E - PROCEEDING - ISST2015

e-Proceeding The 10th International Symposium in Science and Technology 2015

Organized by Chulalongkorn University, Thailand Kansai University, Japan Sirindhorn International Institute of Technology, Thammasat University, Thailand Universiti Sains Malaysia, Malaysia Cheng Shiu University, Taiwan

August 31 – September 2, 2015 Venue : Bangkok & Pathumthani, Thailand

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Organizing Committee Prof. Pirom Kamolratanakul President of Chulaongkorn University Prof. Dr. Mongkol Techakumphu Vice President Research, Chulalongkorn University Asst. Prof. Pomthong Malakul Dean of The Petroleum and Petrochemical College, Chulalongkorn University Prof. Dr. Supot Hannongbua Dean of Faculty of Science, Chulalongkorn University Prof. Munehiro Yoshida Dean of Graduate school of Science and Engineering, Kansai University Prof. Yutaka Maeda Vice President of Kansai University Prof. Hiroshi Tamura Vice Dean of Division of International Affairs, Kansai University Prof. Dr. Yoshinobu Kajikawa Kansai University Prof. Dr. Miki Yamamoto Kansai University Prof. Dr. Somnuk Tangtermsirikul Diretor of SIIT, Thammasat University Prof. Dr. Thanaruk Theeramunkong Head of the School of Information and Computer Technology, SIIT, Thammasat University Prof. Yahya Mat ARIP Deputy Dean (Student Affairs & Networking), Universiti Sains Malaysia Asst. Prof. Wu Te Ko Assistant Dean, Office of International Affairs, Cheng Shiu University Assoc. Prof. Jung-Lang Cheng Cheng Shiu University, Taiwan Prof. Dr. Sung-Tsun Shih Cheng Shiu University, Taiwan Assoc. Prof. Jen-Chou Hsiung Cheng Shiu University, Taiwan

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Local Committee and Technical Committee Asst. Prof. Manit Nithitanakul

Asst. Prof. Sirithan Jiemsirilers

Assoc. Prof. Apanee Luengnaruemitchai

Assoc. Prof. Ratana Rujiravanit

Dr. Chanya Chaicharoenpong

Dr. Uthaiporn Suriyaprapadilok

Dr. Ampira Charoensaeng

Mr. Prasit Srikeaw

Ms. Suchitra Kamnoetjok

Ms.Jitima Preechawong

Mr. Nattakammala Janpetch

Asst. Prof. Dr. Warangkana Saengsoy

Assoc. Prof. Dr. Waree Kongprawechnon

Assoc. Prof. Dr. Chalie Charoenlarpnopparut

Asst. Prof. Dr. Pisanu Toochinda

Assoc. Prof. Dr. Luckhana Lawtrakul

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Materials and Chemistry Page Synthesis, Self-Assembly and Liposomal Encapsulation of Sulfated Cyclodextrin Amphiphiles Tetsuya Furuike*, Hiroshi Tamura Faculty of Chemistry, Materials and Bioengineering, Kansai University

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Synthesis, Structural Investigation, and Some Applications of Nickel Doped Tin Oxide Nanoparticles Myat Myat Thaw¹, Maung Maung Myint²,Soe Myint Maung3, Kyaw Naing4 ¹Lecturer, Dr, Department of Chemistry, University of Yangon ²Lecturer, Dr, Department of Chemical Technology, Defence Services Science and Technology ,Research Centre, Pyin Oo Lwin 3 Professor, Dr, Department of Chemical Technology, Defence Services Science and Technology, Research Centre, Pyin Oo Lwin 4 Pro-rector, Dr, University of Yangon

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Generation of Chitin Fine Flakes with Various Molecular Assemblies on the Solvation of Hydrated Chitin Slurry Seiichi Tokura , Chutima Vanichvattanadecha, Natthakammala Janpech, Ratana Rujiravanit, Petroleum and Petrochemical College, Chulalongkorn University

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Synthesis and Antibacterial Activities of Amino-Containing Cellulosic Materials Monrawat Rauytanapanit a, Rungaroon Waditee-Sirisatthab, Thanit Praneenararata,* a) Department of Chemistry, Faculty of Science, Chulalongkorn University b) Department of Microbiology, Faculty of Science, Chulalongkorn University

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Methanol Oxidation of Pt Particles on Electropolymerized Polypyrrole Apichart Rodchanarowana, Thaneeya Hawisetb, Prachak Inkaewc* a) Department of Materials Engineering, Faculty of Engineering, Kasetsart University b) School of Medicine, Mae Fah Luang University c) School of Science, Mae Fah Luang University

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Determination of Lead by Colorimetric Paper-Based Sensor Nattapong Thuwakham*, Prachak Inkaew School of Science, Mae Fah Luang University

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Development of Functional Fruits Using Composite Film Deposition of Essential Oil May Mee Zin a, Stephan Thierry Dubas a, a) The Petroleum and Petrochemical College, Chulalongkorn University

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Page Thermochromic Response of Polydiacetylene/Zinc Oxide/Natural Rubber Nanocomposite Films Wipawee Panyanuntakit a, KanoktipBoonkerd a*, Nisanart Traiphol b a) Faculty ofScience, Chulalongkorn University b) Research Unit of Advanced Ceramics, Department of Materials Science, Faculty of Science, Chulalongkorn University

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Influence of Phosphorus on the Fusing Temperature of Nickel-base Self-fluxing Alloy Masaaki Inazumi a, Toru Maruyama a a) Graduate School of Science and Engineering, Kansai University

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Life Science Anthelmintic Potential of Psidium guajava against Infective Larvae of parasitic Nematode Yeh Jian Wen a, Nik Ahmad Irwan Izzauddin Nik Hima*, Shaida Fariza Sulaimana, Rahmad Zakariaa School of Biological Sciences, Universiti Sains Malaysia

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The Conservation of Orangutan : The Dilemma between Funding, Profit, Sincerity and Passion Nik Fadzly*, Wan Fatma Zuharah Wan Musthapa, Mashhor Mansor, Aini HasanahAbd Mutalib, Salniza Akmar and Fatin Nabila Nazaruddin School of Biological Sciences, Universiti Sains Malaysia

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The Effect of Temperature and Diet on Immature Stages of Megaselia Scalaris Development and Survival Rate Hadura Abu Hasana, Teo Bee Hsiena and Hamdan Ahmada,b School of Biological Sciences, Universiti Sains Malaysia

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Multifarious role: A study on the feeding behaviour of rove beetle, Paederus fuscipes CURTIS (Coleoptera: Staphylinidae) Maryam Sufiana* and Wan Fatma Zuharaha,b School of Biological Sciences, Universiti Sains Malaysia

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Sublethal Effects on Permethrin and Malathion on The Life History of Aedes Albopictus (Skuse) Mosquitoes Sumayyah Aminodina, Dinesh A/L Rajendran a, Wan Fatma Zuharaha,b School of Biological Science, Universiti Sains Malaysia

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Page Preliminary Investigation on the Diversity of Zooplankton in Muda River, Peninsular Malaysia Azma Hanim Ismail a,*, Asmerlda Abu Hassan a a) School of Biological Sciences, Universiti Sains Malaysia

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Effects of Excessive Light in Changing Life History of Aedes Aegypti Mosquito Wan Fatma Zuharah a*, Liew Yan Hui a, Nik Fadzlyb,c a) Medical Entomology Laboratory,School of Biological Sciences, Universiti Sains Malaysia b) Vector Control Research Unit, Universiti Sains Malaysia c) Centre of Marine & Coastal Studies (CEMACS), Universtiti Sains Malaysia

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Insecticide Residues on Poultry Manures: Comparing the Extraction Methods and Field Efficacy Test on Selected Insecticides in Managing Musca Domestica Population Song-Quan Ong, Abdul Hafiz Ab Majid*, Hamdan Ahmad and Fadhlina Hazwani binti Mohd Fadzlah Household & Structural Urban Entomology Laboratory, Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia,

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A Study on Removal of Pb2+ Ion from Wastewaters by Using Water Cress (Enhydra Fluctuans Lour.)

Khin Sandar Thaw*, Aung Myint Htay** * Lecturer, Department of Chemistry, University of Yangon. ** MSc Student, Department of Chemistry, University of Yangon

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Nutritional Studies and Microbial Analyses of Fermented Flacourtia Rotendifolla (Na-ywe) Leaves.

Moe Moe Aye1, Kyaw Naing2 1 .Lecturer, Dr., Department of Chemistry, University of Yangon 2 .Pro-rector, Dr., University of Yangon

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Information Technology, Electric Performance Evaluation for Simultaneous Perturbation Particle Swarm Optimization Shin Sempukuji a, Hidetaka Ito a, Yutaka Maeda a a) Graduate School of Science and Engineering, Kansai University.

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The Story of Chaotic Circuits and Systems at Sirindhorn International Institute of Technology,Thammasat University Buncha Munmuangsaen, Banlue Srisuchinwong Sirindhorn International Institute of Technology, Thammasat University, Thailand

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Page A Paddy Moisture Measurement Using Hybrid Rectangular and Circular Waveguide Resonator Navapadol Kittiamornkula, Kamon Jirasereeamornkulb, Supaporn Kittisinc, and Kosin Chamnongthai*,b a School of Biomedical Engineering, Christian University of Thailand b Department of Electronics and Telecommunication Engineering, King’s Mongkut University ofTechnology Thonburi c Technology of Information System Management, Mahidol University

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Evaluation of Effect in Initial Setting of Tree Size for GP Kohei Matsumura1*), Yoshiko Hanada2), Keiko Ono3), and Noriaki Muranaka2) 1) Graduate School of Science and Engineering Kansai University 2) Faculty of Engineering Science, Kansai University.

3) Department of Electronics and Informatics, Ryukoku University, Shiga, Japan

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Copyright Protection by Data Embedding to Image Katsuaki Kiwakia*, Hidetaka Itoa, Yutaka Maedaa a) Graduate School of Science and Engineering, Kansai University

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Optimization for Acoustic Characteristics of Wall Based on Three-degree-of-freedom Model Takuya Tanikado1*), Yoshiko Hanada2, Masahiro Toyoda3,and Noriaki Muranaka2 1) Graduate School of Science and Engineering, Kansai University 2) Faculty of Engineering Science, Kansai University 3) Faculty of Environmental and Urban Engineering Departments, Kansai University

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Personal Authentication System Using Pinna Related Transfer Function Tatsuya Miki a*, Yoshinobu Kajikawa a a) Graduate School of Science and Engineering, Kansai University

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By Complex-valued Neural Network using a Simultaneous Perturbation Orbit Control of SCARA Robot Yoichiro Seki a,*, Hidetaka Ito a, Yutaka Maeda a a) Graduate School of Science and Engineering, Kansai University

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FPGA Implementation of pulse Density Complex-values Neural Network Using Simultaneous Perturbation Method Atsushi Tsumoto a,, Yutaka Maeda a,, Hidetaka Itou a, a) Graduate School of Science and Engineering, Kansai University

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Page A Study on 3D Sound Field Analysis for Parametric Array Loudspeaker Hiroaki Besshoa, Chuang Shia, Yoshinobu Kajikawaa a) Faculty of Engineering Science, Kansai University

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Multi-channel Feedforward ANC System Using Microphone Arrays for Noise Source Separation Satoshi Kinoshita a,*, Yoshinobu Kajikawa a a) Faculty of Engineering Science, Kansai University

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A Quantitative Evaluation of Fatigue Based on Brain Activity Measurement Kazuma Mushaa,*, Noriaki Muranakab, Yoshiko Hanada b a) Graduate School of Science and Engineering, Kansai University b) Faculty of Engineering Science, Kansai University

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Measurement of Brain Activity by Intellectual Function under Learning Through NIRS Naoki Shiroyama1*), Yoshiko Hanada2, Noriaki Muranaka3 1) Graduate School of Science and Engineering, Kansai University 2) Faculty of Engineering Science, Kansai University.

3) Graduate School of Social Sciences, Hiroshima University

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Civil Engineering Slump and Compressive Strength of Concrete made with Wet Fly Ash Warangkana Saengsoy a,*, Thuy Bich Thi Nguyen b, Somnuk Tangtermsirikul b a) Construction and Maintenance Technology Research Center (CONTEC), School of Civil Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University b) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University

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Effect of High Free Lime and High Sulfur Trioxide Contents on Volume Change of Cement-FlyAsh Mixtures Adnan Nawaz a, Parnthep Julnipitawong*, b, Pitisan Krammart c, Somnuk Tangtermsirikuld a) School of Engineering and Technology, Asian Institute of Technology b) Construction and Maintenance Technology Research Center, School of Civil Engineering and Technology, Sirindhorn International Institute of Technology,Thammasat University c) Department of Civil Engineering, Rajamangla University of Technology d) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University

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The 10 International Symposium in Science and Technology 2015 Collaboration between Asian Countries in Science and Technology, Aug 31 – Sep 2, 2015, Bangkok, Thailand

Development of Method to Determine Water Retainability of Porous Fine Aggregate Noulananh Lathsoulina,*, Pakawat Sancharoenb, Somnuk Tangtermsirikul a,b, a) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b) Construction and Maintenance Technology Research Center, Sirindhorn International Institute of Technology,Thammasat University

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Advantages of Symbolic Hessian Matrices in Sizing Optimization of Trusses Chantrea Lean a, Pisith Sam a, Pruettha Nanakorn a,* a) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University

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Modeling Dynamics of Traffic States on Urban Road Network Using Artificial Intelligence Mongkut Piantanakulchai a* ,Soknath Mil a ,Sornthep Vannarat b, Kimheang Ly a, Chhivhout Sor a a) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b) Large Scale Simulation Research Laboratory, National Electronics and Computer Technology Center (NECTEC)

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Sustainable Construction Practice in Thailand via Service Life Design Somnuk Tangtermsirikul a*, Chalermchai Wanichlamlerd,b, Krittiya Kaewmanee,b, Taweechai Samranwanichc a) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b) Construction & Maintenance Technology Research Center, School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University c) Department of Civil Engineering, Burapha University

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Deterioration of RC Structure due to Chlorine Attack: A Case Study Pakawat Sancharoen a,*, Tamyot Somyapakdeeb, Somnuk Tangtermsirikul a,c a) Construction and Maintenance Technology Research Center, Sirindhorn International Institute of Technology, Thammasat University b) Repcon Service Company Limited c) School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University

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Energy Dissipation Models for Computing Statistical-Based and Spectral-Based Root-Mean-Square Wave Heights Nga Thanh Duong*,a, Winyu Rattanapitikona, Tawatchai Tingsanchalib a Civil Engineering Program, Sirindhorn International Institute of Technology, Thammasat University b Asian Institute of Technology

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Materials and Chemistry

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Synthesis, Self-Assembly and Liposomal Encapsulation of Sulfated Cyclodextrin Amphiphiles Tetsuya Furuike*, Hiroshi Tamura Faculty of Chemistry, Materials and Bioengineering, Kansai University

ABSTRACT A novel class of amphiphiles involved sulfate groups at C-6 position and palmitoyl groups at C-2, 3 positions have been synthesized smoothly from α, β and γ-cyclodextrins (CDs) as a starting material. These compounds formed stable monolayers with high collapse pressures at the air/water interface. The mixed monolayer behaviors of the 6-O-sulfated CD amphiphiles (SO3-CDC16) in the presence of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol were discussed from the surface pressure-molecular area (π-A) isotherms. The collapse pressures showed the maximum at molar ratios of SO3-CDC16 lower than 10 mol%. Morphological analysis of liposomes with DPPC containing 4 mol% SO3-CDC16 formed in PBS were also performed by a TEM with negative staining, and observed as the vesicles having a diameter of 350-500 nm at the maximum. Moreover, a releasing ability of these liposomes was examined using a fluorescent compound, calcein. It was obviously shown that liposomes containing SO3-CDC16 could release an encapsulated calcein more smoothly than the liposome consisted only of DPPC, and that the release rate depended on the phase transition temperature of each SO3-CDC16 composing the liposome membranes. *furuike@kansai-u.ac.jp INTRODUCTION α-, β-, and γ-Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six, seven, and eight Dglucopyranose residues, respectively, that are linked by α-1,4 glycosidic bonds to form macrocycles. CDs have a hydrophilic outer surface and a hydrophobic central cavity and have the ability to form inclusion complexes with specific guests. Therefore, CDs have the potential to alter the physical, chemical, and biological properties of various organic compounds. The most common pharmaceutical application of CDs is to improve the solubility, stability, and bioavailability of various hydrophobic drugs. Recently, various kinds of CD derivatives have been prepared to extend the physicochemical properties and inclusion capacity of natural CDs as novel drug carriers.

However, the high external hydrophilicity of these CD derivatives results in a lack of affinity of the CD-based supramolecules with biological membranes. This problem is one of the reasons why researchers have been interested in CD derivatives with a relatively hydrophobic exterior. As a result, CD derivatives with increased affinity for biological membranes have been obtained by introducing fatty acids onto the hydroxyl groups of CDs. The monolayer behaviors of synthetic CD amphiphiles have been studied at the air–water interface. The surface pressure–molecular area (πA) isotherms of the monolayers disclose the size of the molecules, as well as the stability and phase behaviors of the monolayer films. The miscibility of mixed monolayers composed of two lipids with various concentration ratios can be elucidated from the π-A isotherms. A correspondence between the ideal line and a plot of molecular area at a fixed surface pressure versus the molar fraction of two lipids shows complete miscibility, whereas deviation from the ideal line represents partial miscibility. Liposomes are artificial cell-like materials and have been considered as a strong candidate for a drug delivery carrier. Drug delivery carriers are required to keep drugs inside their

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vehicle while traveling through the blood stream and to release the drugs after binding to the target cell.

Herein, the miscibility of monolayers composed of SO3-CDC16 with dipalmitoyl phosphatidylcholine (DPPC) and cholesterol was investigated in detail. Moreover, the formation of these liposomes was confirmed by TEM using negative staining. To test the encapsulation ability of the liposomes, 3,3′-bis [N,N-bis(carboxymethyl) aminomethyl]fluoroscein (calcein), an anionic dye, was captured in the liposomes and changes in fluorescence intensity were used to monitor the time dependence of dye leakage.

EXPERIMENTAL A. Materials and Methods Unless otherwise stated, all commercially available solvents and reagents were used witout further purification. 1H and 13C-NMR were recorded at 600 MHz or 150MHz on a BRUKER 600 spectrometer. Assignment of the ring–protons was made by first-order analysis of the spectra, and was confirmed by H-H COSY and HMQC spectra. Chemical reactions were monitored by TLC on precoated plates of silica gel 60F254. Column chromatography was performed on silica gel.

B. π-A Isotherms for Monomolecular Layers The π-A isotherms for monomolecular layers were obtained from experiments performed on a Langmuir-type film balance. A mixture of DPPC and cholesterol containing 0–100 mol% SO3CDC16 10–12 was spread at the air–water interface from mixed chloroform solutions (0.2 mg/mL) by using a microsyringe (Hamilton). After complete evaporation of the organic solvent, the measurement was performed using a Langmuir-type film balance (U.S.I., Japan) located at the airwater interface at 23°C. The temperature was controlled by a thermostated bath. The monolayers were compressed at a rate of 60 mm2/min. The π-A isotherms were determined at least three times.

C. Preparation of Liposomes by Reverse-Phase Evaporation DPPC and cholesterol were added to SO3-CDC16 to increase the stability of the liposomes. The mole ratio of DPPC, cholesterol, and SO3-CDC16 10–12 in the liposomes was 100:10:4. The amphiphilic mixtures dissolved in dichloromethane were put into a 25 mL round-bottom flask, and phosphate buffered saline (PBS, pH 7.4) solution was added in the absence or presence of calcein (0.1 mM) (dichloromethane:PBS = 3:1). The mixtures underwent ultrasonication for 5 min until they formed a homogeneous opalescent dispersion. In these suspen-sions, which were used to form reverse-phase vesicles, dichloromethane was gently removed with a rotary evaporator under reduced pressure. As the majority of the solvent was removed, the material first formed a viscous gel. After destruction of the gel using a vortex mixer for 1 min, dichloromethane was removed completely from the mixture to give a liposome solution. The desired liposomes were separated by column chromatography on a Sephadex G-50 column (30 cm × 1.5 cm i.d.), eluting with PBS at 4°C. D. Determination of Calcein Loading and Releasing Amounts The amount of calcein trapped in the liposomes was measured The membrane integrity of the liposome composed of DPPC containing 10 mol% cholesterol and 4 mol% SO3-CDC16 10–12 after incubation in PBS at 37°C was evaluated by calculating the percentage of retained calcein encapsulated in the liposome. Initially, 0.1 mM calcein in PBS was encapsulated in the liposome.

At different time intervals (up to 48 h), the retention of calcein was estimated by mixing 20 μL

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from each incubation tube with 2.0 mL of PBS (pH 7.4). A fluorescence spectrometer was used to measure the fluorescence intensity of calcein (λex = 490 nm, λem = 520 nm) before (Ftotal) and after (Fin) the addition of 10 mM CoCl2 solution (20 μL). Then, 20% Triton X-100 solution (20 μL) was added to release the entrapped calcein, and the fluorescence intensity was measured again to determine the background fluorescence intensity (Fq). The trapped volume (%) of calcein was calculated according to Equation (1): (1) where r is the dilution factor due to the addition of the CoCl2 and Triton X-100 solutions. Herein, r was 1.02.

RESULTS AND DISCUSSION A. Synthesis of SO3-CDC16 Scheme 1 shows the synthetic method for SO3-α-, β-, and γ-CDC16. First, selective silylation at the primary hydroxyl groups of each CD was carried out with tert-butyldimethylsilyl chloride (TBDMS-Cl) to give silylated CD derivatives 1–3, respectively. The esterification of silylated CDs 1–3 was performed by adding palmitoyl anhydride (2eq.) and DMAP (1eq.) per hydroxyl group in dry pyridine. The reaction mixture was stirred for 48 h at 70°C and gave the desired CD palmitates 4–6.

Next, desilylation occurred in the presence of boron trifluoride etherate (BF3·Et2O) at room temperature for 6 h. Desilylated derivatives 8 and 9 were purified by gel filtration chromatography with a Sephadex LH-20 column. Because the desilylated α-CD derivative 7 was very moisture sensitive and more unstable than the other CD Scheme 1. Reagents and conditions: i) TBDMS-Cl/pyridine, r.t., 8 h; ii) palmitoyl anhydride, DMAP/pyridine, 70ºC, 48 h; derivatives, it was evaporated under iii) BF ·Et O/CH Cl , r.t., 2 h; iv) SO Me N complex/toluene3 2 2 2 3 3 reduced pressure and used in the DMF (1:1), 100ºC, 48 h for 10, 80ºC, 48 h for 11, 12. next step without further purification. Finally, sulfation of the free primary hydroxyl groups of the obtained CD amphiphiles 7–9 was performed with sulfur trioxide-trimethylamine complex in a mixed solvent of toluene and DMF over 48 h under N2 atmosphere, at 80°C in the case of the β- and γ-CD amphiphiles and at 100°C in the case of the α-CD amphiphile. Purification using a Sephadex LH-20 column gave the desired SO3-CDC16 10–12 bearing palmitoyl groups. A shift of ~4 ppm in the low magnetic field for the C-6 position in the 13C-NMR spectra showed that the primary hydroxyl groups were sulfated efficiently. The chemical structures of each CD derivative prepared here were elucidated using 1H and 13C NMR data and elemental analyses.

B. Monolayer Behaviors of SO3-CDC16 at the Air–Water Interface

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The monolayer behaviors of SO3-CDC16 10–12 at the air–water interface were evaluated and the isotherms indicated that each SO3-CDC16 forms a stable monolayer with a high collapse pressure (40 mN/m). The observed molecular areas at the collapse pressure were 2.45, 2.98, and 3.20 nm2 for 10, 11, and 12, respectively. The corresponding mean areas per single lipid chain were 0.204, 0.213, and 0.200 nm2 for 10, 11 and 12, respectively. These values are independent of the size of the CD and close to the calculated hydrocarbon cross-sectional area of 0.200 nm2. This result indicates that the hydrophobic chains of SO3-CDC16 are well-packed and form dense monolayers.

Similarly, the π-A isotherms for the DPPC–cholesterol monolayer (mole ratio of 100:10) containing 10–12 (0 to 50 mol%) were investigated.

Figure 1 shows the π-A isotherms for DPPC– cholesterol, 11, and their mixtures. The films containing between 4 and 8 mol% of 11 formed liquid condensed films with high collapse pressures (max 65 mN/m). This collapse pressure is close to the surface tension of water (72.5 mN/m). With more than 27 mol% of 11–13, the collapse pressures of the mixed films were close to those of pure SO3-CDC16 (~40 mN/m), and Figure 1. π-A isotherms for Langmuir monolayers of the shape of the isotherms was similar. Next, SO3-β-CDC16, DPPC-cholesterol and their mixtures the molecular areas at a pressure of 30 mN/m containing 2 mol% (A), 4 mol% (B), 8 mol% (C), 27 mol% (D), and 50 mol% (E) concentration of SO3-βare plotted as a function of SO3-CDC16 CDC16 spread on the air-water interface. concentration. For most of the mixed ratios for each SO3-CDC16, the plots of the experimental molecule area at 30 mN/m were located on the ideal straight line. However, slight positive deviations from the ideal straight line were observed when the molecular fraction of SO3-CDC16 was less than 8 mol%. The variation of the collapse pressures for 10-12 is shown in Figure 2. The collapse pressures showed maxima between 2 and 8 mol%. These results indicate that SO3Figure 2. Relationships between SO3-CDC16 molar CDC16 forms stable monolayers at low fraction and collapse pressure for mixed monolayers. concentrations by interacting with DPPC– cholesterol. A possible interaction between SO3-CDC16 and the matrix lipids is an electrostatic interaction between the positive charge of DPPC and the negative charge of the sulfonic moieties in SO3-CDC16. For example, ~20 DPPC molecules surround a single CD derivative when the concentration of 11 is 4 mol%. In this work, good miscibility of the monolayers with up to 8 mol% of 10 and 11 supports this suggestion. However, the decrease of collapse pressure at 8 mol% of 12 may be caused by an insufficient number of DPPC molecules to encircle 12. These results show that the mixed monolayers containing no more than 8 mol% of 10 and 11 or less than 8 mol% of 12 have higher collapse pressures than those of only DPPC–cholesterol or only SO3-CDC16, and therefore form more stable monolayers than SO3-CDC16 alone. The more stable mixed films are formed because there are a sufficient number of DPPC molecules to entirely surround SO 3-CDC16.

Thus, various activity measurements were performed with 4 mol% of SO3-CDC16, as this is sufficient for DPPC to encircle SO3-CDC16.

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C. Morphological Analysis of Liposomes Composed of SO3-CDC16 The preparation of liposomes composed only of SO3-CDC16, in the absence of other amphiphiles was carried out by the reverse-phase evaporation method in water. A morphological analysis of the liposomes was performed by TEM after negative staining with a 2% uranyl acetate solution. The TEM images of the liposomes are shown in Figure 3. The liposomes composed of 10 (α-liposome) were observed as spherical particles with a diameter of ~150 nm at the maximum and tended to be smaller than those of the other SO3-CDC16 derivatives.

The liposomes composed of 11 (β-liposome) had a diameter of ~350 nm at the maximum, whereas, the liposomes composed of 12 (γ-liposome) had a diameter of 50–120 nm at the maximum.

D. Release of Calcein Encapsulated in Liposomes Figure 3. TEM images of liposomes of The retention time of vesicles encapsulating calcein DPPC and cholesterol (100:10, mole ratio) was monitored after incubation in PBS for 48 h at containing 4 mol% concentration of 10 (A), 37°C. Figure 4 shows the retention of calcein in the 11(B), and 12 (C). liposome composed of DPPC and cholesterol (100:10, mole ratio) with and without 4 mol% SO3-CDC16.

More than 20% of the total calcein was released from the liposomes within 2 h at 37°C; however, after that, the release rate was slow. The time at which half the total amount of calcein was released was 26, 32, and 42 h for liposomes containing 10, 11, and 12, respectively. Even after 48 h, liposomes that did not contain SO3-CDC16 still retained 60% or more calcein.

