRACT. ii .

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