The liposomal membranes containing SO3-CDC16 are more stable than that consisting only of DPPC. Figure 4. Release of encapsulated calcein However, the retention time of calcein encapsulated in from liposomes composed of DPPC and liposomes containing SO3-CDC16 is shorter. The cholesterol containing 4 mol% SO3-CDC16 relation between liposomal membrane stability and in PBS at 37ºC. Release amounts of the calcein were determined by fluorescence retention capacity is not directly proportional. The spectroscopy; liposomes composed of only release of molecules encapsulated in liposomes is DPPC-cholesterol (○), containing 10 (●), related to the membrane mobility caused by the phase 11(▲), and 12 (■). transition temperature of the compounds. The phase transition temperatures of DPPC, 10, 11, and 12 are 42, 41.0, 37.2, and 36.6°C, respectively. When the phase transition temperature is lower than the temperature of the buffered solution, the release of encapsulated calcein from the liposome is accelerated due to turbulence of the membrane. It has been suggested that the retention capability of the liposome could be controlled by changing the kind of CD.

CONCLUSIONS SO3-CDC16 bearing sulfate groups and long acyl chains were synthesized efficiently using α-, β-, and γ-CDs as a starting material. The obtained amphiphiles formed stable monolayers in the presence of DPPC and cholesterol at the air–water interface, and the collapse pressures were maximized at molar ratios of SO3-CDC16 lower than 10 mol%. Moreover, liposomes with DPPC containing 4 mol% SO3-CDC16 formed in PBS could be observed as vesicles with diameters of

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350–500 nm. The ability of these liposomes to release calcein was also investigated, and the liposomes containing SO3-CDC16 were clearly shown to release encapsulated calcein more easily than the liposomes consisting only of DPPC. It was suggested that the release rate depended on the phase transition temperature of the SO3-CDC16 derivative present in the liposome membrane. We are currently investigating the ability of the liposomes containing SO3-CDC16 as drug delivery carriers, and the results will be reported as soon as possible.

ACKNOWLEDGEMENTS This work was supported by the Kansai University Grant-in-Aid of the Promotion and Upgrading of Education and Research, 2015, “Generation of bio-interactive polymers leading future-oriented medicalsˮ REFERENCES

Sukegawa, T., Furuike, T., Niikura, K., Yamagishi, A., Monde, K. and Nishimura, S. (2002) Chem. Communi. 430-431.

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Synthesis, Structural Investigation, and Some Applications of Nickel Doped Tin Oxide Nanoparticles Myat Myat Thaw1,Maung Maung Myint2 ,Soe Myint Maung3, Kyaw Naing4 a Department of Chemistry, Sagaing University, Sagaing, Myanmar b Department of Chemical Technology, Defence Services Science and Technology, Research Centre, Pyin Oo Lwin, Myanmar c Engineering Science Research Faculty, Defence Services Science and Technology, Research Centre, Pyin Oo Lwin, Myanmar d University of Yangon, Yangon, Myanmar Keywords: Nickel tin oxide nanoparticles, average crystallite size, adsorption, antimicrobial activity, gas sensor ABSTRACT In this research, tin oxide nanoparticles and nickel doped tin oxide nanoparticles were prepared from tin II chlorid by using co-precipitation and hydrothermal method. By using the Debye Scherrer equation, the average crystallite size of the tin oxide nanoparticles is found to be 33.53 nm by using co precipitation method at 500 0Cand 27.55 nm by using hydrothermal method. It is found that the crystal structure of tin oxide is tetragonal structure. The average crystallite size of the nickel doped tin oxide nanoparticles is found to be 19.44 (1%), 23.57nm (2%), and 33.47nm (5 %) respectively. The morphology of SnO2 and Ni doped SnO2 nanoparticles show spherical shape by FE SEM. Application of tin oxide and nickel doped tin oxide nanoparticles is done for adsorption properties and antimicrobial activities in this study. Antimicrobial activity on the tin oxide and nickel doped tin oxide nanoparticles is performed against six microbial strains. It is observed that the highest antimicrobial activity of nickel doped tin oxide nanoparticles is found to be 45 mm on E coli strain by using agar well diffusion method. Characteristic of tin oxide and nickel doped tin oxide nanoparticles is investigated for gas sensor application by using ammonia.

INTRODUCTION Nanoparticles in Waste Water Treatment and Adsorption Technique Adsorption processes are widely used for treatment of polluted surface and ground waters and also play a significant role in advanced wastewater treatment. The adsorption techniques are preferred because of their advantages: low cost, ease of operation, efficiency, simplicity of the equipment and mostly because the adsorbent can be chosen from a wide variety of materials, natural, synthetic and wastes water treatment(Burleson et al.,2004).

Semiconductor gas sensors and its Application areas Tin oxide is the most important material for use in gas sensing applications. Semiconductor metal oxide sensors are playing an important role in the detection of toxic pollutants (CO, H2S, NO, SO2, 1 2

3

4

Associate Professor, Dr, Department of Chemistry, Sagaing University Lecturer, Dr, Department of Chemical Technology, Defence Services Science and Technology ,Research Centre, Pyin Oo Lwin Professor, Dr, Department of Chemical Technology, Defence Services Science and Technology, Research Centre, Pyin Oo Lwin Pro-rector, Dr, University of Yangon

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etc) and combustible gases ( H2,CH4 and flammable organic vapors, et) owing to their advantages of robust nature, less sensitive a to moisture and temperature influence, simple electronic interface, quick response and recovery time. Semiconductors gas sensors can be used for a wide array of applications, ranging from safety equipment ( explosion, leakage, fire, contamination and poisoning protection) up to emission and air quality monitoring(Krishnakumar et al.,2008).

EXPERIMENTAL Preparation of nickel doped tin oxide by co-precipitation method Tin (II) chloride dihydrate was dissolved in distilled water and hydrochloric acid (conc.) solution was added to be clear and heated at sand bath. The clear solution was obtained. The sufficient amount of aqueous ammonia was added into this solution until completely precipitated and stirred for 2 hours and the solution was filtered and then the pH 9.5 was adjusted. The precipitate was collected, washed with distilled water and dried in an oven. The precursor tin oxide was obtained.

The precursor nickel doped tin oxide was calcined at different temperatures 200ºC, 300ºC, 400ºC, 500ºC and 600ºC for 6 hours (Aliahmad & Dehbashi 2013).

Preparation of nickel doped tin oxide by hydrothermal method Tin (II) chloride and nickel (II) chloride were mixed with 5 ml NaOH (5mL). 4.32 g of sodium dodecyl sulphate( SDS) was dissolved in this solution and stirred for 30 min until the suspension was formed and added distilled water ( 50mL) and added into telflon lined stainless autoclave and heated at 1200C for 8 hours and then cooled and filtered and washed with ethanol and distilled water and dried in air at 600C for 12 hours and obtained nickel doped SnO2 nanoparticles (Gnamam and Rajendran 2010).

Figure 1.Teflon-lined stainless steel autoclave (Hydrothermal method) Determination of Antimicrobial Activities of Tin Oxide and Nickel Doped Tin Oxide Nanoparticles In vitro screening, antimicrobial activities of tin oxide and nickel doped tin oxide nanoparticles were carried out against six microbial strains by agar well diffusion method at Central Research and Development Center (CRDC), Yangon, Myanmar. Bacillus subtilis (N.C.T.C-8236), Bacillus pumilus (N.C.I.B-8982), Candida albicans, Staphylococcus aureus(N.C.P.C-6371), Pseudomonas aeruginosa(N.C.T.C-6749) and Escherichia coli (N.C.I.B-8134) were used in this study. Bacteria spore suspension (0.1 g) was added into sterile petri-dish including nutrient agar and autoclaved for 25 minutes at 121 ˚C. It was cooled at 60 ˚C. These nutrient agar plates were made by 10 mm diameter cork borer. Tin oxide and nickel doped tin oxide (0.1 g) was added to these agar plates and incubated at 37 ˚C for 24 hours. The maximum inhibition zone was measured. Standard (0.1 g/ 10 mL) amoxicillin antibiotic was used as control (Amitava et al.,2011).

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Decomposition of Rhodamine B (Basic Dye) and Congo Red (Acidic Dye) on Effect of Contact Time Procedure Decomposition of (10ppm) of Rhodamine B and congo red dye solutions on tin oxide nanoparticles and nickel doped nanoparticles is carried out by varying the different contact time (1, 2, 3, 4, 5, 6 hour). 0.1g of prepared tin oxide nanoparticles and nickel doped tin oxide nanoparticles are added in 100 mL of (10ppm) Rhodamine B and 100 mL of (10ppm) congo red solution. Then, the solutions are shaken with a shaker. After 30 minutes, 10 ml of this solution is taken out, centrifuged and measured the absorbance of clear solution at 553nm for RhodamineB and at 499nm for congo red solution.

Fabrication of gas sensing system by static method Testing chamber unit consists of 11800 ml glass bottle. A multi-pin feed through at the top base of the chamber allows for the electrical connections to attach to the sensor electrodes. Multimeter of type U 232 A True RMS is used to record the variation of the sensor resistance exposed to predetermined air-hydrogen gas maxing ratio. Chamber can be evacuated using vacuum pump (Krishnakumar et al., 2008).

Figure 2. Fabrication of SnO2 sensor coated in ceramic tube

Figure 3.Gas sensor testing system

RESULTS AND DISCUSSION Characterization of Pure Tin Oxide Nanoparticles by XRD Analysis According to the XRD analysis, pure tin oxide nanoparticles are well matched with standard library of PDF 46-1088 of hydrated tin oxide and PDF 77-0447 of cassiterite tin oxide, respectively.

Eight characteristic peaks relative Miller Indices of (110), (101), (200), (211), (220), (310), (112) and (301) are observed. These values are well matched with standard PDF data of tin oxide. The peak positions 2θ values of pure tin oxide in XRD patterns are 24.939, 32.334, 36.364, 50.164, 53.201, 60.363, 63.066 and 64.488. D-spacings (Ǻ) of pure tin oxide nanoparticles are 3.567, 2.766, 2.468, 1.817, 1.720, 1.532, 1.472 and 1.443. All of the peaks can be indexed to be pure tin oxide structure of tetragonal due to the lattice parameters and it agrees with the literature. The average crystallite sizes of nickel doped tin oxide nanoparticles prepared from tin (II) chloride by using two methods are found to be 33.53nm and 27.55 nm by using Debye Scherrer equation.

Dhkl

=

Kλ β hklcosθ

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Figure 4. XRD diffractogram of pure tin oxide nanoparticles at 500ÂşC by co precipitation method

Characterization of Nickel Doped Tin Oxide Nanoparticles by XRD Analysis The X-ray diffractorams of the 1%,2%,5% and 10% nickel doped tn oxide samples are shown in figures 5. It is found that XRD diffractograms of nickel doped tin oxide nanoparticles show four characteristic peaks and its relative Miller Indices of (110, 101, 211, 220, 112 and 301) are observed. The peak position of 2 θ value of nickel doped tin oxide in XRD diffractograms are 26.741, 34.077, 51.901, 54.877, 64.886 and 65.939. D-spacing of nickel doped tin oxide nanoparticles are 3.330, 2.628, 1.760, 1.671, 1.435 and 1.415. All diffraction peaks are well assigned to tetragonal crystallite phase of nickel doped tin oxide with the reference pattern JCPDS 78-0643. It was observed that nickel doping does not change the tetragonal structure of pure tin oxide. It is noted that the intensity of the nickel doped tin oxide peaks decreases with increasing nickel concentration and the full width at half maximum values of nickel doped tin oxide nanoparticles become smaller and two theta values increase when nickel doped tin oxide nanoparticles compared with pure tin oxide nanoparticles. Nickel doping tin oxide samples are done at 1%, 2% and 5% was found to be 19.44nm(1%),23.57nm(2%) and 33.47nm (5%) respectively ( Table 1).

Table 1

Various Average Crystallite Sizes of Nickel Doped Tin Oxide Nanoparticles Sample Average crystallite size (nm) 1% 19.44 2% 23.57 5% 33.47 Figure 6

XRD diffractograms of 1%, 2% and 5% nickel doped tin oxide nanoparticles

Morphological Analysis of Pure Tin Oxide and Nickel Doped Tin Oxide byScanningElectron Microscope and Field Emission Electron Microscope Scanning electron microscope and field emission electron microscope images of pure tin oxide and nickel doped tin oxide nanoparticles are shown in Figure 7 (a) to (c) respectively. The morphologies of pure tin oxide and nickel doped tin oxide nanoparticles show sphereical in shape.

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Figure 7(a) FE SEM micrograph of calcined 1% nickel doped tin oxide sample

Figure 7(b) FE SEM micrograph of calcined 2% nickel doped tin oxide sample

Figure 7(c) FE SEM micrograph of calcined 5% nickel doped tin oxide sampleAntimicrobial Activity of Tin Oxide and Nickel Doped Tin Oxide Nanoparticles by Agar Well-diffusion

maximum inhibition zone (mm)

Antimicrobial activity of tin oxide and nickel doped tin oxide nanoparticles were determined by employing agar well diffusion method on six species of microorganisms. Bacillus subtilis, Staphylococusaureus, Bacillus pumilus, Pseudomonas aeruginosa, Candida albicans and Escherichia coli were carried out at Central Research and Development Center (CRDC) in Insein.

The inhibition zone of tin oxide and nickel doped tin oxide nanoparticles were measured and found on Bacillus subtilis(12 and15mm), Staphylococcus aureus(25 and 30 mm), Pseudomonas aeruginosa(14 and 33 mm), B. pumilius(10 and14 mm), Candida albicans(10 and11 mm) and E. coli ( 28 and 45 mm) respectively. Maximum inhibition zones of E coli on tin oxide and nickel doped tin oxide nanoparticles are shown in Figure 8.

50 0

SnO2

1 2 3 4 5 6 Microbial strains

Nickel doped SnO2

Figure 8. Maximum inhibition zone of different microbial strains 1=Bacillissubtilis2= Staphylococcus aureus3= Pseudomonas aeruginosa 4= Bacillus pumilus5= Candida albicans6= Escherichia coli (E coli) Application of prepared tin oxide nanoparticles and nickel doped nanoparticles for adsorption Properties on Rhodamine B and Congo Red dye solution Adsorption properties of tin oxide nanoparticles and nickel doped nanoparticles are measured by using organic dyes in contaminated water. The wavelengths of maximum absorption of Rhodamine B (basic dye) and congo red (acid dyes) are found at 553nm and 498nm by using UVvisible absorption spectroscopy.

Effect of contact time on adsorption properties of tin oxide and nickel doped tin oxide nanoparticles In this work, adsorption properties of tin oxide nanoparticles and nickel doped nanoparticles for two dyes ( basic and acid dyes) are investigated. It is found that the maximum adsorption capacity for Rh B and congo red dyes were 82.92% , 87.96 %, 73.45% and 78.08% in tin oxide and nickel doped tin oxide nanoparticles. After 6 hours, the degradation process is found to be constantly stable( Figure and ). The oxidation efficiency was expressed by the decolourization degree, DD (%)

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=

where Ct is the color or absorbance value at the indicated wavelength after

oxidation time and Co is the initial color or absorbance of aqueous sample.

Decomposition ( %)

100 SnO2

50 Nickel doped SnO2

0

1

2 Time 3 (4h) 5

6

Figure 9. Decomposition Percent of Rhodamine dye by using SnO2 and Nickel doped Tin Oxide Nanoparticlesat different contact times Determination of gas sensing properties on tin oxide and nickel doped tin oxide nanoparticles Ammonia is a toxic in liquid as a gas that present naturally in the atmosphere in sub part per million (ppm) levels. Increased of amount of ammonia contained in exhaled air can be connected with certain disease, including kidney disorders. The figures [10 (a to d)] show resistance versus time graphs. It is found that the response (40 s) and recovery times (300 s) of SnO2 nanoparticles are observed. The resistance of SnO2 and nickel doped SnO2 nanoparticles decrease when contacted with ammonia gas and increase when removed with the ammonia gas at the same time. It is observed that response time ( 30s and 130s) and recovery time ( 880 s and 650 s) are found by using 8.48 ppm and 4.24 ppm of ammonia gas in nickel doped tin oxide nanoparticles.

Figure 10. (a) Resistance Vs Time Graph for tin oxide nanoparticles with 10 ppm of ammonia

Figure 10. (c) Resistance Vs Time Graph for tin oxide nanoparticles with 8.48 ppm of ammonia

Figure 10. (b) Resistance Vs Time Graph for tin oxide nanoparticles with 8ppm of ammonia

Figure 10. (d) Resistance Vs Time Graph for tin oxide nanoparticles with 4.24 ppm of ammonia

CONCLUSION By using DebyeScherrer equation, the average crystallite size of nickel doped tin oxide nanoparticles is found to be 1.44(1%), 23.57nm (2%) and 33.47nm (5%). According to FE SEM images, pure tin oxide and nickel doped tin oxide nanoparticles appear in spherical shape. It is

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shown that the average crystallite size of the 1 % nickel doped tin oxide nanoparticles decreases compared with pure tin oxide nanoparticles. It is observed that it is noted that the resistance of SnO2 and nickel doped SnO2 nanoparticles has been changed when exposed to 4.24ppm and 8.48ppm ammonia gas. According to the gas sensing properties, it can be realized that nickel doped tin oxide nanoparticles can be used as a sensor material in gas sensor applications.

REFERENCES Aliahmad, M. and Dehbashi, M. (2012), “Experimental Study of Structural and Optical Band Gap of Nickel Doped Tin Oxide Nanoparticles’, International J. Physical Sciences., 7 (37), 5415-5420.

Aliahmad, M. and Dehbashi, M. (2013), “Ni-Doped SnO2 Nanoparticles Synthesized by Chemical Co-Precipitation Method”, IranicaJ. Energy &Environ.,4 (1) Special Issue on Nanotechnology : 4952 Amitava, M., D.I. Mohamned, T.C.Prathna and N.Chandrasekaran. (2011). “Antimicrobial Activity of Alumina Oxide Nanoparticles for Potential Clinical Applications”. Bio Science & Technology, 12, 245-251 Bradley, D.K. (2011), “Nickel Applications & Uses”, China Nickel Conference 8th, 1-3 Burleson, D.J., Driessen, M.D. & Penn, R.L. (2004), “On the Characterization of Environmental Nanoparticles”, J. Environ Science & Health., A 39, 2707-2753 Gnanam, S. and Rajendran, V. (2010), “Synthesis of Tin Oxide Nanoparticles by Sol-Gel Process: Effect of Solvents on the Optical Properties”, J. Sol Gel Science Technology, 53. 555-559 Hall, D.L., Wang, A.A., Joy, K.T., Miller, T.A. and Wooldridge, M.S. (2004), “Combustion Synthesis and Characterization of Nanocrystalline Tin and Tin Oxide Particles”, J. Am. ceram.Soc.,87 (11), 2033-2041 Huggett, J.M. and Shaw, H.F. (1996), “Field Emission Scanning Electron Microscopy a HighResolution Techniques for the Study of Clay Minerals in Sediments”, Clay Mineral.,32, 197-203.

Johari, A., Bhatnagar, M. C. and Rana, V. (2012), “Growth Characterization and IV Characteristics of Tin Oxide SnO2 Bank Wires”, Advanced Materials Letters., 3 (6), 515-518 Kingery, W.D.(1974), “A Note on the Differential Thermal Analysis of Archaeological Ceramics”, Archaeometry., 16, 102-109 Krishnakumar, T., Jayaprakash, R., Singh, V.N., Mehta, B.R. and Phani, A.R. (2008), “Synthesis and Characterization of Tin Oxide Nanoparticle for Humidity Sensor Applications”, J.Nano Research., 4,91-101

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Generation of Chitin Fine Flakes with Various Molecular Assemblies on the Solvation of Hydrated Chitin Slurry Seiichi Tokura, ChutimaVanichvattanadecha, Natthakammala Janpech, Ratana Rujiravanit Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330,Thailand.

E-mail address: tokura1934@gmail.com Key words: Chitin, Solvation, Sonication, Hydration, SEM views

Abstract -Chitin, one of the most productive polysaccharides, has been asked to apply for biomedical materials due to biodegradability and low toxicity in animal body in spite of poor solubility and unstable under drastic condition for chemical modification.

The calcium chloride di-hydrate saturated methanol(CaS solvent) has been investigated to discharge the binary hydrogen bonds to apply for biomedical purposes which were blocked the smooth chemical modification of chitin under mild condition due to rigid crystalline structure. A CaS solvent was demonstrated as a mild solvent for chitin on the destruction of rigid crystalline structure of-chitin. However, chemical modification has little been applied due to too high ionic strength of CaS solvent even if almost of hydrogen bonds were discharged. The hydration was applied for chitin (CHS) as a first step to apply chitin molecule for chemical modification by the addition of large excess of water to chitin/CaS solution successfully. The further affinity study of hydrated chitin molecule(CHS) has not been cleared by sonication and SEM observation that there are variety of organic solvent dependent molecular assemblies and crystallinities due to salvation effect including intermolecular interaction. The ethanol solvated chitin(E-chitin), for example, gave m size of chitin beads with short range of sonication(5min. at 30oC) and blocks with random size, on the other hand, was given by hydrated chitin under similar condition.

The direct salvation of CHS and indirect salvation through E-chitin were found to proceed salvation with various organic solvents, although there are solvent dependent molecular assemblies.

On the sonication of variously solvated chitins, E-chitin has only been converted to beads type product under short range sonication which may suggest the loose packing of linear type chitin molecules in ethanol.

Introduction Chitin, one of rich natural resources on the earth, is widely known as an amino polysaccharide with extremely poor solubility in spite of advanced functions which is one of prospective components of Crustaceans together with proteins and calcium carbonate as shown the chemical structure in Figure 1(1). The advanced functions of chitin are the biodegradability and acceleration of epidermal cell recovery with low toxic property.-Chitin extracted from cuticle of Crab or Shrimp shells, is the highest production on the earth among other types of chitins and the rigid crystalline structure due to the inter- and intra-residual hydrogen bonds. -Chitin does not proceed to dissolution under similar procedure as those for -chitin, even in the DMSO which migrates into crystalline structure of -chitin to make transparent flake, but does not solvate or dissolute at all directly.

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 Figure 1. Chemical structure of chitin and crystalline structures of and-chitins.

Solvation involves different types of intermolecular interactions, hydrogen bonding, ion-dipole, and dipole-dipole attractions or van der Waals forces. The hydrogen bonding, ion-dipole, and dipole-dipole interactions occur only in polar solvents. Ion-ion interactions occur only in ionic solvents. The solvation process will be thermodynamically favored only if the overall Gibbs energy of the solution is decreased, compared to the Gibbs energy of the separated solvent and solid (or gas or liquid). This means that the change in enthalpy minus the change in entropy (multiplied by the absolute temperature) is a negative value, or that the Gibbs free energy of the system decreases.

The conductivity of a solution depends on the solvation of its ions. Hydration also affects electronic and vibrational properties of biomolecules (2).

The application of -chitin is limited due to the specific crystalline structure of chitin in spite of multi-functional groups on the unit sugar, N-acetyl glucosamine (GlcNAc) such as primary and secondary hydroxyl groups, and amide bond. A couple of solvent systems were found to delete intra- and inter- residual binary hydrogen bonds under mild condition, though the preparative condition are limited as usually to prepare the chitin hydrate which are Lithium bromide-DMF(3) and calcium chloride di-hydrate saturated methanol (4). But those systems did not allow the biological and chemical reaction to be well provided due to high ionic concentration and none aqueous condition, even if the production of chitin hydrate has been reported to become highly reactive with slight inclusion of original crystalline structure. The binary processes have been found to make chitin molecule homogeneous gel by solvation of chitin. The first one is ethanol salvation and second is DMSO solvation from chitin hydro slurry (CHS). DMSO solvated chitin seems to be more useful than that of ethanol solvated chitin due to high solubility against wider sorts of compounds, higher skin permeability, lower toxicity than those of ethanol especially on the application for biomedical and cosmetic materials.

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Experimental Chitin: chitin was prepared from Shrimp shells according to the method of Hackman..

Preparation of chitin hydro slurry (CHS): 2%(w/v) chitin solution was prepared in CaS solution by dissolving 20g of chitin powder in 1,000ml of calcium chloride di-hydrate saturated methanol (CaS) solution under refluxing at 80oC for 1 hr. The chitin hydro slurry was precipitated by the addition of large excess of water following to the EDTA treatment of the hydro slurry and dialysis against water.

Preparation of solvated chitins (T-chitin, DMSO-chitin)(I) 100ml of E-chitin suspended ethanol was mixed with 100ml of toluene under stirring at 90oC for several hours followed by centrifugation (1,200 rps X 5min) to collect. (II).The concentrated CHS was, then, diluted by DMSO followed by the heat treatment at 129-140oC under stirring for 1 hour. The DMSO solvated chitin flake was collected by centrifugation (12,000 rpm X 5min), and repeated similar procedures, then supplied for the analyses. A slightly turbid chitin gel was extended on the glass plate by glass rods followed by drying at 170oC under vacuum. (III). CHS was diluted by ethanol and concentrated by centrifugation. The procedure was repeated twice and then refluxed in ethanol for an hour followed by centrifugation to remove extra ethanol. As prepared ethanol solvated chitin (Echitin) was converted to DMSO solvation mode by the dilution of E-chitin with DMSO followed by heat treatment at 140-150oC under stirring to remove ethanol.

Sonication of variously solvated chitins: Variously solvated chitin fine flake suspensions (average concentrations are 5%) were diluted to % by corresponded organic solvent and then sonicated for 30min at room temperature applying Ultrasonic Oscillator (750W). The products were stood for various time intervals at room temperature to observe turbidity of suspension.

Figure 2. Outline of hydration and solvation of chitin.

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Results and Discussion CHS was prepared by the dilution of chitin solution as shown on the Figure 2 showing the outline of hydration and solvation by organic solvents. The key point to prepare E-chitin and variously solvated chitin gels would be the exchange of solvent which was the main reason why E-chitin was picked up as a intermediate to prepare variously solvated chitin gels.

Though the views of variously solvated chitin gels, almost of them were transparent orr slightly turbid gels as shown in Figure 3.

Figure 3. Turbidities of chitin suspensions variously solvated.

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Figure 4. SEM views of solvated chitin gels associated with XRD figures.

SEM observations: The SEM views of variously solvated chitins are shown in Figure 4. As seen in the Figure, solvent dependency is shown clearly on the molecular assembly of chitin flakes together with XRD spectra.

Sonication of solvated chitin flakes; The beads formation of chitin flakes was the typical case in ethanol comparing with that for CHS in water as shown in Figure 5-A and B The beads formation was occurred only by 5 minutes of sonication for E-chitin in ethanol at 30oC But CHS did not give any beads formation at all under similar condition.

Figure 5-A. Sonicated E-chitin in ethanol. B. Sonication of CHS in water. Sonication was done for 5 minutes at 30oC References (1) Gardner K. H. and Blackwell J., Biopolymers, 14, 1581 (1975).

(2) Alirenza, M., Mashaghi et al., Hydration strongly affects the molecular and electronic structure of membrane phospholipids. J. Chem. Phys. 136, 114709 (2012).

(3) Austin, P. R., Z. P. Zikakised., Chitin, Chitosan and Related Enzymes, Harcourt Brace Janovich, New York (1984) p227.

(4) Tamura, H., Nagahama, H., & Tokura, S. (2006). Preparation of chitin hydrogel under mild conditions. Cellulose, 13, 357-364.

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Synthesis and Antibacterial Activities of Amino-Containing Cellulosic Materials Monrawat Rauytanapanit a, Rungaroon Waditee-Sirisattha b, Thanit Praneenararat*,a a

b

Department of Chemistry, Faculty of science, Chulalongkorn University Department of Microbiology, Faculty of science, Chulalongkorn University Keywords : cellulose, cotton, antibacterial surface, chemical modification

ABSTRACT Cellulose is a low-cost, naturally-derived polymer with multiple free hydroxyl groups that can be chemically modified to serve many applications. In this research, a variety of antibacterial cellulosic materials was developed by functionalizing chromatography paper and cotton fabric with aminocontaining molecules such as 3-aminopropyltrimethoxysilane (APS), polyethyleneimine (PEI) and 4,7,10-trioxa-1,13-tridecanediamine (TTD). We also investigated the effect of positive charge on antibacterial activities by appending various alkyl groups onto the amino functionality.

Antibacterial activities of these surfaces were evaluated in both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Enterobacter amnigenus). The results showed that a selected set of the modified surfaces exhibited strong growth inhibition, especially APS-modified cotton with 96.1% and 98.4% inhibitions on Gram-positive and Gram-negative bacteria, respectively.

*thanit.p@chula.ac.th INTRODUCTION Bacterial infection is a major problem for biomedical and food industries, thus causing the development of antibacterial agents and materials from researchers around the world. In this regard, cellulose is one of the most widely used natural materials. It is a versatile starting material for chemical conversions with multiple free hydroxyl groups. Mechanism of antibacterial cellulose can be divided into biocide releasing and contact-active biocidal strategies1. For biocide-releasing surfaces, biocidal agents such as nanoparticles2 are coated on cellulose surface by physical adsorption and leached into surrounding causing bacterial lysis. These type of surfaces showed a temporary activity and might cause an environmental problem. Contact-active biocidal surfaces3-5, on the other hand, contain moieties that are permanently immobilized onto cellulose leading to disruption of bacterial membrane upon contact. In this work, contact-active biocidal on cellulose surfaces were developed by appending various scaffolds containing amino functionalities and tested for antibacterial activities against Gram-positive (Staphylococcus aureus) and Gramnegative bacteria (Escherichia coli and Enterobacter amnigenus).

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EXPERIMENTAL E. Materials All chemicals and reagents were purchased from Sigma-Aldrich Co., Carlo Erba reagents S.p.A., and Acros organics. Whatman® 1 Chr chromatography paper (surface a) and cotton fabric (surface b) were first washed with N,N-dimethylformamide (DMF), deionized water, methanol and dichloromethane. Staphylococcus aureus, Escherichia coli and Enterobacter amnigenus were cultured in Nutrient Broth (Hi-media).

F. Synthesis of APS-modified surfaces (1a and 1b) Surfaces 1a and 1b were prepared by immersing surfaces a and b (6x6 cm2) in a solution of 3aminopropyltrimethoxysilane (APS) (1 mL) in acetone (10 mL) for 5 h with 160-ppm shaking. The surfaces were then washed with acetone to remove unreacted residues, heated at 110 oC for 2 h, followed by immersing in acetone for 18 h and air-drying.

G. Synthesis of PEI-modified surfaces (3a and 3b) Surfaces a and b were first shaken in a solution of sodium meta-periodate (86 mg) and lithium chloride (0.89 g) in milli-Q water for 5 min. The surfaces were then washed with deionized water, methanol and then air-dried. Surfaces 2a and 2b were obtained and then incubated in a solution of polyethyleneimine (PEI) (16 mg), triethylamine (10 mL) and methanol (10 mL) at 45 oC for 24 h.

After the reaction, the surfaces were washed with methanol and subsequently with water to remove unreacted PEI. H. Synthesis of DABCO-modified surfaces (5a and 5b) Surfaces a and b were first reacted with p-toluenesulfonylchloride (3.82 g) in pyridine (15 mL) for 90 min, followed by washing with DMF, methanol and dichloromethane. The surfaces were denoted as 4a and 4b. Next, these surfaces were shaken in a solution of 1,4diazabicyclo[2.2.2]octane in 50% acetonitrile in dichloromethane (10 mL) for 18 h and washed with ethyl acetate, acetonitrile and dichloromethane.

I. Synthesis of TTD-modified surfaces (6a and 6b) Surfaces 6a and 6b were synthesized by immersing surfaces 4a and 4b in a solution of 4,7,10trioxa-1,13-tridecanediamine (TTD) (3.5 mL) in DMF (15 mL) for 18 h. The surfaces were washed with 5.0 M NaOH in methanol, methanol, DMF, water and then heated at 40 oC for 18 h.

J. Antibacterial activities The antibacterial activities of all surfaces were tested against S. aureus, E. coli, E. amnigenus. The bacterial pre-inoculum cultures were grown at 37 oC in 50 mL of NB with continuous shaking at 200 ppm and diluted to concentration 1x107 CFU/mL. Samples (1x1 cm2) were placed in 12-well plates. 100 μL of bacterial suspension were dropped onto each samples and then incubated at 37 oC. After the contact time of 18 h, 900 μL of NB solution was added to samples and incubated at 37 oC for 18 h. Bacterial cell growth was monitored by measuring absorbance at 620 nm. Bacterial inhibition of each surface was calculated by using nonfunctionalized cellulose surfaces as a negative control.

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RESULTS AND DISCUSSION

A. Synthesis of amino-containing cellulosic materials The chemistry of all modification steps is shown in Fig 1. The synthesis of surfaces 1a and 1b from APS consists of 3 steps3 : 1) hydrolysis of methoxysilane to silanol derivatives, 2) silanol adsorption onto cellulose surfaces through hydrogen bonding and 3) condensation between silanol group and hydroxyl group of cellulose (Si-O-C) or two silanol groups (Si-O-Si). Surfaces 2a and 2b were prepared by converting hydroxyl groups of cellulose to the aldehyde group via periodate oxidation. The presence of the carbonyl group was confirmed by 2,4-dinitrophenylhydrazine. These surfaces were then grafted with branched PEI through imine formation. Surfaces 4a and 4b were activated by converting the hydroxyl groups to the tosylate group and finally were reacted with nucleophilic molecules DABCO and TTD. Moreover, the amount of the amino group loaded on surfaces can be determined by reacting each surface with 0.6 M Fmoc-Osu in DMF for 4 h, cleaving Fmoc group with 2% DBU, 20% piperidine in DMF for 15 min and measuring absorbance at 296 nm. The results as shown in Table 1 revealed the higher amount of amino loaded on cotton surfaces.

O

O O Si

O

O O Si

NH2

NH2 O

APS

HO

OH

O n

1a, 1b H2N

H N

N

NaIO4, LiCl

O HO

OH

H2N

O O

O

O

N

PEI

N NH

CH3

N

N

N

OH

4a, 4b

NH

O HO

O HO

NH

N

DABCO

O S O O

TsCl, pyridine

OH

HN

O n

3a, 3b

N

2a, 2b

Chr paper, a Cotton, b

n

N

HN

n

O

n

O O

OH

OH

OH

NH2 m

n

H2N

O

n O

3

NH2

3

5a, 5b

O

NH2

O

TTD

HO

OH

O

6a, 6b n O

CH3I

O O

O HN

O

3

N

HN

3

O HO

OH

O

N H

O O

6b/ C1 n

HO

OH

O

6b/ cap n

Fig 1. The chemical grafting of amino-containing molecules onto cellulose surfaces

23 | P a g e

Table 1. Amount of amino loaded on amino-functionalized surfaces Surfaces

1a

1b

3a

3b

6a

6b

amount of the amino group loading (Îźmol/cm2)

0.443

1.389

1.231

2.064

0.725

0.741

B. Antibacterial activities Antibacterial activities of amino-functionalized surfaces are shown in Fig 2a). Corresponding to the loading results, cotton-modified surfaces displayed higher bacterial inhibitions than did papermodified surfaces. Surfaces 1a and 1b exhibited strong inhibition in all species due to the presence of large silane network and amino alkyl groups. Although surface 1a showed a small amount of amino loading, the surface still retained high activities. Surfaces 3a and 3b, which consist of branched amino polymer, displayed significant inhibition on Gram-positive bacteria. The flexibility and hydrophobicity of PEI might play an important role in peptidoglycan layer binding. Surfaces 5b demonstrated high inhibition against both Gram-positive and Gram-negative bacteria due to the polycationic structure. The positive charge of quaternary ammonium compounds can bind to cellmembrane components via electrostatic attraction. Surfaces 6b showed significant inhibition on Gram-negative bacteria.

b)

a)

c)

Fig 2. Antibacterial activities of amino-functionalized surfaces toward a) E. coli, b) E. amnigenus and c) S. aureus

24 | P a g e

An effect of hidden amino groups was investigated by capping surface 6b with 5% propionic anhydride in DMF. From Fig 3, as the amount of active amino groups decreased, the bacterial growth inhibitions were significantly decreased. Alkylation was also studied by immobilizing surface 6b with iodomethane (C1). The methylation resulted in a large decrease of bacterial inhibition in all species.

Fig 3. Antibacterial activities of modified surface 6b toward E. coli, E. amnigenus, S. aureus CONCLUSIONS In this work, a series of amino-functionalized cellulose surfaces was successfully synthesized via a variety of organic reactions. The resulting modified cellulose surfaces exhibited potent antibacterial activities against E. coli, E. amnigenus and S. aureus. Amino-containing molecules with polycationic structure displayed high bacterial growth inhibition in all species. The modification of branched amino-containing molecules showed significant inhibition on Gram-positive bacterial.

Moreover, amino-containing molecules with long hydrocarbon illustrated great inhibition on Gramnegative bacteria.

ACKNOWLEDGEMENTS This study is financially supported by Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University and the development and promotion of science and technology talented project (DPST).

REFERENCES 1. Siedenbiedel, F. and Tiller, J. C. (2012) Polymers. 4 (1), 46.

2. Knetsch, M. L. W. and Koole, L. H. (2011) Polymers. 3 (1), 340-366.

3. Fernandes, S. C. M., Sadocco, P., Alonso-Varona, A., Palomares, T., Eceiza, A., Silvestre, A. J. D., Mondragon, I. and Freire, C. S. R. (2013) ACS Appl Mater Inter. 5 (8), 3290-3297.

4. Gultekinoglu, M., Tunc Sarisozen, Y., Erdogdu, C., Sagiroglu, M., Aksoy, E. A., Oh, Y. J., Hinterdorfer, P. and Ulubayram, K. (2015) Acta Biomater. 21 44-54.

5. Nakamura, M., Iwasaki, T., Tokino, S., Asaoka, A., Yamakawa, M. and Ishibashi, J. (2011) Biomacromolecules. 12 (5), 1540-1545.

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Methanol Oxidation of Pt Particles on Electropolymerized Polypyrrole Apichart Rodchanarowana, Thaneeya Hawisetb, Prachak Inkaew*c a)

Department of Materials Engineering, Faculty of Engineering, Kasetsart University b) School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand c) School of Science, Mae Fah Luang University, Chiang Rai, Thailand

ABSTRACT Methanol oxidation of Pt paricles on polypyrrole was investigated. Polypyrrole was prepared by electropolymerization of pyrrole onto a gold electrode. A conductive form of polypyrrole was formed, and used as a support for Pt particles. The catalytic activity of Pt particles toward methanol oxidation reaction was evalulated, and compared with commercially available Pt (10% Pt on carbon black). Using polypyrrole as the support enhanced catalytic activity and stability of Pt particles. The ratio of forward and backward peaks of methanol oxidation reaction was used as the indicator for catalytic activity. The ratio from the particles on polypyrrole support was higher than that of commercially available Pt catalyst with the same mass loading. The stability of the catalysts was investigated by chronoamperometry. The catalyst adsorbed onto polypyrrole showed high stability when 0.9 V (vs. Ag/AgCl) was applied to the electrode for 1 hr in methanol oxidation reaction.

Keywords: Methanol oxidation, Electropolymerization, Polypyrrole, Pt particles, Electrocatalysis *prachak.ink@mfu.ac.th

INTRODUCTION Support plays important part in catalytic activity of many catalysts. The support affects the stability and efficiency of the catalyst in the reaction (Lim et al., 2010; Shim et al., 2001). In Polymer Electrolyte Membrane Fuel Cells (PEMFCs) operating below 80 ˚C, oxygen reduction proceeds slowly than that of at higher temperature. To reduce the amount of the catalyst used, the reaction needs to be operated above 80 ˚C. However, at above 80 ˚C, catalytic activity of the catalyst appears to be decreased. The decrease in catalytic activity also was observed in the continuouslyoperated system. This degradation of catalyst is known to be from the contamination of the support.

Pt on carbon is commonly used as the catalyst in PEMFCs and in Direct Methanol Fuel Cells (DMFCs). The degradation of carbon support proceeds via the reaction: C + H2O → CO2 + 4H+ + 4e- (Hoogers, 2010). The degradation of carbon support in long period of use or at higher temperature not only contaminates the catalyst but also desorbs the catalyst from its surface.

Moreover, the desorbed catalyst would form cluster or chunk which reduced the surface area and catalytic activity. Other materials besides carbon were used as Pt catalyst support. Metal, metal oxide, titanium nitride, boron nitride, boron carbide, titanium carbide, silicon carbide, tungsten carbides, and titanium diboride were used as the support for Pt (Lin et al., 2002; Sasaki et al., 2003; Schlange et al., 2010; Shao et al., 2009; Thotiyl et al., 2008; Zhang et al., 2005). Even though those nitride or carbide compounds had high stability as the support, they had low surface area compared to activated carbon or carbon nanotube. The distribution of the catalyst on nitride or carbide surface was not as good as carbon (McCreery, 2008; Sun et al., 2002). Using conductive polymer as the support has potential to resolve the problems encountered from using nitride or carbide and degradation of carbon support at high temperature. A conductive polymer possesses desirable properties to be used as the support; (1) good conductivity (2) stable at high temperature, up to 200

26 | P a g e

ËšC (3) high surface to volume ratio (4) strong absorption for many type of catalysts (5) good distribution for catalyst or particles (Heinze et al., 2010). Nevertheless, the use of conductive polymers as the support in PEMFC or DMFCs was not widely investigated. There were reports of using polyaniline, polypyrrole, polyimide and polythiophene as the support for metals (Asano et al., 2006; Heinze et al., 2010; Shao et al., 2009). Hence, it could be possible to use a conductive polymer as the support for Pt catalyst in PEMFCs or DMFCs. Polypyrrole (PPy) is a conductive polymer with high stability and high conductivity. The conductivity of polypyrrole in the oxidized form is about 500 S/cm (Lee and Park, 2005). Report showed that polypyrrole could be used as the support for Pd, Pb, Cu, Ni, Ru, Sn and Au in many reactions. Polypyrrole was also used as the support for biosensor and chemical sensor in thin film form (Joshi et al., 2000; Qin et al., 2002) and in nanoparticles form (Kim, 2005). The catalytic activity and stability of the metals on polypyrrole surface were enhanced. In this work, polypyrrole was used as the support for Pt particles which were used as the catalyst in methanol oxidation. The stability and catalytic activities of the catalyst were then investigated and compared to commercially available Pt catalyst, Pt particles on carbon support.

EXPERIMENTAL Unless stated otherwise, the materials used in this work were obtained from Sigma-Aldrich (St. Louis, U.S.A.). All materials were in reagent grade and used as received without any further purification. Electrolyte was prepared fresh each day. The triple distillation water was used to prepared aqueous solution. For electrochemical analysis in aqueous solution, a Au electrode (3 mm in diameter) was used as the working electrode, Pt as the counter electrode, and Ag/AgCl as the reference electrode. In non-aqueous solution, Ag/AgNO3 was used as the reference and the potentials were converted to Ag/AgCl scale. All electrodes are from Metrohm Siam (Bangkok, Thailand). Commercial Pt catalyst was Pt 10% on carbon black. Au electrode was polished with alumina particles (0.3 Âľm in diameter) and rinsed with triple distillation water before use.

A. Electropolymerization of polypyrrole Polypyrrole used in this study was prepared by electropolymerization of pyrrole monomer on a gold electrode. A potential of 0.8 V (vs. Ag/AgNO3) was applied to the electrode in 0.1 M pyrrole in acetonitrile + 0.1 M tetrabutylammonium hexafluorophosphate (TBPF6). The potential was applied different periods of time to form a polypyrrole film on the electrode. After the film formation, the electrode was rinsed with acetonitrile to get rid of pyrrole monomer and to clean the polypyrrole film.

B. Deposition of Pt particles on polypyrrole 1 mM K2PtCl6 was used as a precursor for Pt particle preparation. 10 mM NaBH 4 was added to 1 mM K2PtCl6 solution to produce Pt particles. Immediately after obtaining the particles, a desirable amount of Pt particles (colloid) was dropped on the polypyrrole-coated Au electrode (the electrode prepared in part A). The electrode was then left to dry in the air and was rinsed with triple distillation water before use.

C. Deposition of commercial Pt catalyst on Au electrode About 0.0400 g of commercial Pt catalyst was placed into a 10-mL vial. 1 mL triple distillation water was added into such vial. The vial was sonicated in a sonicating bath for 2 min or until the

27 | P a g e

particles distributed equally in the solution. The catalyst was then dropped onto the polished Au electrode and left to dry in the air. The electrode was rinsed with triple distillation water before use.

D. Methanol oxidation of Pt particles Methanol oxidation of Pt particles was carried out in 1 M CH3OH + 0.5 M H2SO4 solutions by a cyclic voltammetry technique at 50 mV/s between 0.0 and 1.0 V (vs. Ag/AgCl). To evaluate the stability of methanol oxidation on the different supporting materials, the current-time curve (chronoamperometry) was performed at 0.90 V in 1 M CH3OH + 0.5 M H2SO4 solution.

RESULTS AND DISCUSSION A. Electropolymerization of polypyrrole Cyclic voltammograms (CV) of polypyrrole formed from electropolymerization were shown in Figure 1. The voltammograms showed the formation of many layers of polypyrrole on the Au electrode. The surface of Au electrode was totally covered with polypyrrole because there was no oxidation peak of Au on the CV scan (Wang, 2006). The expansion of the current peak indicated the thicker films of polypyrrole formed on the electrode (PPy/Au) with longer deposition time. The scans were ceased after the polymer was deposited for 9 seconds to prevent too much polypyrrole on the electrode. The obtained voltammograms were similar to the voltammogram reported in literatures (Heinze et al., 2010; Zhou et al., 2002) for the films prepared by very similar method. In this work, however, the positive limit of the scan was set at 0.8 V to prevent over-oxidation of polypyrrole. There were reports on low conductivity of over-oxidized polypyrrole when the applied potential was over 0.8 V (Heinze et al., 2010).

0.5 mA 9s 7s 4s Increasing deposition time

Current

2s

1s

Increasing deposition time

Potential (V vs. Ag/AgCl) -1

-0.5

0.0

0.5

1

Figure 1. Cyclic voltammograms of polypyrrole prepared from electropolymerization on gold surface. 0.1 M pyrrole in acetonitrile + 0.1 M tetrabutylammonium hexafluorophosphate (TBPF6).

Scan rate 100 mV/s.

B. Deposition of Pt particles on polypyrrole Figure 2 showed the voltammogram of Pt particles deposited onto polypyrrole (Pt/PPy) and the voltammogram of commercial Pt catalyst (Pt/C). The region between -0.2 and 0.2 V is hydrogen adsorption/desorption area which is unique to Pt surface in acid solution (Wang, 2006). From the voltammogram the peaks of hydrogen adsorption/desorption were not easily observed because the amount of Pt loading on the surface was not very high. The capacitive currents for both Pt/PPy and

28 | P a g e

Pt/C, however, were almost as high as those of hydrogen adsorption/desorption currents. The voltammogram of Pt/PPy was stopped at 0.5 V to minimize over-oxidation state of the polymer.

The voltammogram of Pt/C, however, was allowed to be up to 1 V to clean the Pt catalyst.

Current (A)

40 ÂľA

0 Pt/C Pt/PPy

-0.2

0

0.2

0.4

0.6

0.8

1.0

Potential (V vs. Ag/AgCl)

Figure 2. Cyclic voltammogram of Pt particles deposited on polypyrrole (Pt/PPy) and the voltammogram of commercial Pt catalyst (Pt/C). 0.5 M H2SO4. Scan rate 100 mV/s.

C. Methanol oxidation of Pt catalyst The electrochemical performance of Pt particles on polypyrrole (Pt/PPy) was shown in Figure 3.

The oxidation peak of methanol occurred at 0.8 V, which was close to the peak obtained in Pt particles or Pt nanoparticles prepared by other research groups (He, 2009; Kang, 2012). However, there was no reduction peak for the backward scan. In the backward scan, small organic species generated from methanol oxidation were oxidized and produced the oxidation peak. The mechanism of methanol oxidation on Pt catalyst was still controversial. Different pathways have been proposed and are still under investigation by many research groups. The electrochemical performance of Pt particles toward methanol reaction can be compared from the height of the forward oxidation peak and the ratio of the current in the forward (i f) and backward (ib) scan (Edlund, 2011). High electrochemical performance catalyst for methanol oxidation would produce high oxidation current (if) and high if/ib value.

4 3.5

Pt/PPy

Current (mA)

3 2.5

2 1.5 1 0.5 0

Pt/C

-0.5 -1 -0.2

0

0.2

0.4

0.6

0.8

1

Potential (V vs. Ag/AgCl)

Figure 3. Cyclic voltammograms of methanol oxidation on Pt particles on polypyrrole support (Pt/PPy) and commercial Pt catalyst (Pt/C). 1 M CH3OH + 0.5 M H2SO4. Scan rate 100 mV/s. The arrows indicate the direction of the scans.

29 | P a g e

Current (mA)

From Figure 3, the current from the oxidation peak of Pt particles on polypyrrole was about 4 times higher than that of commercial Pt catalyst. The ratio of if/ib for Pt particles on polypyrrole was 1.83 whereas for commercial Pt was 0.80. Hence, it can be concluded that Pt particles on polypyrrole has higher electrocatalytic activity toward methanol oxidation than commercial Pt. The oxidation current and the value of if/ib for methanol oxidation were similar to those of Pt nanoparticles or Pt bimetallic nanoparticles reported from other research groups when the catalyst was loaded at the same amount (the same weight per area). However, the catalysts reported in the literatures were nanoparticles involving complicated preparation steps. The particles in this work were in a colloid form. The enhancement in the catalytic activity could be the effect of the support.

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

Pt/PPy

Pt/C

0

1000

2000

3000

Time (s)

Figure 4. Plots of current versus time of Pt particles on polypyrrole (Pt/PPy) and commercial Pt catalyst (Pt/C). Applied potential = 0.9 V, 1 M CH3OH + 0.5 M H2SO4.

Stability was also very important property for a desirable catalyst. High-stability catalyst could be used for long period of time with very small decrease in the performance or with no decrease in the performance at all. For Pt catalyst in methanol oxidation reaction, plot of current-time was used to evaluate the stability of the catalyst. The desired Pt catalyst should produce high and stable current.

Figure 4 showed the current-time plot of methanol oxidation on Pt particles deposited on polypyrrole and commercial Pt catalyst. From the figure, Pt/PPy had higher current than that of Pt/C when the potential of 0.9 V was applied to the electrode. Moreover, the current decayed slower on Pt/PPy than on commercial Pt catalyst, indicating higher stability of Pt/PPy than commercial Pt catalyst. The decrease in the current of this current-time plot did not mean that Pt was unstable or desorbed from the electrode. A certain or limited amount of methanol was added at the beginning of the reaction. Hence, as the reaction proceeded, the amount of methanol was reduced. For longer period of time, the current would be even less and would reached zero at some point because all methanol had been used up in the reaction.

CONCLUSIONS The methanol oxidation of Pt particles on electropolymerized polypyrrole support was investigated and compared to the oxidation of commercial Pt catalyst, Pt on carbon black. Cyclic voltammetry and chronoamperometry were the techniques used for the investigation. In the same mass loading,

30 | P a g e

Pt particles on polypyrrole showed higher catalytic activity and stability than commercial Pt catalyst. The support plays significant role for such effects.

ACKNOWLEDGEMENTS Financial supports from Mae Fah Luang University and the National Research Council of Thailand (Grant # 57201050015) are greatly appreciated. We also would like to thank the Scientific and Technological Instrument Center (STIC) at Mae Fah Luang University for providing the electrodes and potentiostat used in this work.

REFERENCES Asano, N.; Aoki, M.; Suzuki, S.; Miyatake, K.; Uchida, H.; Watanabe, M. (2006) J. Am. Chem. Soc. 128 (5), 1762-1769.

Edlund, D. (2011). Methanol Fuel Cell Systems. Pan Stanford Publishing, Singapore.

He, W.; Wu, X.; Liu, J.; Zhang, K.; Chu, W. ; Feng, L.; Hu, X.; Zhou, W. Xie, S. (2009) J. Phys. Chem. C. 113 (24), 10505–10510.

Heinze, J.; Frontana-Uribe, B. A.; Ludwigs, S. (2002) Chem. Rev. 110 (8), 4724-4771.

Hoogers, G. (2002). Fuel Cell Technology Handbook; Society of Automotive Engineers . CRC Press, New York.

Joshi, A.; Gangal, S. A.; Gupta, S. K. (2000) Sensors and Actuators B. 156 (2), 938-942.

Kang, Y.; Pyo, J. B.; Ye, X.; Gordon, T.R.; Murray, C. B. (2012) ACS Nano. 6 (6), 5642–5647 Kim, S.; Oh, W.-K.; Jeong, Y. S.; Hong, J.-Y.; Cho, B.-R.; Hahn, J.-S.; Jang, J. (2005) Biomaterials 32 (9), 2342-2350.

Lee, H. J.; Park, S.-M. J. (2005) Phys.Chem. B 109 (27), 13247-13254.

Lim, D. H.; Lee, W. J.; Wheldon, J.; Macy, N. L.; Smyrl, W. H. J. (2010) Electrochem. Soc. 157 (6), B862-B867.

Lin, C. A.; Wu, J. C. S.; Pan, F. J. W.; Yeh, C. T. J. (2002) Catal. 210 (3), 39-45.

McCreery, R. L. (2008) Chem. Rev. 108(7), 2646-2687.

Qin, H.; Kulkarni, A.; Zhang, H.; Kim, H.; Jiang, D.; Kim, T. (2002) Sensors and Actuators B 158 (1), 223-228.

Sasaki, K.; Mo, Y.; Wang, J. X.; Balasubramanian, M.; Uribe, F.; McBreen, J.; Adzic, R. R. (2003) Electrochem. Acta. 48(25-26), 3841-3849.

Schlange, A.; dos Santos, A. R.; Hasse, B.; Etzold, B. J. M.; Kunz, U.; Turek, T. (2010) J. Power Sources. 199(2), 22-28.

Shao, Y.; Liu, J.; Wang, Y.; Lin, Y. J. (2009) Mater. Chem. 19 (1), 46-59.

Shim, J.; Lee, C. R.; Lee, H. K.; Lee, J. S.; Cairns, E. J. J. (2001) J. Power Sources. 102 (1), 172-177.

Sun, Y.-P.; Fu, K.; Lin, Y.; Huang, W. (2002) Acc. Chem. Res. 35(12), 1096-1104.

Thotiyl, M. M. O.; Kumar, T. R.; Sampath, S. J. (2008) Phys.Chem. C. 114 (41), 17934-17941.

Wang, J. (2006). Analytical Electrochemistry. Wiley-VCH, New York.

Zhang, J.; Vukmirovic, M. B.; Xu, Y.; Mavrikakis, M.; Adzic, R. R. (2005) Angew. Chem. 44 (14), 2132-2135.

Zhou, M.; Pagels, M.; Geschke, B.; Heinze, J. (2002) J. Phys. Chem. B 106 (39), 10065-10073.

31 | P a g e

Determination of Lead by Colorimetric Paper-Based Sensor Nattapong Thuwakham, Prachak Inkaew School of Science, Mae Fah Luang University, Chaing Rai, Thailand Keywords: Paper-based sensor, Lead determination, Colorimetric method ABSTRACT Accurate determination of lead in the ppm range is very important. Several methods have been investigated and developed. However, most of them involved complicated sample preparation and expensive equipment. This work reports a simple method for the determination of lead using sodium sulfide. Paper was used as a substrate for preparing a sensor. Sodium sulfide was dropped on the paper to form the detection zone. Lead (II) ions reacted with sulfide ions in the detection zone to form black precipitate. The images of the precipitate were taken with digital camera and analyzed with Photoshop software. The color intensities were then used to plot a calibration graph.

This developed method gave good response for lead concentrations between 100 and 1000 ppm.

The sensor was successfully employed for determining the concentration of lead in acid-digested paint color. *E-mail: nattapong.thu10@lamduan.mfu.ac.th INTRODUCTION Lead is a toxic heavy metal. It has been found in house paint, soil, drinking water and paint-pottery.

Lead may cause muscle and joint weakness or pain, anaemia, headaches, constipation, and effect to central nervous system (Wu et al., 2013). Level of lead allowed depends on the type of product. For example, the United States Consumer Product Safety Commission (USCPSC) indicates that paint should contain no more than 600 ppm of lead. The Thai Industrial Standards Institute sets the maximum limit of lead in paint to be no more than 100 ppm (TISI 2625-2557). Determination of the amount of lead in paint or many sources is important. Techniques for determination of lead in biological and environmental samples are atomic absorption spectroscopy (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS) (Yan & Indra, 2012; Mercan et al., 2015) and electrochemical method (Jovanovski et al., 2015). Although these techniques provide low detection limit and high sensitivity, they need skilled scientist or technician to operate the instrument. Besides, the instruments themselves are expensive to operate and maintain. An analysis using paper as a substrate for a sensor is of interest for many research groups (Cate et al., 2015). Paper has several advantages such as low cost and simplicity of operation. The sensor is generally prepared by embedding or adsorbing the reagent onto the paper and allows the analyte to mix with the reagent to form the detectable species. Colorimetric analysis on paper can be done via color reading employing commercially available Photoshop software (Soldat et al., 2009) or other similar programs. The intensity of the color formed is generally proportional to the concentration of analyte present. In this work, paper-based sensor was developed for the analysis of lead in paint sample. The determined value of lead concentration was compared to stripping analysis to validate the developed technique.

32 | P a g e

EXPERIMENTAL A. Materials and chemicals preparation.

Standard lead solution was prepared by diluting 1000 ppm AAS standard lead solution (ACS grade, Sigma-Aldrich) in triple distillation water (Milli-Q water system, Quantum). Whatman No.1 filter paper (15 cm diameter) was used as the paper substrate. 1 M sodium sulfide solution was prepared by dissolving sodium sulfide (reagent grade, Panreac AppliChem, Spain) in appropriate amount of water. A paint sample was digested by 65% nitric acid (AR grade, Sigma-Aldrich). 0.5 M H2SO4 was prepared by diluting 95-98% H2SO4 (AR grade, Sigma-Aldrich). 1000 ppm AAS standard bismuth solution was from Lobachemie, India. All chemicals were used as received without any further purification.

B. Paper-based sensor preparation.

Template was drawn on a computer and was printed on a filter paper using an inkjet printer. The paper was cut along the printed template as shown in Figure 1(A). The side of square a was 7 mm.

The diameter of circle b was 8 mm. The diameter of circle c was 8 cm. Channels between c and b or c and a were 3 × 8 mm.

Figure 1. (A) Preparation of a template using scissors cutting along the line of the drawing. (B) Formation of the color on the detection zone (b) and blank (a). Pb2+ ions were dropped at (b) and sodium sulfide reagent was added at (c).

C. Color reading of the colorimetric paper-based sensor.

5 μL of 100, 200, 400, 800, 1000 ppm of Pb2+ standard was placed on the circle of the prepared template (area b of Figure 1B) and then a drop of 50 μL of 1 M sodium sulfide was dropped onto the center (area c of Figure 1B). The color formed on the detection zone (area b of Figure 1B) from the reaction between sodium sulfide and lead. The color intensities were read by Photoshop CS5 software. In Photoshop, the color was represented by three numbers; red, green, and blue (RGB).

The values for each color were from 0 to 255. Photographs of the template were taken by a digital camera. The elliptical marquee tool was used to cover the detection zone to determine the color intensity. The intensity of the blank (area a of Figure 1B) was subtracted from the mean intensity, obtained from 7 replicates. The mean intensities were then used to plot the calibration curve for determining the amount of lead.

33 | P a g e

D. Determining the amount of Pb2+ in paint sample by stripping analysis.

Potentiostat (Eco Chemic/PGSTAT 12, Metrohm Siam Ltd.) equipped with glassy carbon (GC) working electrode, reversible hydrogen references electrode (RHE) and Pt wire auxiliary electrode was used for the analysis. [Pb2+] was determined by stripping analysis and standard addition method. 100 ÂľL of 1000 ppm Bi3+ was added to 10 mL of 0.5 M H2SO4 solution. The potential was switched to -0.1 V for 120 s to pre-plate the GC electrode. After preplating, 1 mL of acid-digested sample was added into the electrolyte. The potential was switched to -1 V for 120 s to preconcentrate the sample on Bi-coated electrode, and the stripping was performed and recorded. The series of Pb2+standard was later added to the solution before performing stripping analysis. The heights of the stripping peaks were used to plot the standard addition graph to determine [Pb2+].

RESULTS AND DISCUSSION Figure 2 shows the series of colors formed of [Pb2+] between 100 and 1000 ppm. The black color formed was easy to observe by the naked eyes. Lower concentrations were also tested but the colors formed were hard to observed and were not much different from the blank. Hence, the concentrations lower than 100 ppm were not used in the calibration. For 100 to 400 ppm, the color formed evenly over the detection zone. For 800 and 1000 ppm, however, the formation was not uniform. High intensity was observed at the edge of the paper. The effect of evaporation at the edge could play the part in this color formation at high concentration of lead. To get good reproducibility, the intensity reading was obtained from the whole area of the detection zone. Even though there appeared to be some color formation outside of the detection zone (outside of the circle), their colors were not included in the intensity reading. The formation of the color outside of the detection zone happened because of the wetting of the paper. Some amount of the standard and reagent could flow and reacted outside of the detection zone.

Figure 2. Color formation for different concentration of Pb2+ between 100 and 1000 ppm on the paper-based sensor.

For the convenience of comparison, the images from Figure 2 were cropped and arranged in Table 1. 7 replicates (circle) and 1 blank (square) were obtained from each detection zone of the template.

The values of the intensity read from the detection zone were shown in Table 2. From the reading in different replicates, the values of the intensities were close indicating good reproducibility of the sensor.

34 | P a g e

Table 1. Cropped images of the template (detection zone) from Figure 2.The images were arranged for convenient comparison. blank

Rep.1

Rep.2

Rep.3

Rep.4

Rep.5

Rep.6

Rep.7

100 ppm

200 ppm

400 ppm

800 ppm

1000 ppm

Sample A

Table 2. Intensities of the images in Table 1. The values of the intensities were obtained using Photoshop CS5 software. blank

Rep.1

Rep.2

Rep.3

Rep.4

Rep.5

Rep.6

Rep.7

Average

SD

100 ppm

0.00

2.93

2.92

2.58

1.90

2.19

2.68

2.67

2.55

0.38

200 ppm

0.00

6.28

6.21

6.26

6.42

6.47

6.46

6.24

6.33

0.11

400 ppm

0.00

12.84

12.57

12.09

12.25

12.75

11.97

12.06

12.36

0.35

800 ppm

0.00

21.83

22.60

22.51

22.45

22.49

22.57

22.75

22.46

0.29

1000 ppm

0.00

29.49

29.08

29.54

29.86

29.23

29.11

29.91

29.46

0.34

Sample

0.00

4.82

4.70

4.49

3.60

4.83

4.48

3.70

4.37

0.51

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The plot of the mean intensities versus [Pb2+] was shown in Figure 3. Good linearity was observed for the concentration between 100 to 1000 ppm (R2 = 0.9967). Although the linear range was not low to the part per billion range, this range could be used to determine [Pb2+] in paint where the allowed value was in the hundred part per million range. In Thailand, the government agency surveyed the paint in the market in 2013 and found that some well-known brands had [Pb2+] more than 10,000 ppm while the allowed [Pb2+] in paint was set at 100 ppm. The allowed [Pb2+] was different for each type of materials, and different countries had different allowed values. For example, India set the allowed [Pb2+] in paint to be 600 ppm whereas the United States of America set at 90 ppm. Yan and Erik (2012) used sulfide solution to determine [Pb2+]. Pb2+ ions formed black precipitate or colloids when they reacted with sulfide. The linear range for their work was 0.5-500 ppm. However, they needed to prepare nanoparticles and use spectrophotometer as the tool to determining the [Pb2+]. These requirements were the disadvantages of their method, especially when compared to paper-based sensor which was much sampler and lower cost.

Figure 3. Plot of [Pb2+] versus mean intensities. The values were from Table 2. Mean intensities was the average values of 7 replicates.

The developed sensor was employed for determining the concentration of lead in paint. The sample was acid digested and filtered with filter paper before used. As shown in Table 1, the color of the detection zone of the sample was clearly observed. The mean intensity (Table 2) was used to determine the concentration of lead from the calibration curve (Figure 3). The value obtained from the calibration curve was 114 ppm. This value was close to the value obtained from stripping analysis (120 ppm) indicating the validity of this sensor for the analysis of real sample.

CONCLUSIONS Paper-based sensor was developed for determining Pb2+ concentration. The template was prepared by cutting a filter paper from the ink-jet printed pattern. Na2S was used as the reagent to react with

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Pb2+ to form black precipitate on the detection zone of the paper-based sensor. The developed sensor had a linear response range of 100-1000 ppm. The sensor was employed for the analysis of [Pb2+] in color paint. The value obtained from this sensor was close to that of stripping analysis which was one of the methods frequently used for determining heavy metal concentration.

Moreover, since this sensor is low cost, simple operation and small size, many people will benefit from using this type of sensor as a test kit for the analysis of [Pb2+] in color paint.

ACKNOWLEDGEMENTS Financial supports from Mae Fah Luang University and National Research Council of Thailand 58201050011 are greatly appreciated. We also would like to thank the Scientific and Technological Instrument Center (STIC) at Mae Fah Luang University for providing electrochemical equipment used in this work.

REFERENCES Cate, D. M., Adkins, J. A., Mettakoonpitak, J. and Henry, C. S. (2015). Analytical chemistry.

87 (1), 19-41.

Jovanovski, V., Hrastnik, N. I. and Hocevar, S. B. (2015). Electrochemistry Communications.

57, 1-4.

Mercan, S., Ellez, S. Z., T端rkmen, Z, Yayla, M. and Cengiz, S. (2015). Talanta. 132, 222-227.

Soldat, D. J., Barak, P. and Lepore, B. J. (2009). Journal of Chemical Education. 86, 617-620.

Svehla, G. (1996). Vogel's Quanlitative Inorganic Analysis. pp 62-66. Longman, Singapore.

Wu, F. Y., Zhang, H., Xiao, M. and Han, B. X. (2013). Spectrochimica acta. A. 109, 221-225.

Yan, J. and Indra, E. M. (2012). Analytical chemistry 84 (14), 6122-6127.

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Development of Functional Fruits Using Composite Film Deposition of Essential Oil May Mee Zin a, Stephan Thierry Dubas a, a

The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok, Thailand.

Key words: essential oil, alginate, chitosan, biodegradable, physic mechanical properties

ABSTRACT Composite films of alginate or chitosan mixed with different amount of clay were prepared and use as controlled release membrane. Chitosan and Alginate are two class of biopolymer that are inexpensive and can be used in food related application. Bergamot essential oil was first added to the clay powder in increasing amount and mixed with either chitosan or alginate followed by an ionic crosslink step. Cationic chitosan was cross-linked with sodium sulfate and alginate films were cross-linked with calcium chloride. A series of composite films were prepared using different amount of essential oil, clay and alginate or chitosan. Physicochemical properties such as moisture content, weight loss and swelling behavior were studies. Results showed that 0.1 to 1% of essential oil, 1g to 5g of clay and 1%, 1.5% and 2% of alginate/chitosan were found to be appropriate composition for the preparation of alginate/chitosan-clay-bergamot essential oils composite films.

These films are of interest for the development of fragrance release films with tunable leaching properties.

Corresponding author e-mail Stephan.d@chula.ac.th INTRODUCTION Food is essential for all living creatures, food can be used for nutrition, prevention and treating the illness, Food includes many edible things, but now I am going to focus only on fruits.

Developments of functional fruits are interested now a day. Extraction of essential oil is very popular and very useful in many industries such as food coating. Food coating dates back to ancient times. Coating is an industrial process which consists of applying a liquid or a powder onto the surface of a product of any possible shape to convey new properties. Coating takes much different; depend on the type of industry.

The aim of an edible coating is to extend the shelf-life of fruit to prevent from disfigure and also to prevent form bacterial, to control moisture transfer, gas exchange and to provide an additional protective effect. In some cases edible coatings were not successful because edible coatings totally depend on internal gas control. Quality control must be monitored within the whole storage period.

Change of color, decay ratio, loss of firmness, fermentation and weight loss of edible film coated fruits need to be checked thoroughly.

One major advantage of using edible films and coating is several active ingredients can be incorporated and consumed with food; therefore get safety and nutritional attributes. There are different kinds of edible coating such as proteins, lipids and composite on fresh fruits and vegetables to maximize their post- harvest quality and shelf life. Before coating method was introduced the post-harvest chemical treatment was used but now a day as science and technology is developed in a large scale the preservation methods are change and entered into new era. Now coating method is very popular amongst the fruit industries and many scientists around the world making countless experiments.

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EXPERIMENTAL Preparation of composite film Clay 1g and essential oil 1g were mixed and added with 20 ml of alginate/chitosan 1% (w/v) stirred for 24 hours with magnetic stirrer. The mixture was poured on petri disc and allowed to dry for 48 hours to get dried alginate/chitosan composite film.

Alginate/chitosan crosslink composite film Dried alginate composite films were dipped in calcium chloride 1% for 2 hours to get alginate crosslink composite film.

Dried chitosan composite films were dipped in sodium citrate1% for 2 hours to get chitosan crosslink composite film.

RESULT AND DISCUSSION 5

moisture

4 3 2 1 0 140 150 160 170 180 190 200 210 220 water based ratio

Clay same ratio (0.5g), Concentration of Bergamot oil ratio (150 to 220ÂľL)

5

moisture

4 3 2 1 0 140 150 160 170 180 190 200 210 220

oil ratio

Clay same ratio (0.5g), Concentration of Liquid smoke ratio (150 to 220ÂľL) Moisture Content The amount of water present in films provides an indication of the films hydrophilicity, being the more hydrophilic films those that present the highest values of moisture content (Table 2). These are in agreement with the results obtained for the same chitosan crosslink concentrations reported

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in literature (Casariego et al., 2009). Incorporating bioactive compounds in chitosan crosslink films led to an increase of moisture content. These differences are possibly related with the hydrophilicity of the different peptides. The increase of moisture content in the films with bioactive compounds may also be related with the increase of charged molecules, which present a higher ability to adsorb water (Anker et al., 1999). Water solubility is another parameter that gives indication of the film’s water affinity. The water solubility obtained for the chitosan crosslink film (Table 2) agrees well with results reported in literature (Souza et al., 2009).

30.00% 25.00% 20.00% 15.00% 10.00% 5.00% 0.00%

DSC analyses The DSC curves of 1.5%CTS+1%CIT+1%Bergamot polymer blend. Broad exothermic peaks are observed at various temperatures indicating the crystallization of the polymer blend as well as evaporation of water and decomposition of side chain. The glass transition temperature of 1.5%CTS+1%CIT+1%Bergamot is 250 °C. On comparing the DSC curves of 1.5%CTS+1%CIT+1%Berdamot has higher glass transition temperature.

TG-DTA The TGA thermograms of 1gClay+2%Chitosan+1%Bergamot , has two degradation temperatures which the temperature lower than 100 °C indicating the loss of water. The temperature above 100 ° C indicates the decomposition of pyranose ring structure. The TG of blended polymer shows five decomposition temperatures. Maximum decomposition was observed around from 300 °C, which may be due to the decomposition of side chains.

1g Clay+2%Chitosan+1% Bergamot

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FTIR analyses After crosslinking process in the spectra of all chitosan film samples appears a new band with the hydroxyl groups (-OH) in the 3600-3000 cm-1range, while the carbon-oxygen (C-O) absorption peaks characteristics of primary and secondary alcohols were observed at 1030 and 1100cm-1, respectively. In addition, peaks for CH2–OH bending bands appeared at 1075–1000 cm-1. Bands in the 1460-1649 cm-1 range can be carboxylate group. Bands in the 1425-1583 cm-1 range can be (COO-). A peak at 776.52 cm-1 band showed CH2 rocking. The peak 692.80 cm-1 corresponds to NH bending (Pretsch et al., 2001).

CONCLUSION Application of alginate-clay-essential oil crosslink and chitosan-clay-essential oil crosslink edible coatings was able to improve the quality and shelf life of fruits during storage time at room temperature by reducing weight loss. Overall alginate-clay-essential oil crosslink (Alg:C:Eos=2:1:1%) and chitosan-clay-essential oil crosslink (CTS:C: Eos=2:1:1%) were the best coating formulation as it showed good moisture barrier and condensed heat properties.

ACKNOWLEDGEMENTS This research work was supported by the new researcher from the Thailand Research Fund (TRG 5780163) and from the petroleum and petrochemical College, Chulalongkorn University, Bangkok, Thailand.

REFERENCES Milena Pieróg, Magdalena Gierszewska-Drużyńska, Jadwiga Ostrowska-Czubenko. EFFECT OF IONIC CROSSLINKING AGENTS ON SWELLING BEHAVIOUR OF CHITOSAN HYDROGEL MEMBRANES. Volume XIV, 2009.

Arrieta, M.P., Peltzer, M.A., Garrigós, a.d.C., Jiménez, A., Structure and mechanical properties of sodium and calcium caseinate edible active films with carvacrol, Journal of Food Engineering (2012), doi: http://dx.doi.org/10.1016/j.jfoodeng.2012.09.002 A.M. El-Anany, G.F.A Hassan and F.M Rehab Ali. Journal of Food Technology. Effects of Edible Coatings on the Shelf-Life and Quality of Anna Apple (Malus domestica Borkh) During Cold Storage. Food Technology: 7 (1):5-11, 2009.

Asgar Ali a, Noosheen Zahida, Sivakumar Manickamb, Yasmeen Siddiqui c, Peter G. Aldersond, Mehdi Maqbool . Effectiveness of submicron chitosan dispersions in controlling anthracnose and maintaining quality of dragon fruit. Postharvest Biology and Technology 86 (2013) 147-153.

Azarakhsh, N, Osmen, A, Ghazali, H.M, Tan, CP and Mohd Adzahan, N. The optimization of alginate and gellan based edible coating formulations for fresh-cut pineapples. International Food research Journal 19(1): 279-285(2012).

Bartonomeu G.de S. Medeiros, Ana C. Pinheiro, Maria G-Carneiro-da-Cunha and Antonio A. Vicente. Development and characterization of a nanomultilayer coating of pectin and chitosan Evaluation of its gas barrier properties and application on 'Tommy Atkins' mangoes BMC Biotechnology - Preparation of alginate coated chitosan microparticles for vaccine delivery.

Journal of food Engineering 110(2012) 457-467.

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Chin-San Wu. A comparison of the structure, thermal properties and biodegradability of polycaprolactone/chitosan and acrylic acid grafted polycaprolactone/chitosan.journal of polymer 46(2005)147-155.

Elere-Velickora Gleonara Wiakelhaugen, Vitor D.Alven, Margoride molodas-Martins. Impact of chitosan beewax edible coatings on the quality of fresh strawberries under commercial storage conditions. LWT-food science and Technology xxx (2013) 1 to 13.

Jafarizadeh Malmiri, H., Osman, A., Tan, C.P. and Abdul Rahman, R. Development of an edible coating based on chitosan-glycerol to delay. 'Berangan' banana (Musa sapientum cv. Berangan) ripening process. Food Journal: 18 (3): 989-997 (2011).

Qiu-Ping Zhong and Wen-Shui Xia. The Physicochemical Properties of Edible and Preservative films from Chitosan / Cassara strach/ Gelatin Blend Plasticized with Glycerol. Journal of Food Technol. Biotechnol. 46(3) 262-269 (2008).

Yong Ling Song, Lei Lin, Huixing Shen, Juan You, Yong Kang, Luo. Effect of sodium algine to based edible coating containing different anti-oxidants on quality of shelf-life of refrigerated bream. Food control 22(2011) 608-615.

Z.S Hu, M-Zhang and S-Wang. The effect of high Pressure (HP) argon (AV) treatments on the quality of fresh-cut apple at cold storage. Food control journal 23 (2012) (20-127).

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Thermochromic Response of Polydiacetylene/Zinc Oxide/Natural Rubber Nanocomposite Films Wipawee Panyanuntakit a, KanoktipBoonkerd *a, Nisanart Traiphol b a Department of Materials Science, Faculty of Science, Chulalongkorn University b Research Unit of Advanced Ceramics, Department of Materials Science, Faculty of Science, Chulalongkorn University Keywords : Polydiacetylene, Natural rubber, Thermochromic ABSTRACT Polydiacetylene (PDA) is the conjugated polymer which can be prepared by UV polymerization of diacetylene (DA) monomer. PDA exhibits color transition from blue to red when subjected to external stimuli, which is of interest for sensing applications. Previous studies by one of the authors have attempted to improve properties of PDA by incorporating ZnO nanoparticles. The PDA/ZnO nanocomposite exhibits reversible thermochromism. The PDA/ZnO nanocomposite is more stable than PDA. It can be dispersed in different organic solvents and mixed with polymer matrices. In this work, we try to develop thermochromic sensing material in the form of solid film by using natural rubber (NR) as a matrix. The 10,12-pentacosadiynoic acid (PCDA) was used as a diacetylene monomer. Mixtures of the poly(PCDA)/ZnO nanocomposites in toluene and NR solution were fabricated into films by drop casting. The result showed that NR can potentially be used as a matrix to prepare poly(PCDA)/ZnO nanocomposite sensor films. This was not only because the color transition of the poly(PCDA)/ZnO nanocomposites embedded in the natural rubber still can clearly be observed by naked eyes but their reversible thermochromic behavior still also exists. Moreover, the poly(PCDA)/ZnO/natural rubber nanocomposite films exhibit elastic and flexible properties, which can extend the utilization of the material in various applications. * k.boonkerd@gmail.com INTRODUCTION The development of sensing technology from polymer has received much more attention in worldwide. Polydiacetylene (PDA) is one of the conjugated polymers that can exhibit colorimetric response after applying external stimuli such as temperature or solvent. PDA is produced by the UV irradiation of self-assembled diacetylene (DA), without the initiators or catalysts. Due to the backbone possessing long conjugation length, PDA exhibits a deep blue color with the maximum from blue to purple or red, depending on degree of perturbation. One of the proposed mechanisms for color transition of PDA due to the external stimuli is the change of molecular conformation of side chain and backbone affecting the electronic states of PDA, thus causing the change of its optical absorption properties (Patlolla et al., 2009). Although the color change of PDA can be clearly seen by naked eye, its thermochromic bahavior is an irreversible process (Ahn et al., 2009).

Several studies have attempted to improve the thermochromically reversible property of PDA based on the addition of metal oxide or metal nanoparticles into the system. Zinc oxide (ZnO) is one of metal nanoparticles used as a nano-substrated for DA self-assembled to prepare PDA/ZnO nanocomposites. It was reported that by comparison with the pure PDA, the PDA/ZnO nanocomposites are more stable, have high color stability, and show color transition at higher temperature. Moreover, the thermochromic bahavior of PDA/ZnO nanocomposites is reversible which is important for the development of sensing technology (Rungruangviriya at el., 2010). The

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thermochromic behavior of PDA/ZnO nanocomposites exhibits two-steps reversible and irreversible color transition. First step of reversible color transition is blue-to-purple and the second step of reversible color transition is purple-to-red. Furthermore, PDA/ZnO nanocomposites can be dispersed in various organic solvents while they still remain blue color and color transition property. From this advantage, PDA/ZnO nanocomposites can be fabricated into the thin films by mixing with different polymer matrices such as polystyrene, poly(methyl methacrylate) and polyethylene (Toommee at el., 2015). Although PDA/ZnO nanocomposites immobilized on the polymer still preserved color transition and reversible thermochromics behavior, the crack of film was found. Therefore, the quality of the film needed to be further improved. Searching the new polymer that can suitably be used as a matrix to prepare the PDA/ZnO nanocomposite sensor film is challenged.

For this study, we are interested to use natural rubber (NR), cis-1,4-polyisoprene, as a matrix. The reason behind the selection of NR is not only the abundant availability, but also its properties including high elasticity, flexible and mechanical strength.

EXPERIMENTAL A. Materials The 10,12-pentacosadiynoic acid (PCDA) used as a diacetylene monomer in this study was purchased from Fluka. The ZnO nanoparticles with diameter in the range of 20-200 nm were commercially available at Nano Materials Technology, Thailand. All of the organic solvents (AR grade) were purchased from RCI Labscan Co., Ltd. Natural rubber (STR-5L grade) was purchased from Tana Para Co., Ltd. and used as obtained.

B. Preparation of poly(PCDA)/ZnO nanocomposites The PCDA monomers were dissolved in chloroform and then filtered using 0.45 μm pore size nylon membrane to remove polymerized molecules. Then, the PCDA solution was heated at about 50ºC to slowly evaporate chloroform. The ZnO nanoparticles were dispersed in deionized water by using probe sonication for 10 min. The ZnO suspension was added into the purified PCDA monomers. The concentration of PCDA monomers was kept at 0.5 mM, whereas the ratio of ZnO/PDA was fixed at 10 wt.%. The suspensions were sonicated at about 80ºC for 60 min. The suspension was kept at ∼4ºC for ∼24 h to allow the self-assembling. The PCDA/ZnO nanocomposite was polymerized by UV light irradiation (λ∼ 254 nm, 10 W) for 5 min. The obtained poly(PCDA)/ZnO nanocomposite aqueous suspension was dried at 45ºC for 48 h.

C. Preparation of poly(PCDA)/ZnO/natural rubber nanocomposite films To prepare poly(PCDA)/ZnO/natural rubber nanocomposite films, natural rubber (NR) was first dissolved in toluene. The NR solution was then filtrated using a stainless sieve to remove the NR gel. At the meantime, the poly(PCDA)/ZnO nanocomposites were dispersed in toluene. The poly(PCDA)/ZnO nanocomposite suspension and NR solution were mixed at 1:1 and 1:2 v/v ratios and stirred until the mixtures became homogeneous. The mixtures were dropped on glass slide and left to dry in ambient conditions.

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D. UV/vis absorption characterization UV/vis absorption spectra of poly(PCDA)/ZnO nanocomposite suspension, poly(PCDA)/ZnO /NR nanocomposite suspension and poly(PCDA)/ZnO/NR nanocomposite film were measured using Analytik Jena Specord S100.

E. Color photographs of poly(PCDA)/ZnO/natural rubber nanocomposite films To study the colorimetric response to temperature of poly(PCDA)/ZnO/NR nanocomposite film, photographs of the obtained nanocomposites film heated in an air oven from 35ยบC to 200ยบC in a step-wise process, followed by consecutive cooling to room temperature were taken.

RESULTS AND DISCUSSION A. Optical properties For this study, we are interested in developing polydiacetylene-based sensor into the solid thin film by using natural rubber (NR) as a matrix. Therefore, the influence of NR on optical properties of poly(PCDA)/ZnO nanocomposites was first discussed. Fig. 1 shows the absorption spectra of poly(PCDA)/ZnO nanocomposites in different environments. As seen in Fig. 1, poly(PCDA)/ZnO nanocomposites in toluene exhibits a maximum absorption peak of blue phase at 643 nm with broad shoulder peak at 588 nm and a low-energy band at about 675 nm. When poly(PCDA)/ZnO nanocomposites in toluene was added into the NR solution at 1:1 and 1:2 v/v ratios, the mixtures were still blue. No agglomeration of poly(PCDA)/ZnO nanocomposites was observed. From Fig. 1, it was clearly seen that the absorption peak of poly(PCDA)/ZnO nanocomposites now surrounded with NR chains was comparable to that of poly(PCDA)/ZnO nanocomposites in toluene. Therefore, it can be inferred that for the suspension state, the presence of NR chains did not disturb the conjugation length of poly(PCDA) in the nanocomposites. Next, the absorption characteristic of poly(PCDA)/ZnO/NR nanocomposite film was discussed. As mentioned earlier, fabrication of polydiacetylene-based sensor to thin film sensor is a key to widen its application. However, it is essential to prepare the sensor film in its blue-phase before an enviornamental perturbation is applied. In this work, the films were obtained by drop casting poly(PCDA)/ZnO nanocomposite suspension in the NR solution at 1:1 and 1:2 v/v ratios on glass slides. It was observed that the obtained poly(PCDA)/ZnO/NR nanocomposite films were in blue. To confirm that the use of NR as a matrix did not disturb the color stability of poly(PCDA)/ZnO nanocomposites, the absorption peak of poly(PCDA)/ZnO/NR nanocomposite film was determined and as shown in Fig. 1. The result showed that the absorption peak of poly(PCDA)/ZnO nanocomposites embedded in the NR solid matrix was similar to that of the poly(PCDA)/ZnO nanocomposites in toluene. Changing the environment from toluene to both NR solution and NR solid matrix did not change the optical property of poly(PCDA)/ZnO nanocomposites. Therefore, it can be concluded that the NR can be a good candidate to fabricate the poly(PCDA)/ZnO nanocomposites sensor film.

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Fig. 1 Absorption spectra of (a) poly(PCDA)/ZnO nanocomposites in toluene (b) 1:1 v/v poly(PCDA)/ZnO nanocomposites in NR solution (c) 1:2 v/v poly(PCDA)/ZnO nanocomposites in NR solution (d) 1:1 v/v poly(PCDA)/ZnO/NR nanocomposites film and (e) 1:2 v/v poly(PCDA)/ZnO/NR nanocomposites film.

B. Thermochromisms in NR matrix It was reported that the color of the dried poly(PCDA)/ZnO nanocomposites deposited on a glass slide changed from blue to purple and purple to red at about 80 and 160 ºC, respectively (Chanakul, A., 2013). Temperatures at which the dried poly(PCDA)/ZnO nanocomposites showed partial color reversibility and color irreversibility were about 160 and 180 ºC, respectively. To study thermochromic behavior of poly(PCDA)/ZnO nanocomposites embedded in the NR matrix, the obtained films prepared from poly(PCDA)/ZnO nanocomposites in NR solution at 1:1 and 1:2 v/v ratios were heated from 35 to 200 ºC in an air oven, then followed by cooling to room temperature.

The color transition of poly(PCDA)/ZnO/NR nanocomposite films are exhibited in Fig. 2. In general, even though consisting of different poly(PCDA)/ZnO nanocomposite loadings, both films showed the blue-to-purple and purple-to-red color transition at almost the same temperature which were at about 80 and 180 ºC, respectively. Moreover, both films only showed the color irreversibility when subjected to temperature at about 180 ºC or higher. The difference in thermochromic behavior between these two films was temperature exhibiting partial color reversibility. The poly(PCDA)/ZnO/NR nanocomposite film consisted of higher poly(PCDA)/ZnO nanocomposite loading showed partial color reversibility at 160-180 ºC which was higher temperature than that of the one consisted of lower poly(PCDA)/ZnO nanocomposite loading.

Regardless of poly(PCDA)/ZnO nanocomposite loading, it was found in this study that the temperature at which the poly(PCDA)/ZnO/NR nanocomposite film exhibited the blue-to-purple transition was similar to that of the poly(PCDA)/ZnO nanocomposite. However, the temperature at which the poly(PCDA)/ZnO/NR nanocomposite film exhibited the purple-to-red transition and color irreversibility was 20 ºC higher than those of the poly(PCDA)/ZnO nanocomposite.

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Fig. 2 Color photographs of (A) 1:1 v/v poly(PCDA)/ZnO/NR nanocomposites films (B) 1:2 v/v poly(PCDA)/ZnO/NR nanocomposites films were taken at different temperatures and also after cooled down to room temperature.

CONCLUSIONS The fabrication of poly(PCDA)/ZnO nanocomposites into the solid films using NR as a matrix was successfully carried out by a drop casting method. Since the most important property of polydiacetylene-based sensor is its unique blue-to-red transition taking place upon stimulation, it is essential to prepare the sensor in blue phase before external stimulation is applied. The result from this study showed the NR was a good material to use as a matrix for preparing the poly(PCDA)/ZnO nanocomposite sensor film. The poly(PCDA)/ZnO/NR nanocomposite film was blue. No agglomeration of poly(PCDA)/ZnO nanocomposites in the NR matrix was observed.

Moreover, the films almost showed thermochromatic bahavior similar to that of the poly(PCDA)/ZnO nanocomposites.

ACKNOWLEDGEMENTS The study was supported by Special Task Force for Activating Research (STAR), Ratchadaphiseksomphot Endowment Fund, Chulalonhkorn University.

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REFERENCES Patlolla, A.; Wang, Q.; Frenkelc, A.; Zunino, J.; Skelton, D. and Iqbal, Z. (2009) Material Research Society Proceedings, 1190.

Ahn, D. J.; Lee, S. and Kim, J. (2009) Adv. Funct. Mater. 19, 1483-1496.

Rungruangviriya, N. and Traiphol, N. (2010) J. Metals, Materials and Minerals, 20, 35-41.

Traiphol, N.; Rungruangviriya, N.; Potai, R. and Traiphol, R. (2011) J. Colloid Interface Sci. 356, 481-489.

Chanakul, A.; Traiphol, N. and Traiphol, R. (2013) J. Colloid Interface Sci. 389, 106-114.

Toommee, S.; Traipholb, R. and Traiphola, N. (2015) Colloids and Surfaces A: Physicochem.

Eng. Aspects, 468, 252–261.

Chanakul, A. (2013) Doctoral dissertation, Department of Materials Science, Faculty of Science, Chulalongkorn University

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Influence of Phosphorus on the Fusing Temperature of Nickel-base Self-fluxing Alloy Masaaki Inazumi a, Toru Maruyama a a

Graduate School of Science and Engineering, Kansai University, Japan Keywords : Thermal spray coating, fusing treatment, nickel-base self-fluxing alloy

ABSTRACT Nickel-base self-fluxing alloys are used thermal spray material to coat a surface of wear resistance parts, such as and steelmaking rolls. After thermal spraying of the alloy, a treatment that is called fusing to remelt coatings is performed. In the treatment, the coating becomes dense and adheres strongly to the substrate, and wear resistance is improved due to distribution of hard compounds in the coating. However, because fusing treatment is performed at a high temperature of 1050ยบC, it is concerned about physical changes like melting and thermal deformation of the substrate. Therefore, in order to perform the fusing treatment without the physical change, it is necessary to decrease the fusing temperature. From a binary phase diagram of nickel-phosphorus alloy, it is known that the solidification temperature of nickel is decreased by alloying with phosphorus. In this study, influence of addition of phosphorus on the fusing temperature of nickel-base self-fluxing alloy was investigated. As a result, the solidification temperature range of nickel-base self-fluxing alloy was decrease with phosphorus content. It was revealed that the liquidus temperature of nickel solid solution of the alloy of 3.0mass%P is decreased by about 100ยบC compared with the phosphorus-free alloy. *k918352@kansai-u.ac.jp INTRODUCTION A steelmaking roll is damaged by wear and oxidation due to contact with hot ingot. For cost reduction related to replacement of the rolls, the long-life of the rolls is required. In the methods for achieving the long-life of the roll, there are various methods such as changes in operating conditions and equipment. As a method for achieving the long-life of the roll at low cost, there is a surface treatment for changing the surface condition of the rolls. Thermal spray coating of the nickel-base self-fluxing alloy is used for one of the surface treatment. After thermal spraying of the alloy, a treatment that is called fusing to remelt nickel solid solution in coatings is performed. In the treatment, the coating becomes dense and adheres strongly to the substrate, and wear resistance of the coating is improved due to distribution of hard compounds in the coating. However, because fusing treatment is performed at a high temperature of about 1050ยบC, it is concerned about physical changes like melting and thermal deformation of the substrate. Therefore, in order to perform the fusing treatment without the physical change, it is necessary to decrease the fusing temperature.

From a binary phase diagram of nickel-phosphorus alloy, it is known that the solidification temperature of nickel is decreased by alloying with phosphorus. In this study, influence of addition of phosphorus on the fusing temperature of nickel-base self-fluxing alloy was investigated. And, distribution of the alloying elements using EPMA and hardness of the alloy also were investigated.

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EXPERIMENTAL The solidification temperature of the nickel solid solution of nickel-base self-fluxing alloy containing phosphorus was calculated by thermodynamic calculation using CALPHAD method.

SGTE2011 was used in the database for the calculation. The solidification temperature range of the nickel solid solution was measured from cooling curve of melting alloy. The nickel-base selffluxing alloy of JIS-NiCrCuMoBSi 69 15 3 3A and Ni-13mass%P alloy were used as raw materials for the melting alloy. Table 1 is shown the chemical composition of the raw materials. The specimens with phosphorus in content of 0.3, 1.5, and 3.0mass% was prepared by using the raw materials. These raw materials were melted in an electric furnace by heating to 1400째C in argon atmosphere, and the melted alloy was cooled in the furnace. Temperature of the alloy during solidification was measured during cooling for thermal analysis. Distribution of elements in the alloy after solidification was measured using EPMA. Hardness of the alloy was measured by vickers hardness tester with a load of 10kgf. Five times measurement were performed to evaluate an average value.

RESULTS AND DISCUSSION A. Calculated phase diagram Fig. 1 shows a calculated phase diagram of isocompositional section of Ni-P-3.50%B-4.30%Si-0.8%C16.5%Cr-3.00%Fe-3.00%Cu3.00%Mo alloy(% is mass%). A blue part in Fig. 1 is the solid-liquid coexistence temperature range of nickel solid solution of nickel-base self-fluxing alloy. From Fig. 1, the liquidus temperature was gradually decreased with increasing phosphorus content. The solidus temperature was drastically decreased by over 100째C with increasing phosphorus content by 0.01mass%P. And, the temperature was gradually decreased with increasing phosphorus content over 0.01mass%. From this result, it was suggested that phosphorus contributes to decreasing of the fusing temperature.

B. Thermal analysis curve and EPMA (the P-free alloy)

1600

1065℃

1200

1192℃

1000 0 -0.2 -0.4 -0.6 -0.8 0.02 0.01 0 -0.01 -0.02 0.9

977℃

2

dT/dt, K/s

b) nM=11

nD=21

2

2

dT /dt , K/s

2

r

a) (n=2sec.)

954℃

1400

nM=11

c)

nD=21 nDD=21

Temperature, K

Fig. 2 shows thermal analysis curves of a nickel-base selffluxing alloy. Fig. 3 shows thermal analysis curves of nickelbase self-fluxing alloy containing 3.0mass%P. The (a) in Fig. 2 and Fig. 3 show temporal change in temperature. The (b) in Fig. 2 and Fig. 3 show relation between time and temporal change in differential of temperature between a defined interval. The (c) in Fig. 2 and Fig. 3 shows relation between time and temporal change in differential of temperature between a defined interval. The (d) in Fig. 2 and Fig. 3 show temporal change of contribution rate in the differential (a) in Fig. 2 and Fig. 3. From Fig. 2, it was found that these are four phase transformations in the P-free alloy.

However from Fig. 3, it was found that these are five phase transformations in the 3.0mass%P alloy. Fig. 4 shows element distribution of in the P-free alloy measured by EPMA. From Fig. 4, it was found that the boride consisting of chromium, molybdenum, and boron and the carbide consisting of chromium and carbon are existed. The boride was surrounded by the dendrite-like structure shown in Fig 4(1) consisting of nickel, iron, copper, and chromium. The dendrite-like structure was surrounded by the structure shown in Fig. 4(2) consisting of nickel and boron. The

Temperature, K

50 | P a g e

d) nM=11

0.95

nD=21

1 0

500

Time, s

1000

Fig. 2 Thermal analysis curves of nickel-base self fluxing alloy. a) Relation between time and temperature b) Relation between time and dT/dt, K/s 2 2 2 c) Relation between time and dT /dt , K/s d) Relation between time and r 2

r

2

2

2

dT /dt , K/s

2

dT/dt, K/s

Temperature, K

structure shown in Fig. 4(2) was surrounded by the structure 1600 a) shown in Fig. 4(3) consisting of nickel and silicon. From Fig. 970℃ 954℃ 905℃ (n=2sec.)

1400 1 and Fig. 4, it is considered that the peak of latent heat 818℃ 1200 1113℃ release of 1192°C in Fig. 2 showed crystallization of the 1000 boride and the carbide. According to Fig. 1, it was found to 0 nM=11 b) crystallize boride follow by carbide from liquid phase around -0.5 1200°C. However, the only one peak of latent heat release at nD=21 around 1200°C of thermal analysis curve in Fig. 2 was found.

-1 c) 0.02 nM=11 Therefore, because supercooling occurs, it is suggested that 0 crystallization of these two compounds were detected as one nD=21 peak. It is considered that the peak of latent heat release of -0.02 nDD=21 1065°C in Fig. 2 showed crystallization of the dendrite-like 0.9 d) structure shown in Fig. 4(1). It is suggested that the dendritenM=11 0.95 nD=21 like structure was crystallized on the boride as nucleus. It was suggested that the peak of latent heat release of 977°C in Fig. 1 0 500 1000 2 showed a eutectic reaction of the phases shown in Fig. 4(1) Time, s Fig. 3 Thermal analysis curves of nickel-base and Fig. 4(2). These two phases are adjacent to the dendriteself fluxing alloy containing 3.0mass%P. like phases. Therefore, it is considered that the eutectic a) Relation between time and temperature b) Relation between time and dT/dt, K/s reaction of these two phases was occured after crystallization 2 2 c) Relation between time and dT /dt , K/s 2 2 of dendrite-like phase. It is suggested that the peak of latent d) Relation between time and r heat release of 954°C in Fig. 2 showed a ternary eutectic reaction of the phases shown in Fig. 4(1), Fig. 4(2), and Fig. 4(3). These three phases are adjacent to the eutectic phase which is considered to be crystallized at 977°C. These three phases are

51 | P a g e

less part adjacent to the dendrite-like phase shown in Fig. 4(1). Therefore, it is considered that these three phases is crystallized after the eutectic phase which is considered to be crystallized at 977°C. Hirose et al. performed thermal analysis of Ni-15.0% Cr-3.0% B-4.0% Si-0.7% C-3.2% Fe alloy(% is mass%). As a result, firstly, M2B was crystallized at 1277°C. Secondly, M7C3 was crystallized at 1257°C. Thirdly, nickel solid solution was crystallized in the dendrite at 1027°C. Subsequently, nickel solid solution and M2B was crystallized as the eutectic phase at 977°C, Finally, nickel solid solution, Ni3(C, B), and Ni3Si was crystallized as the eutectic phase at 947°C, and solidification of the alloy was finished. They were reported as described above. Akebono et al. performed thermal analysis of Ni-17.0%Cr-3.3%B-4.2%Si-0.9%C-4.0%Fe-3.0%Cu-2.0%Mo alloy(% is mass%). As a result, firstly, CrB was crystallized at about 1420°C. Secondly, Cr7C3 was crystallized at about 1300°C. Thirdly, nickel solid solution was crystallized in the dendrite at about 1050°C. Subsequently, nickel solid solution and Ni3B eutectic was crystallized as the eutectic phase at about 990°C. Finally, nickel solid solution, Ni3B, and Ni3Si was crystallized as the eutectic phase at about 980°C, and solidification of the alloy was finished. They were reported as described above. The results of this study shown in Fig. 2 and Fig. 4 are the same sort of results as their reports. Therefore, it was suggested that the alloy was crystallized in the order of boride and carbide, nickel solid solution, nickel boride, and nickel silicide.

C. Thermal analysis curve and EPMA (the 3.0mass%P alloy) From Fig. 5, it was found that boride, carbide, and dendrite-like structure shown in Fig. 5(1) was existed in the alloy containing 3.0mass%P. It is considered that these structures in the alloy containing 3.0mass%P is the same structure as the P-free alloy. By the addition of phosphorus, it was found that the structure consisting of nickel and phosphorus shown in Fig. 5(2) and the structure consisting of nickel, silicon, and phosphorus shown in Fig. 5(3) were existed. From Fig. 1 and Fig. 5, it was suggested that the peak of latent heat release of

52 | P a g e

1113°C in Fig. 3 showed crystallization of the boride and the carbide. Therefore, because supercooling occurs as the P-free alloy, it is considered that crystallization of these two compounds were detected as one peak. It was suggested that the peak of latent heat release of 962°C in Fig. 3 was showed crystallization of the dendrite-like structure shown in Fig. 5(1). As the P-free alloy, it was suggested that the dendrite-like structure was crystallized on the boride as nucleus. However, the remaining peak in Fig. 3 is not revealed whether the peak that corresponds to which phase because the structure in the 3.0mass%P alloy was more complex than the P-free alloy. Further analysis of the alloy containing 3.0mass%P is required.

D. Relationship between each transformation temperature of nickel-base self-fluxing alloy and phosphorus content.

Fig. 6 shows a relationship between each transformation temperature of nickel-base self-fluxing alloy and phosphorus content. From Fig. 6, the four phase transformations in the 0.3mass%P alloy and the 1.5mass%P alloy were found. The five phase transformations in the 3.0mass%P alloy were found. Because phosphorus content in these alloys is less than the 3.0mass%P alloy, it is considered that the phase transformation in 818°C in Fig. 3 was not detected. According to Fig. 6, each transformation temperature was gradually decreased with increasing phosphorus content. Therefore, it was suggested that the addition of phosphorus to the alloy was contributed to decrease of the transformation temperature. While the liquidus temperature of the phase containing nickel of the P-free alloy was 1065°C, that of the 3.0mass%P alloy was 970°C. From these results, it is suggested that the fusing temperature can be decreased to 970°C below.

53 | P a g e

E. Relationship between vickers hardness of the alloy and phosphorus content.

Fig. 7 shows a relation between vickers hardness of nickel-base self-fluxing alloy and phosphorus content.

The hardness of the alloy was increased with increasing phosphorus content. According to Fig. 4, phosphorus was existed with nickel and silicon in the alloy. Therefore, it is suggested that the hardness of the alloy was increased by solid solution hardening of phosphorus.

CONCLUSIONS Influence of phosphorus on the property of the nickel-based self-fluxing alloy was investigated.

The results are as follows: (1) It was found that the boride consisting of boron, Chromium, and molybdenum, the carbide consisting of chromium and carbon, nickel solid solution, nickel boride, and nickel silicide was existed in the nickel-base self-fluxing alloy.

(2) Phosphorus was existed with nickel, or nickel and silicon in the alloy.

(3) Each transformation temperature was gradually decreased with increasing phosphorus content.

(4) While the liquidus temperature of the phase containing nickel of the P-free alloy was 1065째C, that of the 3.0mass%P alloy was 970째C. It is suggested that the fusing temperature can be decreased to 970째C below.

(5) The hardness of the alloy was increased with phosphorus content because of solid solution hardening of phosphorus.

REFERENCES

Hirose, M et al, (2005), Development of Metal Lining Steel Pipes Using Centrifugal Method and Induction Heating for Wear and Corrosion Resistance, Reports of Fukuoka Industrial Technology Center, 78-81.

Akebono H et al, (2004), Fatigue Properties of Thermally Sprayed Steel with Ni-Based SelfFluxing Alloy and Its Controlling Factor, Doctoral Dissertation, Keio university.

54 | P a g e

Life Science

55 | P a g e Anthelmintic Potential of Psidium guajava against Infective Larvae of parasitic Nematode Nik Ahmad Irwan Izzauddin Nik Him a*, Yeh Jian Wen a, Shaida Fariza Sulaimana, Rahmad Zakariaa a*

School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang Keywords: Ethnoveterinary, parasitic nematode, Psidium guajava, anthelmintic

ABSTRACT Parasitic nematode infection in livestock animals is a global issue. The use of anthelmintic drugs not only kills the gastrointestinal worms but also contribute to emergence of drug resistance.

Anthelmintic resistance is one of the problems that normally occur in the livestock industries.

Because of that this study was carried out to determine the phytochemical constituents of Psidium guajava and to investigate the potential use of it leave and peel extracts against infective stage of parasitic nematodes. In-vitro study of leaves and peel water extract shows that both extract significantly reduced the motility and inhibit the migration of the infective larvae at all concentrations tested (5, 10, 20, 50 and 100 mg/ml)(p<0.05). Results suggested the presence of flavanoids, tripertenes, steroids, alkaloids in leave water extract and addition tannins and saponin in the peel water extract might contribute to the efficacy of the extract against parasitic nematodes. *nikirwan@usm.my INTRODUCTION Parasitic nematodes infection in livestock animals is one of the major causes for the loss in livestock industries. It has been reported to be occurred worldwide. The indication of the infection such as diarrhea, decrease in milk yield, poor reproductive performance and poor feed utilization will bring to sudden death to the animals. The most common method to control the infection is through the use of the anhelmintic drugs. The heavy usage of anthelmintic drugs to kill the parasite not only minimise the infection but also create the nematode population that resistant to the treatment which lead to the requirement of the higher dose. In Malaysia, many studies had reported the emergence of resistance towards more than two anthelmintics from different drug groups (Chandrawathani et al., 2013). In such a situation, the alternative methods of parasitic nematode control such as rotational grazing or herbal remedies was suggested can be used to reduce the helminthiasis problem. Psidium guajava, commonly known as guava previously has been used as traditional medicine. It is used in folk medicines as therapeutic agent for the treatment of numerous diseases in East Asian and other countries. It had proven that Psidium guajava has antidiabetic effect (Oh et al., 2005) and antiinflammatory effect (GutiĂŠrrez et al., 2008). Recent study has shown that a hexane fraction of Psidium guajava leaves also can induces anticancer activity (Ryu et al., 2012). In addition, a study by Ismail et al. (2012) showed that the butanolic extract of leaves of Psidium guajava caused paralysis and subsequently death in Indian earthworm (Pherithima posthuma). Interestingly, the result had also revealed that when the extraction is increased in concentration, the butanolic extract of Psidium guajava is more potent than reference control piperazine citrate. Looking into this possibility, this study was carried out to evaluate the effectiveness of Psidium guajava water extracts against parasitic nematode in-vitro study.

56 | P a g e

EXPERIMENTAL A. Sample collection and preparation of aqueous extract Sample of Psidium guajava leaf and fruit was obtained from Tropical Fruit Farm in Penang (5.414716N, 100.2170E). Both leaves and fruits were washed with running tap water and left to dry in an oven at 40°C. The leaves were left to dry in the oven approximately for 2 days. 50 g of the ground plant material was dissolved in 250ml distilled water in a conical flask. The conical flask was kept for 24 hours at 50°C. After 24 hours, filtered the dissolved plant material and centrifuged at 2500 rpm.

The aqueous filtrate was kept in a sterile bottle and kept in refrigerator at 4°C.Stock solution of different concentrations (5 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml and 100 mg/ml) were prepared for both Psidium guajava leaves and peels.

B. Phytochemical constituents test Test for alkaloids Dissolved 2 g of iodine and 6 g of potassium iodide into 100 ml of distilled water to prepare Wagner’s reagent. 2 drops of Wagner’s reagent was added into the aqueous extract. Reddish brown precipitate shows the presence of alkaloids (Raaman, 2006).

Test for flavonoids 1 ml of 10% ammonia was added into 1 ml of aqueous extract. Formation of yellow coloration indicates the presence of flavonoids (Raaman, 2006).

Test for saponins 1 ml of extract solution was mixed with 20 ml of distilled water and was shook in a graduated cylinder for 15 minutes. Development of stable foam shows the presence of saponins (Devmurari & Jivani, 2010).

Test for tannins 1 ml of 5% ferric chloride solution was added into 1ml of warm aqueous extract. Dark green or dark blue colour developed shows the presence of tannins (Devmurari & Jivani, 2010).

Test for tripertenes and steroids 1 ml of aqueous extract was dissolved into 1 ml of chloroform, added with 1 ml of concentrated sulfuric acid. Formation of reddish brown at interface shows the presence of terpenoids (Ayoola et al., 2008).

C. Faecal culture To obtain the larvae for the test, fresh sample of faeces were collected from the goat‟s farm located in Kampung Tobiar, Penang. All fresh samples were collected directly after defecation of goats. Samples were then stored in the ice box and brought to the lab for further analysis. In the laboratory, the faeces were used to estimate the number of egg per gram by using McMaster counting techniques to determine the level of infection. The remaining faeces samples were then used for larvae culture as described by Harada and Morri (1955). The test tubes were incubated at room temperature for 7- 10

57 | P a g e days. The L3 stages were collected and transferred into a 15 ml conical flask and kept in refrigerator prior to use.

D. Motility assay For the motility assay, a little modification of techniques described by Stepek et al (2005) was applied. The infective larvae were incubated in the aqueous extract of leaves and peels of Psidium guajava at elevated concentrations (5, 10, 20, 50 and 100 mg/ml). Five L3 were randomly selected and were transferred 1 ml distilled water in a 24-well test plate. 1 ml of Psidium guajava leaves at different concentrations were added into each well and left to incubate at 27°C for 24 hours. The motility of L3 nematode larvae were recorded for 24h to 48 hours. Distilled water was used for positive control. The experiment was repeated for 3 times.

E. Larvae migration inhibition assay To test the migration of larvae, standard method as described by Molan et al. (2000) was used with a little modification. 5 L3 larvae were placed in 1 ml distilled water in a 24-well test plate. 1 ml of Psidium guajva leaves at different concentrations (5 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml and 100 mg/ml) were added into each well and left to incubate at 27°C for 2 hours. After 2 hours of incubation, the incubated L3 larvae were transferred onto muslin cloth (34 μm) and left to incubate for another 2 hours at 27°C to enable the active larvae to migrate through the muslin cloth for counting.

The number of L3 larvae which had migrated through the muslin cloth was counted under the dissecting microscope. 2 replicates were run for each concentration with negative and positive control.

100% methanol was used for negative control, while distilled water was used for positive control. The experiment was repeated for 3 times.

RESULTS AND DISCUSSION A. Prevalence of parasitic nematodes infection McMaster technique has been applied to determine the level of parasitic infection in goats farm in Kampung Tobiar, Penang. From the analysis, the number of egg was recorded higher at 5958 epgs.

According to Hutchinson (2003), the epgs more than 500 are considered as highly infected and the whole farm required treatment. Early analysis shows that the requirement of the treatment in this farm to reduce the parasitic burden in animals. A total of 50 larvae were collected randomly after 7 days of incubation. In this study, there are 4 nematode species found which are Haemonchus contortus, Oesophagostomum sp., Cooperia sp. and Trichostrongylus sp. The most abundant nematode species in this study was H. contortus, which recorded 62%±0.58 and followed by Oesophagostomum sp. which has 28% ±0.58. Cooperia sp. and Trichostrongylus sp. has 8% ±0.58 and 2% ±0.58 respectively. H. contortus can be identified by its narrowly rounded head and medium length of tail.

Oesophagostomum sp. has broad and rounded head and filamentous tail. Cooperia sp. has squared head and medium length of tail while Trichostrongylus sp. has tapered head and short cone tail.

B. Phytochemical constituents test The phytochemical constituent tests were carried out and it shows that the leaves of P. guajava contain flavonoids, tripertenes and steroids, and alkaloids. While for peel of P. guajava, it contains flavonoids, tripertenes and steroids, alkaloids, tannins and saponins. Results on phytochemical study were shown in Table 1.

58 | P a g e Table 1: The result of phytochemical constituent test for both leaves and peel of Psidium guajava Test Flavonoids Tripertenes and Steroids Alkaloids Tannins Saponins Presence (+) Absence (-)

Psidium guajava leaves + + + -

Psidium guajava peel + + + + +

C. Psidium guajava significantly reduced the motility of the L3 After 24h, there was a significant reduction of the motility of the L3 compared to control in the Psidium guajava leaf and peel extracts. At 5 mg/ml of Psidium guajava leaf extract, the motility of nematode larvae were reduced from 44± 0.24% at 24 hours incubation to 0% at 48 hours of incubation. For 10 mg/ml, the motility of nematode larvae also shown to be reduced from 24 ± 0.18% at 24 hours incubation to 0% at 48 hours incubation. While for 20 mg/ml, the motility of nematode larvae also shown to be reduced from 9 ± 0.24% at 24 hours incubation to 0% at 48 hours incubation.

However, at 50 mg/ml and 100 mg/ml of Psidium guajava leaf extracts, 24 hours of incubation was enough to kill all tested L3 nematode larvae (Figure 1). The same results were observed for the peel extract (Figure 2).

Figure 1: The effect of the aqueous extract of Psidium guajava leaves on larval motility assay. Results represented the percentage of mean ± standard error of larvae motility based on 3 separated experiments. *p <0.05 = significant

Figure 2: The effect of the aqueous extract of Psidium guajava peels on larval motility assay. Results represented the percentage of mean ± standard error of larvae motility based on 3 separated experiments.

59 | P a g e *p <0.05 = significant D. Migration of L3 were inhibited in different concentrations of extract After 24h incubation, the migration of the L3 was shown to be inhibited in all concentration of the leaves extract tested (Figure 3). The same observation was observed for the peel water extract.

However the effect of leaves water extract is greater compare to the peel. The reduction of the migration was significantly different compare to the control group (p<0.05).

Figure 3: The percentage migration of nematode larvae against different concentration of Psidium guajava leaves and peel. Distilled water was used as positive control and methanol was used as negative control. Graph represented the percentage of mean Âą standard error of larvae migration based on 3 different experiments. *p<0.05 = significant Based on this assay, the leaves and peel water extracts reduced the motility and inhibits the migration of active L3 stage in-vitro which suggesting that this plant contained ethnoveterinary properties.

Anthelmintic properties maybe due to the synergetic effects of the active phytochemical constituents such as alkaloids, saponins, tannins, flavonoids, tripertenes and steroids that are present in the leaves and peel water extract. All these phytochemical constituent previously was shown to contain anticancer and anti-inflammatory activity (GutiĂŠrrez et al., 2008; Ryu et al., 2012). Study on tannins suggested that the mode of action of tannins as anthelmintic is attributed to their capacity to bind to some proteins of the metabolism or larva's organs and muscles causing a change in their functions and resulting in the paralysis or death to the L3 larvae (Wahab et al., 2010). Whereas, for flavonoids is known for its antioxidant properties by uncoupling the oxidative phosphorylation and hindering the energy production in helminths (Higdon, 2005). According to Wagner (1999), alkaloids exhibit the ability to inhibit or to activate enzyme which destroy enzymes that involved in the breakdown of fats and carbohydrates. It also affects nucleic acid and protein synthesis. It might explain the reduction of the motility and migration of the L3. However, further research is needed to confirm and to investigate the exact mechanism on how these extract effects the fitness of the L3.

60 | P a g e CONCLUSIONS In this study, Psidium guajava can be seen as potential source for the treatment of parasitic nematode in ruminants. The work so far has demonstrated significant in-vitro anthelmintic activity of water extract of leaves and peel of Psidium guajava. In-vivo study should be carried out to investigate the full potential of Psidium guajava as an alternative which might provide a safer and low cost treatment.

Quantitative analysis of this plant should be investigated in more detail.

ACKNOWLEDGEMENTS The authors express their sincere thanks to the Tropical Fruit Farm in Penang for providing the plant for the study. The authors are grateful to Universiti Sains Malaysia (Research University Grant (1001/PBIOLOGI/811275) for financing the project and providing the laboratory facilities.

REFERENCES Ayoola, G., Lawore, F., Adelowotan, T., Aibinu, I., Adenipekun, E., Coker, H., and Odugbemi, T. (2008). Chemical analysis and antimicrobial activity of the essential oil of Syzigium aromaticum (clove). African Journal of Microbiology Research, 2, 162-166.

Chandrawathani, P., Premaalatha, B., Nurulaini, R., Erwanas, A., Zaini, C., Aizan, M., Ramlan, M., and Khadijah, S. (2013). Severe Anthelmintic Resistance in Two Free Grazing Small holder Goat Farms in Malaysia. Journal of Veterinary Science & Technology, 4(137), 2.

Devmurari, V., & Jivani, N. (2010). Phytochemical screening and antibacterial activity of ethanolic extract of Artemisia nilagirica. Annals of Biological Research, 1(1), 10-14.

Gutiérrez, R. M. P., Mitchell, S., and Solis, R. V. (2008). Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology, 117(1), 1-27.

Harada, Y., and Mori, O. (1955). A new method for culturing hookworm. Yonago Acta Med, 1(3), 177-179.

Hutchinson, G. W. (2003). Gross Pathology of Ruminants. Pathology and diagnosis of internal parasites in ruminants, University of Sydney, pp. 309 - 338.

Ismail, M., Minhas, P., and Khanum, F. (2012). Anthelmintic Activity of Guava Psidium guajava.

International Journal of Research in Pharmaceutical and Biomedical Sciences, 3(1).

Molan, A., Hoskin, S., Barry, T., and McNabb, W. (2000). Effect of condensed tannins extracted from four forages on the viability of the larvae of deer lungworms and gastrointestinal nematodes. The Veterinary Record, 147(2), 44-48.

Oh, W. K., Lee, C. H., Lee, M. S., Bae, E. Y., Sohn, C. B., Oh, H., Kim, B. Y., and Ahn, J. S. (2005). Antidiabetic effects of extracts from Psidium guajava . Journal of Ethnopharmacology, 96(3), 411-415.

Raaman, N. (2006). Phytochemical Techniques. New Delhi: New India Publishing Agency, pp. 19 24.

Ryu, N. H., Park, K.-R., Kim, S.-M., Yun, H.-M., Nam, D., Lee, S.-G., … Ahn, K. S. (2012). A Hexane Fraction of Guava Leaves (Psidium guajava L.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells.

Journal of Medicinal Food, 15(3), 231–241.

Stepek, G., Buttle, D., Duce, I., Lowe, A., and Behnke, J. (2005). Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro. Parasitology, 130(02), 203-211.

Wagner, H. (1999). Medicinal Phytochemistry. Environmental Health Perspectives, 107(10).

61 | P a g e The Conservation of Orangutan : The Dilemma between Funding, Profit, Sincerity and Passion Nik Fadzly*, Tan Sue Han, Wan Fatma Zuharah Wan Musthapa, Mashhor Mansor, Aini Hasanah Abd Mutalib, Salniza Akmar School of Biological Sciences, Universiti Sains Malaysia, 11800, USM, Penang, Malaysia. *corresponding author: nfadzly@usm.my; nroselnik@outlook.com Abstract An iconic species to Malaysia and Indonesia, Orangutans represents as an example of why conservation of critically endangered animals is important in ASEAN countries. There are several rehabilitation centers, zoos and governmental and non-governmental agencies that are involved in protecting these magnificent creatures. However, although the goal of conservation is the same, the methods are not always agreed upon by all. This paper examines the status, management style and goals of a few selected rehabilitation centers from Malaysia and Indonesia.

Introduction Orang utans (Pongo pygmaeus pygmaeus) are one of the largest primates in the world. Orang utans are known for their distinctive orange red hair that blends when with the sunlight which resulted in them being well camouflaged amongst the forest trees. Orang utans mature sexually at a very late age period; around the age of twelve to fifteen. (Galdikas, 1988) Their rate of reproduction is very slow as the female will take 7 – 8 years to take care of her offspring and refuses to mate with any male.

Hence, it takes them a long time to replace the individuals who died. Due to the vast development in urban cities and illegal logging activities, orang utans‟ habitats have been severely disturbed. As a result, this species is classified as endangered. Also, in some countries, the orang utans are hunted for their meat whereas the infants of orang utans are captured and being kept as pets (Corlett, 2007).

To bring back the orangutans from the brink of extinction, conservation efforts have been carried out.

Eco-tourism, captive breeding are among the ways that have been carried out towards this aim.

Captive breeding, in particular has been carried out mostly in zoos. However, with only limited number of orangutans in each zoo, this presents a problem: lack of genetic variation (Muir et al.

1995). So, an exchange of DNA of orangutans between these zoos is encouraged whenever possible to allow “genetic drift” among these orangutans and preventing one‟s population from becoming “stagnant”.

Eco-tourism plays a very important role in conservation as it helps to generate income for the expenditure needed in the conservation of orangutans. One of the successful programmes is the Mount Leuser National Park in Indonesia whereas in Malaysia, the Sepilok Orangutan Rehabilitation Centre in East Malaysia and also the Bukit Merah Orang Utan Island in Perak in West Malaysia have been showing some promising results. Educating the public is also very important. Those who are exposed to more knowledge on a particular animal being conserved provide more support towards the orangutans‟ conservation of it (Tisdell & Wilson, 2004).

Funding for conservation projects has always been a critical issue. Luckily, there multiple grants available internationally catered specifically for Orangutan. Whilst there is available funding, we predict that the financial support will decline in the future. One of the main factors that will be mostly affected by funding would be the management of the conservation project. One of the main concerns would be regarding the salary and maintenance of the ground staff. This paper discusses about how

62 | P a g e these two components are intricately linked and proposes several ideas on how to mitigate the problem.

Materials and methods This paper is a compilation of review and a short interview question to several key person involved in the management of Orangutan conservation from government and non-government organization. We asked 5 simple question through email. The questions were: 1. In your opinion, what is the perfect conservation plan for Orangutan?

2. How is your organization funded? And how does funding is obtained?

3. What are your opinion concerning using Orangutan as a display asset for ecotourism?

4. How passionate do you think your staff are? How do you keep them motivated and passionate?

5. In a theoretical situation, you need to hire a staff (that deals directly with Orangutan). You are presented with two candidates. The first candidate had shown experience, great interest and passion during interview, but asks for a higher salary. The second candidate lacks experience and is quite naĂŻve concerning Orangutan, but is willing to work for half the pay. Which candidate would you choose? What are your suggestions to mitigate/handle the selected candidate in the future?

We also asked the respondent whether they would like to remain anonymous or not. Further mention of their name and organization will only be mentioned by their consent.

Results and discussion Unfortunately, we did not managed to get many responses from the organizations. NGOâ&#x20AC;&#x;s were much more cooperative and transparent in their answers but would rather remain anonymous. The contents and discussion points in this paper is quite sensitive since it involves several parties/organizations with different objectives. Therefore, in order to remain neutral, we will not mention any specific names or example. Delgado and Van Schaik (2000) have outlined the distribution pattern, behaviour and conservation of Orangutan in Borneo and Sumatra. The initial step to conserve Orangutan was by setting up rehabilitation centers by national and international organizations. This in turn will promote ecotourism that can produce revenue to continue the conservation efforts and provide local people with additional income.

Throughout the years, there have been multiple rehabilitation centers that had been set up by various organizations, both national and international. We categorized the centers into three categories: Category Government Private

Non-profit NGO

Description Rehab centers set up by the national government with full government funding Rehab centers set up by private individual/company with links and funding by corporate sponsors Rehab centers set up by private NGO, funding obtained through international, national grants

63 | P a g e and donations.

It must be mentioned subtly here that, eventhough all the centers aims for the conservation of Orangutan, there are disagreement on certain levels between the respected parties. For our short interview question one, most NGO‟s and academician would prefer a simple and applicable conservation plan. There is a specific focus needed for habitat conservation (reduce logging, land convertion to palm oil plantations, etc. An emphasis is given that conservation cannot be treated as political and commercial tool. For question two, concerning funding, most NGO‟s obtain their funds through international grants. Those with university links usually apply through national and university grants. We have no information from the private and government category. In reality, reducing forest deforestation or preventing land conversion is a tricky business. Tata et al. (2014) has outlined through several economic models that reducing or stopping forest conversation will incur several drawbacks to the country. Without a doubt that the species value is important, however, the likelihood of the locals would also needed to be taken into account. This, in turn is intricately connected to the commercial and political situation of the country. A more diplomatic approach suggested is the carbon storage/carbon footprint payment by the companies as suggested by Tata et al. (2014) and Nantha and Tisdell (2009).

For question three, the concept of ecotourism and display asset is viewed differently from various organization viewpoint. Conservationist would argue that captive and semicaptive animals cannot be termed as asset for ecotourism or entertainment business. However, it must be noted that respondents do agree with the need of public awareness and education. In general, the acceptable concept of ecotourism must follow specific regulation and requirements as outlined by Macfie and Williamson (2010). Currently, this stands to a moot point as different person have a different opinion.

For question four and five, this relates to the passion and sincerity of the ground staff. While the key person of any conservation plan is known to be very passionate about the job, the same could not be said for the staff or ground workers. Conservation work is a very tough, challenging and often a rewardless (in terms of monetary). Several NGO‟s and even national rehab centers offers a voluntary work option to cater for lack of manpower. In a way, it is the workers who pay to work with the animals. Most NGO‟s and academic would prefer experience. It is too time consuming to train new staff, and the general consensus is that its better to keep a small select number of experienced staff rather than to have a fleet of inexperienced trainees. Another useful suggestion from the respondent is to rely and respect the local people. To keep the staff motivated, one the suggested ways is to empowere the staff with their decision and management. Encourage the staff to do research, analyse data and present the data as their own.

To conclude, this short paper serves as a preview for a much more complex analysis of the management of conservation projects, especially for Orangutans. The important message at the end of the day is that whilst it is important to safeguard Orangutan, it is also important to support and take care the welfare of those involved in the conservation project.

References Corlett, Richard T. (2007). The impact of hunting on the mammalian fauna of tropical Asian forests.

Biotropica, 39(3), 292-303.

Delgado, R. A., & Van Schaik, C. P. (2000). The behavioral ecology and conservation of the orangutan (Pongo pygmaeus): a tale of two islands. Evolutionary Anthropology: Issues, News, and Reviews, 9(5), 201-218.

64 | P a g e Galdikas, BirutĂŠ MF. (1988). Orangutan diet, range, and activity at Tanjung Puting, Central Borneo.

International Journal of Primatology, 9(1), 1-35.

Macfie, E. J., & Williamson, E. A. (2010). Best practice guidelines for great ape tourism (No. 38).

IUCN. Muir, C, Galdikas, BMF, & Beckenbach, AT. (1995). Genetic variability in orangutans The Neglected Ape (pp. 267-272): Springer.

Nantha, H. S., & Tisdell, C. (2009). The orangutanâ&#x20AC;&#x201C;oil palm conflict: economic constraints and opportunities for conservation. Biodiversity and Conservation, 18(2), 487-502.

Tata, H. L., van Noordwijk, M., Ruysschaert, D., Mulia, R., Rahayu, S., Mulyoutami, E., ... & Dewi, S. (2014). Will funding to Reduce Emissions from Deforestation and (forest) Degradation (REDD+) stop conversion of peat swamps to oil palm in orangutan habitat in Tripa in Aceh, Indonesia?.

Mitigation and Adaptation Strategies for Global Change, 19(6), 693-713.

Tisdell, C & Wilson, C (2004). The public's knowledge of and support for conservation of Australia's tree-kangaroos and other animals. Biodiversity & Conservation, 13(12), 2339-2359.

65 | P a g e Determination of Potential Producers and Consumers in Tropical River Ecosystem using Stable Isotope Analysis Suhaila Ab Hamid* and ‘Amila Faqhira Zainodin School of Biological Sciences, Universiti Sains Malaysia ABSTRACT In freshwater ecosystem, different food sources may be used by different animals due to their digestibility or because of hydrology that affects their availability to consumers. Present study utilized stable isotope markers of carbon-13 (δ13C) and nitrogen-15 (δ15N) to indicate existing food web in tropical river. The mean δ13C values of all producers in all study sites were ranged from – 26.00 to – 31.11 ‰. The highest δ13C value was obtained from the tipulids (Insecta: Diptera) in Batu Kurau River with an average of - 15.03 ± 0.022 ‰, while the lowest was from the heptageniids (Insecta: Heptageniidae) in Jelai River with - 33.08 ± 0.210 ‰. The zenarchopterid fish in Ara River had the highest δ15N value recorded with an average of 9.68 ± 0.020 ‰. Nitrogen isotopes are useful indicators of trophic status of the consumers. Mean δ15N values of organic matter ranged from 3 to 6 ‰ were measured for aquatic macrophyte, leaf packs and algae in all four study sites. The δ 15N values of aquatic insects ranged from 2.59 (Coleoptera: Elmidae) to 8.11 ‰ (Hemiptera: Nepidae).

Elmids exhibited the lowest average dietary trophic position value of 0.81 on leaf packs, while the consumption of cyprinid fish prey on elmids were assigned to trophic level 3.62. The fish families: Cyprinidae, Syngnathidae and Zenarchopteridae recorded the highest δ15N values ranged from 6.90 to 9.68 ‰. In accordance with these δ13C and δ15N values, it indicated that the major trophic chain in this freshwater ecosystem began with the organic matter followed by the aquatic insects and ended with fish.

Keywords: food web, tropical river, stable isotope * corresponding author: ahsuhaila@usm.my

INTRODUCTION Rivers arise in the mountain ranges where their flow is torrential, slowing down and meander their way to the sea. As rivers flowing to low reaches, their substrate, water quality and food sources for aquatic fauna changes too. Different food sources may be consumed by different animals due to their morphology, digestibility and the hydrology that affects their availability to consumers. The stable isotope approach has become widely used in ecology, providing the possibility of obtaining objective and repeatable measures of trophic position, food chain length omnivory (Cabana and Rasmussen, 1994). The stable isotope approach is based on the isotopic concentration in the consumers‟ tissues resembles the stable isotopic composition of their diet (De Niro and Epstein, 1978, 1981; Peterson and Fry, 1987), which allows the establishing of the relative contributions of isotopically different sources to the diet of consumers (Fry, 2006). Carbon (δ13C) changes relatively as trophic level change and is used to identify the ultimate source of carbon, or the primary energy source for an ecosystem or group of organisms (Fry and Sherr, 1984). Nitrogen (δ15N) becomes enriched when transferred throughout a food web by means of feeding and predation (Peterson and Fry, 1987). Therefore, this study aimed to identify the food sources of aquatic insects in the freshwater ecosystems and to determine the trophic structure of the food web in all study sites.

66 | P a g e EXPERIMENTAL A. Study sites Samples for stable isotope analysis were collected from four selected rivers in Bukit Merah catchment area in Kerian, Perak, Malaysia: Batu Kurau River (N 04o 54‟ 17.4” E 100o 49‟ 59.9”), Jelai River (N 05o 00‟ 49.8” E 100o 48‟ 37.4”), Ara River (N 05o 05‟ 25.5” E 100o 51‟ 10.7”) and Ayer Hitam River (N 05o 01‟ 33.3” E 100o 83‟ 49.9”).

B. Samples collection and preparation Samples of plants, aquatic macroinvertebrates and fish were collected randomly from all four selected rivers for stable isotope analysis in order to compare the trophic levels in the food web. All stable isotope samples of plants, aquatic macroinvertebrates and fish were oven dried at 50 – 60 o C for two days or until the samples were sufficiently dried and the tissues were ground into a homogenous, fine powder (Jardine et al., 2003) using a mortar and pestle. Ground samples were placed back in small vials and stored in the freezer until they were ready to be analysed (Salas and Dudgeon, 2001).

C. Stable isotope analysis Powdered samples were sent to Doping Control Centre (DCC) in Universiti Sains Malaysia (USM), analysed for carbon and nitrogen stable isotopes δ13C and δ15N by using an elemental analyser (EA) that is connected to an isotopic ratio mass spectrometry (IR-MS) following standard procedures.

D. Relative trophic position calculation Relative trophic position estimation was calculated using the following equation: Trophic position = λ + In this equation, λ is the trophic level of the base (λ = 1 if the base is primary producers), δ 15Nsc is the nitrogen isotopic composition of the consumer (secondary consumer or any higher consumer), δ15Nbase is that of the food base and Δn is an estimated of the average increase in Δ15N per trophic level (Post, 2002).

RESULTS AND DISCUSSION Results on δ13C and δ15N of present study showed various degrees of carbon and nitrogen isotopes in different land-use river ecosystem. In freshwater ecosystems, the δ13C can indicate the relative importance of allochthonous and autochthonous sources of carbon. Mixed allochthonous leaf litters which were found in all four study sites, were predicted to be the main local primary producers for the aquatic insects, besides algae and aquatic macrophytes. The mean δ13C values of all producers in all study sites were ranged from – 26.00 to – 31.11 ‰ (Figure 1). Leaf packs contained the lowest carbon signatures among the food sources, as autochthonous carbon had more 13C-enriched than allochthonous sources (Salas and Dudgeon, 2001), and this explained most aquatic insects consumed coarse particulate organic matter (CPOM) and fine particulate organic matter (FPOM) (Merrit and Cummins, 1996) instead of directly consuming on leaf packs (Palmer et al., 1993). Leaf litters in the stream will undergo decomposition process with aids from shredders and other microorganisms to break them down into smaller particles (Anderson and Sedell, 1979). Studies done by Dhiya Shafiqah (2014), Lester et al. (1995), Thorp et al. (1998) and Bunn et al. (1999) also observed a similar δ13Cenrichment of autochthonous sources compared to allochthonous material. The highest δ13C value was obtained from the tipulids in Batu Kurau River with an average of - 15.03 ± 0.022 ‰, while the lowest was from the heptageniids in Jelai with - 33.08 ± 0.210 ‰.

67 | P a g e The application of δ15N as a tracer of organisms‟ trophic position as well as organic matter source information (Peterson, 1999) had eliminated many problems to estimate trophic position (Vander Zanden et al., 1997). In this study, the nitrogen isotopic signature values of allochthonous food source were slightly higher than authochthonous food sources. Hicks and Laboyrie (1999) proved that leaves conditioned for 28 days had δ15N values that were 0.7 to 3.0 ‰ higher than fresh leaves. Mean δ15N values of organic matter ranged from 3 to 6 ‰ were measured for aquatic macrophyte, leaf packs and algae in all four study sites. The δ15N values of other aquatic insects on the other hand, ranged from 2.59 (Elmidae) to 8.11 ‰ (Nepidae). The zenarchopterid fish in Ara River had the highest δ15N value recorded with an average of 9.68 ± 0.020 ‰. This explained the feeding mechanisms of aquatic insects from the same functional guilds were based on similar sources that would place them in the same trophic structure. Elmids exhibited the lowest average dietary trophic position value of 0.81 on leaf packs, while the consumption of cyprinid fish prey on elmids were assigned to trophic level 3.62.

Distribution of nitrogen isotope has been proved to be an indicator of trophic position as the 15N increases consistently with the increase of trophic level of consumers (Fry, 1988; Peterson and Fry, 1987). The fish families:Cyprinidae,Syngnathidae and Zenarchopteridae, which were the top underwater predator, recorded the highest δ15N values (6.90 to 9.68 ‰) than any other aquatic insects as the values of δ15N increased with increasing trophic level. In this study, three trophic levels were identified generally in all four study areas: the producer, primary consumer and predator similar to results reported by Dhiya Shafiqah (2014) and VanDongen et al. (2011).

CONCLUSIONS In conclusion trophic interactions do occurred between the terrestrial (riparian) and aquatic (lotic) habitats in freshwater ecosystem. These two habitats are contiguous and share the same energy sources and display similar community interactions. Organic matter, both within riparian zone or wetted channel, represents a significant energy source for terrestrial and aquatic macroinvertebrates.

In addition, aquatic insects are also an important component in the fish diet. In all four reaches, regardless of riparian condition, has showed strong connectivity and energy flow between terrestrial and aquatic zones. This was the principally evident in the organic matter  aquatic insects  fish pathway.

ACKNOWLEDGEMENTS We thank the School of Biological Sciences for providing facilities to carry out this study. This study was financially supported by USM Research University Grant - RUI (1001/PBIOLOGI/811247).

68 | P a g e

Aquatic insects

Organic matter

δ15N (‰)

Fish

δ13C (‰) Figure 1: Stable isotope analysis value in all four studied rivers in Bukit Merah, Kerian, Perak.

REFERENCES Anderson, N. H., & Sedell, J. R. (1979). Detritus processing by macroinvertebrates in stream ecosystem. Annual Review of Entomology. 24 (1), 351 – 377.

Bunn, S. E., Davies, P. M., & Mosisch, T. D. (1999). Ecosytem measures of river health and their response to riparian and catchment degradation. Freshwater Biology. 41, 333 – 345.

Cabana, G., & Rasmussen, J. B. (1994). Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes. Nature (Lond.). 372, 255 – 257.

De Niro, M. J., & Epstein, S. (1978). Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta. 42, 495 – 506.

De Niro, M. J., & Epstein, S. (1981). Influence of diet on the distribution of nitrogen isotopes in animals. Geochim Cosmochim Acta. 45, 341 – 351.

Dhiya Shafiqah, R. (2014). Habitat characterization, trophic position and seasonal influence on aquatic insects in the selected feeder streams of Belum–Temengor Forest Complex (BTFC), Perak.

MSc thesis, Universiti Sains Malaysia, Penang.

Fry, B. (1988). Food web structure on Georges Bank from stable C, N and S isotopic compositions.

Limnology and Oceanography. 33 (5), 1183 – 1190.

Fry, B. (2006). Stable isotope ecology. New York, NY: Springer.

Fry, B., & Sherr, E. B. (1984). δ13C measurements as indicators of carbon flow in marine and frashwater ecosystems. Contrib. Mar. Sci.. 27, 13 – 47.

Hicks, B. J., & Laboyrie, J. L. (1999). Preliminary estimates of mass–loss rates, changes in stable isotope composition, and invertebrate colonisation of evergreen and deciduous leaves in a Waikato, New Zealand, stream. New Zealand Journal of Marine and Freshwater Research. 33, 221 – 232.

Jardine, T. D., McGreachy, S. A., Paton, C. M., Savoie, M., & Cunjak, R. A. (2003). Stable Isotopes in Aquatic Systems: Sample Preparation, Analysis and Interpretation. (2656). Canadian Manuscript Report of Fisheries and Aquatic Sciences, Canada.

Lester, P. J., Mitchell, S. F., Scott, D., & Lyon, G. L. (1995). Utilization of willow leaves, grass and periphyton by stream macroinvertebrates: a study using stable carbon isotopes. Arch. Hydrobiol..

133, 149 – 159.

69 | P a g e Merritt, R. W., & Cummins, K. W. (1996). An Introduction to the Aquatic Insects of North America, 3rd ed. Kendall/Hunt Publishing Company, Dubuque.

Palmer, C., O'Keeffe, J. A. Y., Palmer, A., Dunne, T., & Radloff, S. (1993). Macroinvertebrate functional feeding groups in the middle and lower reaches of the Buffalo River, eastern Cape, South Africa. I. Diet variability. Freshwater biology. 29 (3), 441 – 453.

Peterson, B. J. (1999). Stable isotope as tracers of organic matter input and transfer in benthic food webs: A review Acta Oecologia. 20 (4), 479 – 487.

Peterson, B. J., & Fry, B. (1987). Stable isotopes in ecosystem studies. Annu Ref Ecol Syst. 18, 293 – 320.

Post, D. M. (2002). Using stable isotopes to estimate trophic position: models, methods and assumptions. Ecology. 83, 703 – 718.

Rosenfeld, J. S., & Roff, J. C. (1992). Examination of the carbon base in southern Ontario streams using stable isotopes. J. N. Amer. Benthol. Soc.. 11, 1 – 10.

Salas, M., & Dudgeon, D. (2001). Stable–isotope determination of mayfly (Insecta: Ephemeroptera) food sources in three tropical Asian streams. Fundamental and Applied Limnology. 151, 17 – 32.

Sprules, W. G., & Bowerman, J. E. (1988). Omnivory and food chain length in zooplankton food webs. Ecology. 69: 418 – 426.

Thorp, J. H., Delong, M. D., Greenwood, K. S., & Casper, A. F. (1998). Isotopic analysis of three food web theories in constricted and floodplain regions of a large river. Oecologia. 117, 551 – 563.

Vander Zanden, M. J., Cabana, G., & Rasmussen, J. B. (1997). Comparing trophic position of freshwater fish calculated using stable nitrogen isotope ratios (δ15N) and literature dietary data.

Canadian Journal of Fish and Aquatic Sciences. 54, 1142 – 1158.

VanDongen, J., Poczekaj, K., & Snyder, E. (2011). Understanding nutrient pathways and reciprocal subsidies between stream and riparian zones using stable isotope analysis. In: SFS 2012 Annual Meeting of Freshwater Stewardship: Challanges and Solutions. Louisville, KY; 2012 May 20 – 24; Grand Valley State University.

70 | P a g e The Effect of Temperature and Diet on Immature Stages of Megaselia Scalaris Development and Survival Rate Hadura Abu Hasan*,a, Teo Bee Hsien a, Hamdan Ahmad a,b a School of Biological Sciences, Universiti Sains Malaysia b Vector Control Research Unit, Universiti Sains Malaysia ABSTRACT Mass production of small flying insects is currently under demand for the swiftlet farming industry.

The preference diet to small Diptera has now increased by domesticated swiftlet and the continuous production of Megaselia scalaris is proposed to achieve the level of this requirement. Abiotic factor such as temperature is likely to exert a major effect on the development of this species and may influence the quality of its production in industry. In this study, the effect of different incubation temperatures and diet to the developmental time and survival rate of immature stages of M. scalaris were evaluated. Under laboratory condition, three different incubation temperatures (19˚C, 27˚C and 35˚C) and three different diets (chicken food, soya meal and oat with milk powder meal) were tested at 70 ±5 % relative humidity with constant light condition. The ideal temperatures for rearing the larvae were 27˚C and 35˚C while the optimum temperature for the development of pupae was 27˚C. Among three different diets, the consumption of chicken food by M. scalaris larvae showed the shortest developmental time and therefore, produced the highest survival rate. However, larval diet type has no effect on the developmental time and survival rate of M. scalaris pupae. Such information is beneficial to provide the effective mass-rearing of this species especially in swiftlet farming industry.

Keywords: Megaselia scalaris, temperature, diet, survival rate, developmental time *hadura@usm.my INTRODUCTION Megaselia scalaris (Loew) (Diptera: Phoridae) is a small and cosmopolitan fly, which is also recognized as “phorid fly” or “scuttle fly” (Idris et al., 2001). It has brown yellowish colour with some dark markings on the abdomen and its body length is about 2 mm long. This species has been greatly used in forensic studies (Disney, 2008; Varney & Noor, 2010; Zuha & Omar, 2014), studies on myiasis in humans (Thevan et al., 2010; Nazni et al., 2011), experimental species in genetics and developmental biology (Harrison Cooper, 2003; Rasmussen & Noor, 2009). This species is an omnivorous and consuming a variety of food ranging from dead or alive animals, living plants, fruits, fungi, discarded food waste and culture media (Disney, 2008; Varney & Noor, 2010). Importantly, this species also can be a feedstuff to other organisms. Previous study by Lourie & Tompkin (2000) showed that small-bodied diptera has been identified to be one of the most organisms consumed by swiftlets. Therefore, the best candidate for such criteria is the scuttle fly, M. scalaris.

To mass-rearing of this species in the laboratory, shortening the developmental time and decreasing mortality of M. scalaris are the fundamental criteria that should be considered. Disney (2008) documented that data on the temperature is required to obtain relevant information on the duration of development of this species at various temperature. There are some differences in the literature regarding the duration of M. scalaris development at different temperatures as summarized by Disney (2008). These data showed the development of M. scalaris in the laboratory cultures under almost

71 | P a g e constant temperature regimes. Other data such as diet used for the development of M. scalaris in the laboratory is also important to provide the effect on this criteria (Idris et al., 2001; Zuha et al., 2012).

M. scalaris has the sponging mouthpart and it feeds only on fluid diets (Sukontason et al., 2003).

Therefore, the variation in diet quality such as moisture level of the organic materials are likely to affect the growth and survival rate of this insect species.

The objective of this current study is to investigated the effect of different incubation temperatures (19˚C, 27˚C and 35˚C) and diets (chicken food, soya meal and oat with milk powder meal) on the survival rate and developmental time of larvae and pupae of M. scalaris in the laboratory. Thus, the conditions for mass-rearing of M. scalaris in the laboratory could be optimized by shorten the developmental time and minimize the mortality rate which could increased the production of M. scalaris in short period of time.

MATERIALS AND METHODS A.Production of M. scalaris Laboratory colony of M. scalaris was established and maintained in Swiftlet Research Centre, Kampung Paya Jaras Hilir, Selangor under the Department of Veterinary Service Malaysia at 70 ±5 percentage relative humidity. The colony has been maintained by rearing one hundred pupae in a cage (24 cm x 19.5 cm). After the pupae had developed into adults, females and males were allowed to mate for 24 hours. Commercial broiler chicken starter mixed with water (1:1.5) was provided daily in petri dish (9 cm diameter) for female oviposition. After three days of oviposition, all the adults in the cage were killed in a freezer. Eggs that found on the culture media inside the petri dish were transferred to a new cage and added with fresh culture media. They were reared until became pupae and all these pupae were then removed from culture media and transferred to a cage for adult emergence.

B. Effect on variety of diets and different temperatures Three types of diets were prepared in the laboratory. Each type of diet were mixed with different water ratios: 1:1.5 (commercial broiler chicken starter : water), 1:1:4 (soya bean: flour: water) and 12:4:9 (oat: milk powder: water). In this study, 10 g of commercial broiler chicken starter was placed into a plastic container (4 cm x 6 cm) with 1 cm diameter hole on the lid which was covered with muslin clothes. Six newly hatched larvae were put into the plastic container and placed in an incubator at three different temperatures (19±1˚C, 27±1˚C and 35±1˚C). Five replicates were tested for each temperature. Constant light of 24 hours was provided and larvae were examined every 24 hours until they become into pupae. The time taken for each larva to become into pupa was recorded. The frequency of daily pupation was also recorded. The pupae which were formed in plastic containers were removed and put in the microcentrifuge tubes which were separetd by cotton wool. The pupae were examined every 24 hours until these immature stages developed into adults. The time taken for each pupae to develop into adults and the total number of adult emergence were also recorded daily.

Adults which were failed to emerge after thirty days were assumed as dead. The experiments were repeated using other different diets such as soya meal and oat with milk powder meal.

C. Statistical analysis The larval and pupal developmental time (mean ± SE), survival rate of larvae and pupae (%) were recorded. Data were then analysed by two-way ANOVA followed by Tukey HSD test for multiple comparisons of means at P < 0.05 using SPSS 11.5 software.

72 | P a g e RESULTS AND DISCUSSION A.Effect of temperatures on larval developmental time and survival rate There were significant differences between three different temperatures on the larval developmental time (F = 156.679, P < 0.05) and larval survival rate (F = 8.938, P < 0.05) of M. scalaris. Results showed that 27°C and 35°C had faster developmental time and higher survival rate compared to 19°C on the development of M. scalaris larvae. This results suggested that the development time and survival rate of immature stages of M. scalaris were faster at higher temperature. As reviewed by Disney (2008), the temperature recorded ranging from 15 °C to 37.3 °C for the development of M. scalaris in the laboratory. Previous study by Zuha et al. (2012) showed a similar temperature range as in this current study. Their results showed that eggs and larva development time of M. scalaris cultured in liver agar were faster at 33°C compared to 27°C and 30°C in the laboratory. However, to compare this current results to those previous results as summarized by Disney (2008) is seemingly not feasible because the colony of M. scalaris and the laboratory procedures used were different.

B. Effect of temperature on pupal developmental time and survival rate The pupal developmental time (F = 347.084, P < 0.05) and survival rate (F = 32.773, P < 0.05) of M. scalaris varied significantly with temperatures. The results showed the shortest pupal developmental time and highest survival rate at 27°C compared to 19°C and 35°C. In this current study, most of the pupae were unable to develop at 19°C while some adults which were developed from these pupae suffered from deformation. On the other hand, most of the adults that reared at 35°C were unable to remove their puparium completely during emergence and eventually caused the fatality in this present study. Greenberg & Wells (1998), in their laboratory study also documented that the developmental rate gradually increased at inclining temperature but pupa developmental time and adult emergence were longer in 35°C than 29°C. Although Trumble & Pienkowski (1979) indicated that increasing temperature is able to enhanced the development rate of M. scalaris, Greenberg & Wells (1998) suggested that the growth rate of M. scalaris at particular developmental stages was not necessarily shortened when the temperature increased.

C. Effect of diets on larval developmental time and survival rate There were significant differences between three different diets on the developmental time (F = 16.956, P < 0.05) and survival rate (F = 172.964, P < 0.05) of M. scalaris larvae. Chicken food showed the fastest larval growth rate and highest survival rate of M. scalaris followed by soya meal and oat with milk powder meal. Corn and soya that present in the chicken food which create spaces in between of the two ingredients allowed retaining an appropriate water contents for the larvae to survive until the end of the experiment. While for the presence of the flour with the soya, it can reduce the space in between of these two ingredients and as a result decreasing the water retention of the soya meal. As for the oat with milk powder meal, sticky condition was existed in which both ingredients able to absorb large quantity of water until expanding and no space was found in between the milk powder and oat. Hence, the water content in the meal dehydrated quickly as shown in the study of Pastor et al. (2011) on houseflies, Musca domestica.

D. Effect of diets on pupal developmental time and survival rate The pupal developmental time (F = 1.786, P > 0.05) and survival rate (F = 0.705, P > 0.05) were not significantly different between three different diets. Pupae is a stage that did not consume any food.

Therefore, it has insignificant effect of the poor pre-feeding. Similar finding was also documented by Idris et al. (2001). Disney (1994) also revealed that diets type had no influence on the development of pupae from puparation to adult emergence. Furthermore, study by Saastamoinen & Rantala (2013)

73 | P a g e indicated that there was no effect of the poor larval feeding to the pupal developmental time in the butterfly, Melitaeacinxia. Consistent result was found in the study by Patel et al. (1986) which also mentioned that larval feeding have no effect on the pupal growth rate of cotton leafworm, Spodopteralitura.

E. Interaction between temperatures and diets on the larval developmental time and survival rate of M. scalaris Two-way ANOVA analysis showed that there was no interaction between temperatures and diets on the larval developmental time of M. scalaris (F = 1.770, P > 0.05). Study by Wolesensky & Logan (2007) documented that the temperature can caused the foremost effect on the insects development without any interaction with other factors. On the other hand, there was interaction between temperatures and diets to the survival rate of the larvae of M. scalaris (F = 22.571, P < 0.05).

According to the study by Mullen & Arbogast (1979), low temperatures can sluggish metabolic activities of the larvae such as movement and feeding. Food in poor water retention is able to intensify the situation by triggering the desiccation to the larvae. As a result, larvae will loss energy and ultimately suffer from mortality. While at the high temperature, the evaporation of water from the food is high causing the larvae unable to survive. Thus, low or high temperature with poor water retention from food can increased the mortality rate of the larvae.

F. Interaction between temperature and diets on the pupal developmental time and survival rate of M. scalaris Results on two-way ANOVA analysis showed that there was interaction between temperatures and diets on the developmental time (F = 22.571, P < 0.05) and survival rate (F = 22.571, P < 0.05) of M. scalaris pupae. This current study showed that the results seemed to have contradict the result obtained for M. scalaris larval developmental time and survival rate. The temperature is seemed to have interaction with previous larvae feeding and therefore influence the result. For example, interaction between temperature and water content (Vanâ&#x20AC;&#x;t Hof & Martin 1989) and dietary protein concentration (Kingsolver & Woods, 1998) affected the growth rate of tobacco hornworms, Manduca sexta. In contrast, the study by Park (2011) indicated that there was no interaction between temperature and food quality on the survival rate of grasshopper, Camnulapellucida.

CONCLUSIONS This current study showed that the temperature influence the larval and pupal development time and survival rate of M. scalaris in the laboratory. Larval development time and survival rate also influenced by different types of diets but this factor has insignificant effect on the pupae stage of M. scalaris. These findings provide insight to a better standardize protocol in mass-rearing of M. scalaris and therefore provide a beneficial information for the effective production of M. scalaris in the laboratory. This study is important in order to perform the greatest production of M. scalaris as edible insects particularly for a better profitability in swiftlet farming industries.

ACKNOWLEDGEMENTS The authors expressed a greatest gratefulness to the Director General, Department of Veterinary Services Malaysia.

74 | P a g e REFERENCES Disney, R.H.L. (1994). Scuttle Flies: The Phoridae. London: Chapman & Hall.

Greenberg, B., & Wells, J. D. (1998). Forensic Use of Megaselia abdita and M. scalaris. Journal of Medical Entomology, 35(3), 205-209.

Harrison, D. A., & Cooper, R. L. (2003). Characterization of development, behavior and neuromuscular physiology in the phorid fly, Megaselia scalaris. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 136(2), 427-439.

Idris, A. B., Abdullah, M., & Lin, T. P. (2001). Effect of various diets on the development of scuttle fly, Megaselia scalaris (Loew)(Diptera: Phoridae), larvae and pupae and percent of adult emergence and longevity. Pakistan Journal of Biological Sciences, 4(3), 321-325.

Kingsolver, J. G., & Woods, H. A. (1998). Interactions of temperature and dietary protein concentration in growth and feeding of Manduca sexta caterpillars. Physiological Entomology, 23: 354-359.

Mullen, M.A., & Arbogast, R. T. (1979). Time temperature mortality relationships for various storedproduct insect eggs and chilling times for selected commodities. Journal of Economic Entomology, 72: 476-478.

Nazni, W. A., Jeffery, J., Lee, H. L., Lailatu-Amar, M. N., Heo, C. C., & Sadiyah, I. (2011).

Nosocomial nasal myiasis in an intensive care unit. Malaysian Journal of Pathology, 33(1), 53-6.

Park, K. S. (2011). The effect of temperature and diet on the growth and mortality of Camnulapellucida (Orthoptera: Acrididae). Practicum in Field Environmental Biology.1-15.

Pastor, B., Cickova, H., Kozanek, M., Martinez-Sanchez, A., Takac, P., & Rojo, S. (2011). Effect of the size of the pupae, adult diet, oviposition substrate and adult population density on egg production in Musca domestica (Diptera: Muscidae). European Journal of Entomology, 108:587-596.

Patel, I.S., Rote, N.B., Shah, A.H., Patel, U.G., & B. K. Patel (1986). Biology of cotton leafworm Spodoptera litura Fb. (Noctuidae:Lepidoptera) on cotton. Gujarat Agricultural University Research Journal, 11(2): 67-68.

Rasmussen, D. A., & Noor, M. A. (2009). What can you do with 0.1× genome coverage? A case study based on a genome survey of the scuttle fly Megaselia scalaris (Phoridae). Bmc Genomics, 10(1), 382.

Saastamoinen, M., & M.J. Rantala (2013). Influence of developmental conditions on immune function and dispersal-related traits in the Glanville Fritillary (Melitaeacinxia) Butterfly. PLOS ONE. 8(11): 111.

Sukontason, K., Sukontason, K. L., Piangjai, S., Boonchu, N., Chaiwong, T., & Vogtsberger, R. C. (2003). Mouthparts of Megaselia scalaris (Loew)(Diptera: Phoridae). Micron, 34(8), 345-350.

Thevan, K., Disney, R. H. L. H., & Ahmad, A. H. (2010). First records of two species of Oriental scuttle flies (Diptera: Phoridae) from forensic cases. Forensic Science International, 195(1), e5-e7.

Trumble, J. T., & Pienkowski, R. L. (1979). Development and survival of Megaselia scalaris (Diptera: Phoridae) at selected temperatures and photoperiods. Proceeding of the Entomological Society of Washington, 81(2), 207-210.

Van‟t Hof, H. M., & Martin, M. M. (1989). Performance of the tree-feeder Orgyialeucostigma (Lepidoptera: Liparidae) on artificial diets of different water content: a comparison with the forbfeeder Manduca sexta (Lepidoptera: Sphingidae). Journal of Insect Physiology, 35: 635–641.

Varney, R. L., & Noor, M. A. (2010). The scuttle fly. Current Biology, 20(11), R466-R467.

Wolesensky, W., Joern, A., & Logan, J. B. (2005). A model of digestion modulation in grasshoppers, Ecological Modelling.188:358-373.

75 | P a g e Zuha, R. M., & Omar, B. (2014). Developmental rate, size, and sexual dimorphism of Megaselia scalaris (Loew)(Diptera: Phoridae): it's possible implications in forensic entomology. Parasitology research, 113(6), 2285-2294.

Zuha, R. M., Razak, T. A., Ahmad, N. W., & Omar, B. (2012). Interaction effects of temperature and food on the development of forensically important fly, Megaselia scalaris (Loew)(Diptera: Phoridae).

Parasitology research, 111(5), 2179-2187.

(Huey & Stevenson 1979, Crowder & Magnuson 1983, Karasov 1984, Zimmerman & Tracy 1989, Yang & Joern 1994; Harrison & Fewell 1995,).

76 | P a g e Multifarious Role: A Study on The Feeding Behaviour of Rove Beetle, Paederus Fuscipes CURTIS (Coleoptera: Staphylinidae)

a

Maryam Sufian a and Wan Fatma Zuharah*,a,b Medical Entomology Laboratory, School of Biological Sciences, Universiti Sains Malaysia b Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia

ABSTRACT Rove beetles (Paederus spp.) are known as medically important insects which caused dermatitis linearis to humans. Although being a major pest in human settings, they are substantial biological control agent of insect pests in the rice field of Southeast Asia. Under laboratory conditions, we evaluated the prey preference of this aggressive predator when it encounters three main agriculture pests of rice plant, the leafhoppers Nephotettix virescen (Distant), Recilia dorsalis (Motschulsky) and the planthoppers Nilaparvata lugens (StĂĽl). Paederus fuscipes displayed a Type II functional response for all prey species, but at high prey densities they predominantly consumed more on R. dorsalis presented both dead and alive. A significantly higher attack rate and lower handling rate time was observed for P. fuscipes predation on dead R. dorsalis prey. When various density preys was tested simultaneously, the predator showed greater preferences towards R. dorsalis and Ni. lugens even in treatment with lower number of individuals. Paederus fuscipes behavioural response of; (1) searching, (2) capturing, and (3) feeding on its prey items presented dead and alive conditions, showed that highest number of searching and capturing attempts was most frequently displayed on the alive Ni. lugens prey species. Whereas P. fuscipes spent significantly higher feeding time in consuming the dead prey compared to the alive prey. Hence due to its multifarious feeding behaviour, P. fuscipes showed desired qualities of a promising natural predator in handling numerous pest species in the rice agro-ecosystem.

Keywords: Feeding behaviour, functional response, Paederus, predation, prey preference.

INTRODUCTION Paederus fuscipes CURTIS is widely distributed around the world for instance in Europe, Asia, India, New Guinea, Africa and Australia except Antarctica (Frank and Kanamitsu 1987; Singh and Ali 2007). Paederus species inhabit mostly in moist environments such as in marshes, riverine and crop fields. In Malaysia this beetle is predominantly distributed in the West part of the Peninsular and is mostly found in the rice field area (Manley 1977). In the rice field, staphylinid beetles P. fuscipes is a predator and play a role in controlling insect pest populations. This predator species preyed upon the leafhoppers (Manley 1977) thrips, planthoppers and other rice pests species (Kalshoven and Van der Laan 1981). Therefore, P. fuscipes are beneficial to farmers in agriculture sector as they help to control rice pests, which normally start to attack the rice crop from the nursery until the harvesting season. The aim of our study is to investigate P. fuscipes feeding behaviour on three factors base on the (1) functional response study, (2) prey preferences, and (3) the predatorâ&#x20AC;&#x;s behaviour in response to three common types of prey species (Nephotettix virescen, Recilia dorsalis and the planthoppers Nilaparvata lugens) commonly found in the rice field area.

77 | P a g e MATERIALS AND METHODS A. Collection site of P. fuscipes and prey species The collection site for both predator and prey species was located at the rice field areas at Jalan Sejahtera Indah, Telok Air Tawar, Butterworth Penang (N 5° 29' 9.3171" E 100° 23' 1.3012").

B. Sampling Methods To capture P. fuscipes beetles, four UV blacklight traps was set up, Fay-Prince Blacklight Trap (John W. Hock) along the rice field boundary (a walkway dry area where no rice plant is planted / also known levee). UV blacklight traps were used to capture P. fuscipes as black rays are found to be most attractive to the adult of P. fuscipes. Paederus fuscipes are nocturnal insects and attracted to the light at night (George and Hart 1990). The blacklight traps were set up just before the nightfalls around 7.00 pm and the first collection of P.fuscipes started from 8.15 pm until 10.15 pm. The adult of rice insect pests, (1) the green leafhopper, N. virescen, (2) zig-zag leafthopper, R. dorsalis and, (3) brown planthopper, Ni. lugens were captured in abundance using conventional light trap method.

Conventional light trap is a trap with a light bulb which generated using a portable generator and hang in front of large white sheeth. White sheet acts as a reflector of the light, a resting and collection site for these insects. Sampling was repeated for the fresh batches of prey a day prior to the experiment.

Insects were collected by using a mechanical insect aspirator. Collected P.fuscipes and rice pests were then brought back and reared in the laboratory.

C. Rearing method of P. fuscipes The collected adult P. fuscipes were reared in the Medical Entomology Laboratory, Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia (5° 21' 21.913" N, 100° 18' 4.575" E) at a temperature of 28 ± 2 °C , 63.8 ± 2.0 % RH and a photoperiod of 12:12 (L:D). All subsequent experiments were conducted at the same laboratory condition. Paederus fuscipes beetles were reared in a plastic container of 16.5 cm height x 11.5 cm width in size and covered with muslin cloth to allow air ventilation as well as to prevent the beetles fom escaping. A moist cotton ball was provided as a water source on one side of the plastic container.

D. Functional Response Study Paederus fuscipes were offered with different densities of prey at 1, 3, 5 and 10 individuals in separate experiments. Two conditions of prey were also tested; (1) using dead prey (carcass) and (2) alive prey. Each P. fuscipes beetle was placed singly in a plastic container measuring 5.6 cm in height x 4.6 cm in width with a moist cotton ball on one side of the plastic container. Paederus fuscipes was starved for 24 hours prior to the experiment. On the other side of the container, prey item of mentioned densities were offered for 24 hours. After 24 hours, the remaining prey item was counted and recorded. The equation which best describes the results as no prey was replace during the course of the experiment is the “random-predator” equation of Rogers (1972): Ne = N [1 – exp [α (Th Ne – T)] ] where Ne is the number of prey eaten, N is the is the initial number of prey available (number of prey offered), α is the instantaneous attack rate, T is the total available time, and T h is the „handling time‟ (or the time required to handle a prey item). The attack coefficient, α and handling time, T h were estimated from the functional response data using a standard non-linear least squares technique. Twoway ANOVA was conducted to compare the differences between the number of prey (N. virescen, Ni.

78 | P a g e lugens and R. dorsalis) and condition of prey (dead and alive) that were consumed by the P. fuscipes beetles.

E. Preference of Prey Paederus beetles were placed singly in a 5.6 cm in height x 4.6 cm in width plastic container. In this experiment, a moist cotton ball was placed at one end of the plastic container, whereas a specific mixture of pest species was offered on each side of the plastic container. All preys were acclimatized for 10 minutes, prior to introduction of P. fuscipes predator. In this experiment, P. fuscipes beetles were presented with a mixture of; 1) adults of N. virescen and Ni. lugens, 2) adults of N. virescen and R. dorsalis, 3) adults of Ni. lugens and R. dorsalis at ratios of 0:6; 1:5; 2:4; 3:3; 4:2; 5:1; 6:0.

Experiment was repeated separately using two conditions of prey (1) dead prey (carcass) and (2) alive prey. Paederus fuscipes was starved for 24 hours prior to the experiment. After 24 hours of exposure, the remaining prey item was counted and recorded. Prey preference of predator was calculated based on Manly‟s preference index by using Manly‟s α (Manly 1974). This index takes into account the reduction of prey due to predation with Chesson (1982) modification for the description of prey depletion: α=

n (NN - CN ) (NN ) ((NN – CN ) n (NN – CN ) NN )

where N is the initial number and C is the consumed number of Ni. lugens (Ni) and N. virescen (N).

The predator‟s preferences (α) on the prey item can be predicted by using the attack constant from the functional response experiment on this mathematical model: αn =

αN

αN αN – (αN αN)

where αn was the predicted preference for Ni. lugen, αNi and αN were the attack constants for Ni. lugen and N. virescen respectively. The same equation was then used once again for the predicted preference of R. dorsalis that were consumed by the predator, P. fuscipes. A t test was performed to determine any significant difference in the preference of predators for these two species.

F. Predator’s Behaviour This experiment was set up to determine the predator‟s behaviour in capturing its prey. Three behaviours of P. fuscipes predator was observed; 1) searching rate (duration of time spent in foraging to locate the prey item for the first time), 2) prey capturing (number of attempt spent to approach the selected prey to start feeding), and 3) prey ingestion (duration of time spent in feeding). Paederus fuscipes were offered with single dead (carcass) or alive prey of the adult of N. virescen, Ni. lugens or R. dorsalis in separate experiments. Behaviour data were recorded following predator‟s behaviour as describe above for an hour period. Paederus fuscipes was starved for 24h prior to the experiment. The predator‟s behaviour of searching, capturing and ingestion were all examined using the two-way analysis of variance (ANOVA). Independent variables were types of prey species (N. virescen, Ni. lugens or R. dorsalis) and prey conditions (dead or alive).

G. Statistical Analyses Data analyses were computed using SPSS version 20.0. Normality test was conducted for all data at the 0.05 significance level by using Shapiro-Wilk test prior to two-way ANOVA analyses. If the criteria for normality are not met, the data were normalize using a log transformation.

79 | P a g e RESULTS AND DISCUSSION A. Functional Response Study The functional response of P. fuscipes predator on the consumption of different prey species N. virescen, Ni. lugens and R. dorsalis was significantly different (F = 10,461, df = 2,234, p = 0.000).

Greatest consumption was seen on the dead prey items (p < 0.05). The number of dead prey consumed

by P. fuscipes predator was 3.7 ± 0.26 of R. dorsalis, 2.65 ± 0.26 of Ni. lugens and 2.35 ± 0.37 of N. virescen per day. However, the average maximum number of alive prey that can be consumed by P. fuscipes predator was only 2.2 ± 0.25 (± SE) of R. dorsalis, 2.1 ± 0.31 of Ni. lugens and 1.0 ± 0.26 of N. virescen per day. On whole, P. fuscipes predator consumed significantly higher number of R. dorsalis prey presented alive and dead compared to other prey species. Kundu et al. (2014) stated that the preference towards a given prey type also depends on the density and the morphology of the prey species in an ecosystem. Essentially, if a prey item contributes only a small part of the energy needed to sustain the predator, the predation rate increases and the predator must consume or kill more preys to stay alive (Vézina 1985). Our study showed that the time taken to handle the dead green leafhopper, N. virescen was considerably higher compared to other prey species. This is due to N. virescen is generally bigger in size compared to Ni. lugens and R. dorsalis. Thus, P. fuscipes took greater time to consumed N. virescen. Overall, P. fuscipes preferentially consumed Ni. lugens and R. dorsalis preys predominantly more compared to N. virescen. Highest attack rate of live and dead preys presented to P. fuscipes predator was prominently seen on Ni. lugens (0.24 ± 0.02) and R. dorsalis (0.43 ± 0.03) respectively.

B.Preference of Prey Furthermore, our results on prey preference study proven that P. fuscipes preferred mostly on R. dorsalis followed by Ni. lugens when they were offered choices between Ni. lugens vs. N. virescen, Ni. lugens vs. R. dorsalis and R. dorsalis vs. N. virescen. Stephens and Krebs (1986) stated under the „optimal foraging theory‟ which anticipated that predators will select prey that provides the best energy in returns. Hence, P. fuscipes may have not wasted their energy in handling for a relatively larger prey such as N. virescen. They require a lot of energy in handling larger prey which is relatively stronger as they have hard and thick stratum than the smaller prey. When Ni. lugens was offered together with N. virescen, significantly greater preference was observed on the Ni. lugens prey species instead of the N. virescen species (t = 3.59, df = 138, p = 0.000). Paederus fuscipes consumed significantly more on Ni. lugens even when there were proportionally fewer Ni. lugens in relative to N. virescen. Similar situation was also observed when P. fuscipes was given a choice between R. dorsalis and N. virescen prey species. However, when Ni. lugens and R. dorsalis preys were offered together to the P. fuscipes predator. Recilia dorsalis was predominantly preyed upon by the predator even at low number of individuals but no statistically significant was found between this two species (t = 1.29, df = 138, p = 0.201). Hence, P. fuscipes preferred R. dorsalis > Ni. lugens > N. virescen respectively.

C. Searching behaviour of P. fuscipes on its prey Paederus fuscipes exhibited significantly higher searching rate on the live prey items compared to the dead (carcass) prey (p < 0.05). Our study found that P. fuscipes will immediately attacked the dead preys after short searching period of time once they were placed in the containers. Therefore, less searching attempts by the predator was found when facing the dead prey. This suggests that P. fuscipes normally regulate their predation behaviour accordingly when confronted with its prey.

Interestingly, the least searching behaviour displayed by P. fuscipes predator was on the alive prey of green leafhopper, N. virescen with a mean number of 11.1 ± 3.33. This indicates that P. fuscipes was

80 | P a g e able to capture the live N. virescen without difficulty as it displayed little movement due to its larger size which increases P. fuscipes sight ability to detect it easily, making it more susceptible to predation.

D. Capturing behaviour of P. fuscipes on its prey Paederus fuscipes predator displayed significantly more number of attempts on the alive prey items compared to the dead preys (p < 0.05). Predators need to make more effort on catching and ambushing on the presented alive prey items before they can finally started to feed. The highest number of attempts was displayed on the alive prey of Ni. lugens species with mean number of 4.6 Âą 1.15. When comparing the capturing activities of P. fuscipes on the dead prey items, the greatest attempt rate was on the N. virescen with a mean of 3.3 Âą 0.4. Basically, the number of time spent on searching decreased and the attempt rate increased if the predators are famished and vice versa.

E. Feeding behaviour of P. fuscipes on its prey Paederus fuscipes are voracious feeders which feed on alive and dead prey items. Significantly less feeding time was needed by P. fuscipes in consuming alive prey. It is likely that P. fuscipes spent more time focusing and feeding on the presented dead prey item as they do not need to spend time foraging for their prey. Although when they might finally capture on the alive prey item, they need to scuffle on consuming the alive prey as it is also struggling to escape. Consequently causing the feeding process time of P. fuscipes declined for the alive prey. Thus, considerable significant differences in P. fuscipes predation rates were found between prey conditions presented dead or alive (F = 91.043, df = 1, 54, p = 0.000). Overall, P. fuscipes was significantly observed to spent more time feeding on the dead prey items compared to the alive preys (p < 0.05).

CONCLUSIONS In conclusion, our results demonstrated the Type II functional response curve which indicates the ability of P. fuscipes beetle in regulating phytophagous pest populations by killing them at low density. This is one of the important factors which will control the initial colonization of insect pests once they start invading the rice crop especially after rice seeds were sowed in the rice field.

However, we believe that several other aspects need to be considered for a successful biological control programs. Functional response is expected to be different in the field than in the laboratory.

Still, the results obtained from the laboratory functional response studies are useful in understanding the feeding behaviour of an individual P. fuscipes species. This study showed that P. fuscipes predator is polyphagous in their feeding habits but preferred mostly to feed upon small and soft bodied insect preys. Further studies are necessary to evaluate the predation potential of P. fuscipes beetles as biological control agents in the field. Since actual field study is difficult to conduct, simulated field study can be conducted to produce more realistic conclusions and a better understanding of P. fuscipes predation.

ACKNOWLEDGEMENTS We are grateful to all staffs from School of Biological Sciences, Universiti Sains Malaysia who were involved in this project. This study received financial support from Research University Grant USM (1001/PBIOLOGI/811284). Sincere thanks to The Ministry of Higher Education, Malaysia and Universiti Sains Malaysia Fellowship Scheme for providing postgraduate financial support to Maryam Sufian.

81 | P a g e REFERENCES Chesson, J. 1982. Estimation and analysis of parasitoid search and attack parameters from field data.

Environ. Entomol. 11: 531–537.

Frank, J. H., & Kanamitsu, K. 1987. Paederus sensu lato (Coleoptera: Staphylinidae): natural history and medical importance. J. Med. Entomol. 24: 155-191.

Kalshoven, L. G. E., & Van der Laan, P. A. 1981. Pests of crops in Indonesia. (Revised).

Manley, G. V. 1977. Paederus fuscipes (Col.: Staphylinidae): a predator of rice fields in west Malaysia. Entomophaga 22: 47-59.

Manly, B. F. J. 1974. A model for certain types of selection experiments. Biometrics 30: 281-294.

Rogers, D. J. 1972. Random search and insect population models. J. Anim. Ecol. 41: 369–383.

Singh, G., & Ali, S. Y. 2007. Indian J. Dermatol. Venereol. Leprol. 73: 13–15 Stephens, D. W., & Krebs, J. R. 1986. Foraging Theory. Princeton University Press, Princeton.

Vézina, A. F. 1985. Empirical relationships between predator and prey size among terrestrial vertebrate predators. Oecologia. 67: 555-565.

Kundu, M., Sharma, D., Brahma, S., Pramanik, S., Saha, G. K., & Aditya, G. 2014. Insect predators of mosquitoes of rice fields: portrayal of indirect interactions with alternative prey.

82 | P a g e Sublethal Effects on Permethrin and Malathion on The Life History of Aedes Albopictus (Skuse) Mosquitoes Sumayyah Aminodina, Dinesh A/L Rajendran a, Wan Fatma Zuharaha,b a

Medical Entomology Laboratory, School of Biological Science, Universiti Sains Malaysia b Vector Control Research Unit, Universiti Sains Malaysia

ABSTRACT Aedes albopictus is an important dengue vector in rural and sub-urban areas in Southeast Asia. The use of excessive insecticide in control dengue control program may cause changes in normal mosquitoesâ&#x20AC;&#x; life history. Here, we examined the effect of 0.2% permethrin and 1.5% malathion on three strains Aedes albopictus mosquitoes. Using impregnated paper in bioassay test-tube, the surviving mosquitoes after 24 hour were used for subsequent tests for sublethal effects of Ae. albopictus mosquitoes. Our study found that Ae.albopictus in Penang are still susceptible to permethrin, but have started to develop resistance towards malathion as the mortality rate was at a range of 57%-58% for wild urban and sub-urban strains and 93% for laboratory strains. The longevity of the treated Ae.albopictus were found to be unaffected by both insecticides but have higher rate of fecundity. However, the high longevity and fecundity rate has caused the decrease in fertility rate of both treated mosquitoes strains. These insecticides did not influence the sex ratio of Aedes albopictus as the male to female ratio. It can be seen that resistance towards insecticides has been started in Ae.albopictus in Penang and might pose a full insecticide resistance threat in the future.

Keywords : Aedes albopictus, life history, malathion, permethrin, sublethals.

INTRODUCTION Aedes mosquitoes is considered to be medically important as a vector for some deadly arboviruses like dengue with an amount of 2.5 billion people are residential in highly potential area and there are 390 million infections in a year (Bhatt, 2013). Between 2001 and 2008, 1.2 million cases were reported in 4 countries of Western Pacific region with highest number of cases and deaths as Malaysia included, with 4798 deaths (WHO, 2009).Due to such nuisances and complications, mosquito control approaches were taken in plenty of ways in an attempt to reduce contact between man and mosquito thus reducing human suffering.The unavailability of vaccines and failure in existing dengue control strategies, researchers are looking for proper measurements and techniques in controlling viral transmission. Unfortunately, heavy reliance on insecticides has brought up a rise to insecticide resistance problem (Mallet, 1989). Among the chemicals that are used in controlling mosquitoes are malathion and permethrin which used in thermal fogging to control adult mosquito population.

Malathion is among the insecticide that has high toxicity to fish though it has a low mammalian toxicity (Sanjoy, 2012). Meanwhile, permethrin a is from pyrethroids that have wide range usage and commonly used as contact poison (Heather, 2003).The purpose of this study is to determine the sub lethal effects that occur in Aedes albopictus mosquitoes with the usage of common use of insecticides.

EXPERIMENTAL A.

Obtaining eggs and culturing techniques of Ae. albopictus

83 | P a g e To obtain wild strain of Ae. albopictus, ovitraps were set up at Sungai Dua,Penang for urban strain and Batu Maung for suburban strain. The ovitraps were placed at the intended locations and were left for 3 to 4 days to allow female mosquitoes to lay eggs. Whereas, the laboratory strain was obtained from Vector Control Research Unit, Universiti Sains Malaysia.

B. Adult bioassay for Ae. albopictus using 0.2% permethrin and 1.5% malathion A total of 20 Female Ae. albopictus mosquitoes aging from 2 to 5 days old of each strain were prepared and tested using bioassay kit test tube for 1 hour. The impregnated paper with concentration 0.2% permethrin and 1.5% were used. The number of knockdowns of the mosquitoes was collected at an interval of 1 minute throughout the 60 minutes. After an hour all 20 mosquitoes were then collected and placed in a paper cup with cotton bud soaked in sucrose and was left for 24 hours and mortality was recorded. Three replicates were conducted for each treatments and mosquito strains.

C.

Longevity and fecundity of Ae. albopictus after treated with 0.2% permethrin and 1.5% malathion.

Mosquitoes of each strain and chemical from bioassay were provided blood . The blood-fed mosquitoes were the place individually in separate paper cup and was supplied with 10% sucrose solution. A moist filter paper in cone shape was placed inside each paper cups to facilitate the oviposition substrate. The number of eggs laid by mosquitoes was checked daily till died. Three replicates were conducted for each treatment.

D. Fertility of Ae. albopictus after treated with 0.%2 permethrin and 1.5% malathion Eggs obtained from above were then immersed in separate plastic containers filled with 600 ml of chlorine-free water. The larvae hatched from the eggs were counted daily. The percentage of the egg hatchability was calculated with the following formula:

E. Sex ratio of Ae. Albopictus adult after treated with 0.2% permethrin and 1.5% malathion.

The sex ratio of male and female was calculated with the following formula:

RESULTS A. Mortality rate of Ae. albopictus after treated with permethrin and malathion No knockdowns were observed in the 60 minutes after treated with both insecticides for all the strains.

The mean mortality rate after 24 hours for urban, suburban and laboratory strains of Ae. albopictus treated with 0.2% of permethrin was 71.67% ± 8.82, 73.33% ± 10.14 and 66.67% ± 13.64 respectively . There was no significant difference found between the strains (F=0.144, df=2, P>0.05; Table 1) for treatment using 0.2% of permethrin.

There was an increasing trend on the mortality rate between urban, suburban and laboratory strain of Ae. Albopictus when treated with 1.5% malathion which was 56.67% ± 1.67 for urban, 58.33% ± 6.01 for suburban and 93.33% ± 6.67 for laboratory strains. Mortality rate of urban and suburban strains is significantly lower than laboratory strain (F=14.48, df=2, P<0.05).

84 | P a g e B. Effects of insecticides on longevity of Ae. albopictus.

Comparing between strains, the mean longevity of urban, suburban and laboratory strain of Ae. albopictus showed no significant difference after being treated with 0.2% of permethrin (F=2.48, df=2, P>0.05). As for treatment with malathion 1.5% the suburban strain seems to have highest mean longevity at 15.16 ± 1.44 when compared with the other strains but showed. No significance between the strains (F=0.83, df=2, P>0.05). Meanwhile in control treatment, urban strains seems to have the highest mean longevity (11.22 ± 0.57 days), followed by suburban strain with a mean of (10.95 ± 0.57 day) and laboratory strain with 9.50 ± 0.45 day. (F=2.53, df=2, P>0.05; Table 1).

C. Effects of insecticides on fecundity of Ae. albopictus In permethrin 0.2%, laboratory strain of Ae. albopictus has the highest mean fecundity with a mean of 37.67 ± 9.95 while suburban strain has the least mean fecundity of 17.56 ± 9.49 with no significant differences (F=2.51, df=2, P>0.05). Meanwhile, in malathion 1.5%, there is a clear increasing trend where urban strain fecundity was 19.46 ± 6.01, suburban strain with 26.16 ± 8.00, and laboratory strain with 33.25 ± 9.95. It was also observed that there was no significant difference in any of the strains that were treated with malathion 1.5% (F=0.26, df=2, P>0.05). In control treatment, laboratory strain laid significantly more egg with 22.28 ± 3.54 eggs ,while suburban strain has the least mean fecundity in control with 13.37 ± 3.45. (F=4.06, df=2, P<0.05; Table 1).

D. Effects of insecticides on fertility of Ae. albopictus Based on permethrin 0.2%, suburban strain has the highest fertility rate among the three strains with a mean percentage of 61.44% ± 31.05, followed urban strain with a mean of 31.22% ± 16.74 and finally laboratory strain with a mean of 23.78% ± 4.94. (F=2.31, df=2, P>0.05; Table 1). In malathion 1.5%, no significant difference was also found between the strains (F=1.01, df=2, P>0.05). In control, suburban strain seem to have the highest fertility rate with a mean of 76.48% ± 3.07, followed by urban strain with a mean of 75.22% ± 2.92 and finally laboratory strain with a mean of 66.85% ± 3.17. Similar to the previous treatments, there was no significance seen in between the strains on the control (F=2.80, df=2, P>0.05).

E. Effects of insecticides on sex ratio of Ae. albopictus There was no significant difference on the all treatments of the adult sex (P>0.05). The sex ratio of mosquitoes of all treatment is very close to 1:1 ratio.

Table 1 : Summarize table on the effects of permethrin 0.2% and malathion 1.5% on three strains of Aedes albopictus.

PERMETHRIN 0.2% Mortality rate Longevity Fecundity

Urban 71.67% ± 8.82a 14.18 ± 1.41a 22.06 ± 9.12a

Suburban 73.33% ± 10.14a 11.44 ± 1.34a 17.56 ± 9.49a

Laboratory 66.67% ± 13.64a 10.50 ± 0.61a 37.67 ± 8.75a

Fertility Sex ratio

31.22% ± 16.74a 1: 1.02a

61.44% ± 31.05a 1: 0.99a

23.78% ± 4.94a 1: 1.12a

56.67% ± 1.67 a 12.58 ± 1.11a 19.46 ± 6.01a

58.33% ± 6.01a 15.16 ± 1.44a 26.16 ± 8.00a

93.33% ± 6.67b 11.50 ± 0.65a 33.25 ± 9.95a

MALATHION 1.5% Mortality rate Longevity Fecundity

85 | P a g e Fertility

23.45% Âą 9.44a

32.53% Âą 6.04a

50.93%Âą 25.12a

Sex ratio 1: 0.94a 1: 0.93a 1: 1.26a *Same small alphabet following each value in every row shows that there was no significant difference between the strains DISCUSSION In this study, all strains of Ae. albopictus identified were still susceptible when tested with lower dose of 0.2% permethrin with mortality rate ranging between 66.67%-73.33% . Aedes albopictus in Penang was found still susceptible to permethrin as reported by previous researchers (Chan et al., 2011; Intan et al., 2015).Susceptibility towards permethrin is due to less the consumption of the permethrin insecticides used in vector control program. Malathion seems frequently used in residual spray and thermal fogging (Muda, 1985). The average use of insecticides for both chemical was 256 803 kg for malathion and 45 509 kg for permethrin used worldwide which permethrin was less used than malathion (Zaim, 2007). Hence this excessive usage of malathion in vector control program caused high resistance among the mosquitoes in Penang (Lee, 1997). This was the reason for the significance on the mortality rate between the strains that were treated with malathion 1.5%.

Both insecticides have not fully affected the longevity of the Ae. albopictus. Most Aedes mosquitoes have an average lifespan of 2 weeks to a month (Maricopa, 2006) and most of the treated mosquitoes are around the range but longer than reference laboratory strain especially when treated with 1.5% malathion. Besides, the possible reason happens due to excessive malathion usage worldwide. The increase in longevity strain may also have occurred due to the mosquito changing its physiology process like increasing its longevity with price of reducing its fertility rate when exposed to insecticides (Hoffmann, 2009).

In terms of fecundity rate of suburban wild strains treated with showed the highest rate compared to urban wild and laboratory strains. This finding is in contrast with research by Lee (1996), where the fecundity rate of field strain seems to have lower fecundity rate than laboratory strain. suburban strains has higher fecundity rate possibly because of this strain have experienced high longevity rate which give them chances to produce more eggs. High longevity rate can be accounted in the high fecundity rate of an insect (Robinson, 1979). This can also be due the principle of allocation of the mosquito to increase its fecundity rate in expense of its fertility rate (Hoffmann, 2009).

This study found that higher fecundity in treated wild strains Ae. albopictus female mosquitoes were not permitted to higher fertility after treated with both permethrin and malathion insecticides. The reduction in the fertility rate was most can be associated with the influence of the insecticides which the subsequent effects might be carried by female adults and passed to their eggs. Previous research

by Ngufor (2014) showed that insecticide can affect mosquitoes by reducing fertility rate such as in Anopheles progeny. It can be suggested the eggs that was laid by the treated mosquitoes might have pesticide transference by the mosquito causing it to show embriocidal effects, thus reducing fertility (Irigaray, 2010).

Exposure to both permethrin and malathion insecticides does not affect on the sex ratio of the Ae.albopictus mosquitoes. The sex ratio did not differ was may be due to the decrease of the fertilized eggs induced after treated with insecticides (Delpuech, 2002).

86 | P a g e CONCLUSIONS Fecundity rate of the suburban (wild) mosquitoes seems to be higher than in laboratory (susceptible) mosquitoes due to increase in longevity rate which caused the mosquitoes to change their physiological process. Increase in fecundity rate is to ensure the survival of the species. The increase in longevity and fecundity rate has caused the fertility rate to take the toll through the principle of allocation as the fertility rate was reduced in the insecticide treated mosquitoes as compared to the control. In a nutshell, heavy usage of insecticides like permethrin and malathion may have started to turn Ae. albopictus to develop resistance on them in factors like longevity, fecundity and the fertility of the mosquitoes. In conclusion, this study indicated that both insecticides, malathion and permethrin is still working well for Aedes mosquitoes strains collected from urban and suburban areas as well as the susceptible laboratory strains.

ACKNOWLEDGEMENTS This project was funded under Fundamental Research Grant Scheme (FGRS) by Ministry of Higher Education and Universiti Sains Malaysia (203/PBIOLOGI/6711359). We also thanked the staff and member of the Medical Laboratory, Vector Control Research Unit and School of Biological Sciences,USM for all assistance during this project.

REFERENCES Chan, H.H., Mustafa, F.F.W., Zairi, J., (2011). Accessing the susceptibility status of Aedes albopictus on Penang island using two different assays. Tropical Biomedicine 28(2), 464-470.

Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W., et al. (2013). The global distribution and burden of dengue. Nature, 496, 504-507.

Delpuech, J.M., Meyet J. (2002). Reduction in Sex Ratio of the Progeny of a Parasitoid Wasp (Trochogramma brassicae) Surviving the Insecticide Chlorpyrifos. Arch.Environ. Contam. Toxicol., 45, 203-208.

Heather, I. (2003). Environmental Fate of Permethrin. 1-12.

Hoffmann, G.R. (2009). A perspective on the scientific, philosophical and policy dimensions of hormesis. Concentration- Response, 7, 1-51.

Lee, C.W., Yap, H.H., Chong, N.L. (1996). Comparison of selected biological parameters of laboratory susceptible and field collected strains of the German cockroach, Blatella germanica (L.) (Dictyoptera: Blattellidae). Malaysian Journal of Science, 17A, 37-38.

Intan, H.I., Zairi, J., Hilary, R., Charles, S.W. (2015). Contrasting patterns of insecticide resistance and knockdown resistance (kdr) in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia. Parasites & Vectors 8, 13p.

Irigaray, F. J. S.-D.-C., Moreno-Grijalba, F., Marco, V., PĂŠrez-Moreno, I. (2010). Acute and Reproductive Effects of AlignÂŽ, an Insecticide Containing Azadirachtin, on the Grape Berry Moth, Lobesia botrana . Journal of Insect Science, 10, 33, 1-11.

Mallet, J. (1989). The Evolution of Insecticide Resistance: Have the Insects Won. TREE, vol 4 (11), 336-340.

Maricopa County Environmental Services. (2006). Lifecycle and information on Aedes aegypti mosquitoes. Maricopa County, AZ. Muda A.R. (1985) .Pest problem and the use of pesticide in grain storage in Malaysia. Australian Centrte for Agricultural Research, 11-16.

87 | P a g e Ngufor, C., et. al. (2014). Olyset Duo速 (A Pyriproxyfen and Permethrin Mixture Net): An Eperimental Hut Trial against Pyrethroid Resistant Anopheles gambiae and Culex quinquefasciatus in Southern Benin. 10p.

Sanjoy, D., Rita, M. (2012). A Study on the Effect of Organophosphorus Pesticide Malathion on Hepato-Renal and Reproductive Organs of Heteroptenus fossilis (Bloch). The Science Probe, Volume 1, 1-13.

World Health Organization,. (2009). Dengue: guidelines for diagnosis, treatment, prevention and control. World Health Organization.

Zaim, M., & Jambulingam, P. (2007). Global Insecticide Use for Vector-Borne Disease Control, 3rd edition. World Health Organization. WHO Pesticides Evaluation Scheme. 89p.

88 | P a g e Preliminary Investigation on the Diversity of Zooplankton in Muda River, Peninsular Malaysia Azma Hanim Ismail *,a,, Asmerlda Abu Hassan a a School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia Keywords: zooplankton, Rotifera, Cladocera, Copepoda, Malaysia ABSTRACT The present study aimed to survey the three main groups of freshwater zooplankton (Rotifera, Cladocera and Copepoda) in the Muda River which is the most important water resources for agriculture and water supply in northwestern part of Peninsular Malaysia. A total of 26 taxa were recorded and Rotifera contributes the highest number of species. Among the Rotifera families recorded, Brachionidae and Lecanidae showed greater number of species, in contrast with lower species richness of Cladocera and Copepoda. This checklist is the first insights of zooplankton distribution in Muda River to serve as a basis for future reference of Malaysian zooplankton. *azma_hanim@yahoo.com INTRODUCTION Zooplankton play an important role as a main function for primary and secondary links in the food chain of aquatic ecosystems. The freshwater zooplankton community is largely dominated by three major groups which are Rotifera, Cladocera and Copepoda (Wetzel, 1983). Zooplankton communities are influenced by physico-chemical factors of the river such as temperature, flow rate, dissolved oxygen, pH and nutrient content of the water body (Jafari & Alavi, 2010). Even though they are only few millimeter in length and known as permanent swift water streams organisms, some species of zooplankton can be used as biological indicator to measure the changes of water quality and environmental health in an aquatic ecosystem (Rissik & Suthers, 2009). Thus, the trophic status can be identified by determining the composition, abundance and also the diversity of zooplankton in the ecosystem. Unfortunately, research on zooplankton community structure in the river is still lacking and far less investigated in Malaysia. Therefore, this current study was conducted to determine the zooplankton species distribution and abundance in Muda River. Hence, results of this study are expected to be new information and knowledge on zooplankton community for river management of Muda River and to serve as a basis for future reference of Malaysian zooplankton.

MATERIAL AND METHODS A. Study Site The research was conducted in Muda River which is located in the state of Kedah, northern Peninsular Malaysia (Figure 1). It is the longest river in Kedah with the length of about 180 km and has a drainage area of 4210 km² (Julien et al., 2010). It is a major source of water supply for the people in northern state of Kedah. The major tributaries of Muda River system are the Ketil River, the Sedim River and the Chepir River. At the end of the upstream of this river is a Muda Dam. The amount of annual rainfall in the Muda River is about 2000 - 3000 mm (Ghani et al. 2010). The Muda River has been affected by unsustainable human activities of the urban development which causes floods almost

89 | P a g e every year. This indicates that human activities have led to the perturbations of natural riverine system of Muda River.

B. Field Sampling Field sampling was conducted on February 2015. The weather during the sampling was hot and windy. Zooplankton samples were collected using a conical plankton net opening diameter of 20.5 cm and mesh size of 35 Îźm. The net was towed at six different locations along the river (Figure 1) for a distance of 15 m at 0.1 m from the surface water. Then, the filtrate samples of three replicates were kept in polyethene bottles and preserved with 70% ethanol.

Muda

Figure 1: Location of 6 sampling stations at Muda River (Source: Bing Maps) C. Laboratory Analysis Zooplankton samples were identified and counted using Sedgwick - Rafter counting cell under a compound microscope equipped with a camera at various magnifications. Three sub-samples of 1 ml were analyzed and this procedure was conducted for each sample replicate. Zooplankton species was identified based on Idris (1983) and Shiel (1995).

RESULTS AND DISCUSSION A. Zooplankton Species Composition During the present study, Rotifera was the dominant group in Muda River followed by Cladocera and Copepoda. Table 1 indicates the distribution of zooplankton species composition which was recorded at Station 1 until Station 6 of Muda River. The total number of zooplankton taxa that was registered across the 6 stations in Muda River was 26 with 4 orders and 10 family members. For Rotifera, 3 orders and 8 families were recorded. The most representative Rotifera were Brachionidae which included Brachionus forficula, Brachionus caudatus, Brachionus sp., Keratella cochlearis, Keratella sp., Anureopsis coelata and Anureopsis sp. Among the Cladocera, Bosminidae was the most diverse family while Cyclopoida was the only order observed for Copepoda.

90 | P a g e Rotifera are a common feature in zooplankton communities that occurring almost exclusively in freshwater environments and also a dominant group in freshwater ecosystem (Melo et al., 2014).

Their diverse feeding habits lead this group to occur at high density in most aquatic systems (Gosselain et al., 1998). Meanwhile, Cladocera and Copepoda contribute a small composition in the present study. This is probably due to fish predation that reduces the densities of the larger cladocerans and copepods taxa compared to the smaller sized of zooplankton such as rotifers (Jack & Thorp, 2002).

Table 1: Species composition of zooplankton at each sampling station in Muda River Station Taxa/Order

Family

Species 1

2

3

4

5

6

Lecane bulla

+

-

-

-

-

+

Lecane curvicornis

-

+

-

-

+

-

Lecane hamata

-

-

+

-

-

+

Lecane stenroosi

-

-

-

+

-

-

Lecane leontina

-

-

-

-

-

+

Lecane sp.

+

+

+

+

+

+

Brachionus forficula

-

-

-

+

+

-

Brachionus caudatus

-

-

-

-

+

-

Brachionus sp.

-

+

+

+

+

-

Keratella cochlearis

+

-

+

-

-

+

Keratella sp.

+

+

+

+

+

+

Anuraeopsis coelata

-

-

-

-

+

-

Anuraeopsis sp.

-

-

-

-

+

+

Trichotriidae

Macrochaetus sp.

-

-

-

+

-

-

Proalidae

Proales sp.

-

-

+

-

-

-

Proalides sp.

+

-

-

-

-

-

Asplanchnidae

Asplanchna sp.

+

+

-

+

+

-

Trichocercidae

Trichocerca sp.

-

+

+

+

-

+

Synchaetidae

Polyarthra sp.

+

+

+

+

+

+

Rotifera Ploimida

Lecanidae

Brachionidae

91 | P a g e

Flosculariacea

Filiniidae

Synchaeta sp.

+

+

+

+

+

+

Filinia sp.

+

+

-

-

-

-

+

-

+

+

-

+

Bosmina longirostris

-

+

-

-

-

-

Bosmina sp.

-

+

-

-

-

-

Diaphanosoma sp.

-

+

-

-

-

-

+

+

-

+

+

+

Bdelloidea Cladocera Bosminidae

Sidiidae Copepoda Cyclopoida Note: „+‟ = presence, „-‟ = absence

B. Zooplankton Abundance Figure 2 illustrates the relative abundance (%) of zooplankton groups in the study site. Rotifera contributed with 96.3% followed by Copepoda (2.6%) and Cladocera (1.1%). The lower abundance of Cladocera might be due to less vegetation and open water condition of the study sites that make the cladocerans easy vulnerable to the predators. According to Czerniawski & Pilecka-Rapacz (2011), high abundance of crustacean was observed in rivers which were relatively covered by macrophytes or vegetation coverage. This condition will enable them to escape from fish and hiding into the dense vegetation of macrophytes. Although cladocerans usually are less abundant than rotifers, they often make up an important part of the community due to their larger size (Pennak, 1989).

96.3%

1.1%

Rotifera

2.6%

Copepod a

Figure 2: Relative abundance (%) of zooplankton groups in Muda River

Figure 3 shows the mean value of zooplankton abundance in the study sites from Station 1 until Station 6. The highest mean value of zooplankton abundance occurred at Station 2 (1578 ± 198 ind/m³) while the lowest mean value was recorded in Station 6 (844 ± 146 ind/m³). The values were comparable to those reported in Black Volta River, Ghana (Alhassan et al., 2015) and Haraz River, Iran (Jafari et al., 2011) with organisms ranging between 160 – 1911 ind/m³ and 805 – 1126 ind/m³ respectively. Based on the result of one-way ANOVA, there was a significance difference of zooplankton abundance between sampling stations (p<0.05) in the present study.

92 | P a g e

Mean abundance (ind/m³)

1800 1300 800 300 -200

1

2

3

4

5

6

Station Figure 3: Mean zooplankton abundance at each sampling station in Muda River C. Ecological Indices The values of ecological indices of zooplankton in the present study are shown in Table 2. According to Zar (1999), ecological indices signify as richness and how the species abundances are distributed among the species. The values of Shannon-Weiner index in the present study fluctuated between 1.561 and 2.264. Mason (1988) points out that Shannon-Weiner index values in the range of 1 to 2 are characteristics of moderate polluted condition while the value above 3 express stable environmental conditions.

The values of Margalef index were between 2.46 and 3.12. The larger the index value, a more healthy the body of water (Paul & Nandi, 2005). Thus, this index indicates that Station 6 was the richest station in terms of the species richness. Pielou‟s evenness index values varied from 0.628 to 0.911 and the values which are more than 0.5 indicated that the zooplankton ecology is balance during the study period (Frutos et al, 2009).

Table 2: Ecological indices at all sampling stations during the study period Station

Shannon-Weiner index, H‟

Margalef index, R1

Pielou‟s evenness index, J

1

1.724

2.66

0.719

2

1.561

2.80

0.628

3

2.170

2.46

0.905

4

1.888

2.92

0.788

5

2.137

2.96

0.860

6

2.264

3.12

0.911

CONCLUSIONS Species composition generated in the present study will contribute to the understanding on geographical distribution of freshwater zooplankton particularly in Malaysian river as well as benefit

93 | P a g e the decision-makers on conservation efforts. Other than that, a species list of zooplankton diversity is an important biological indicator of environmental change in river ecosystem. Further research on zooplankton community dynamics in this river is necessary by increasing temporal and spatial sampling in order to gain most accurate results. Detail sampling on abiotic and biotic factors also required which may influence the distribution and abundance of zooplankton community in the ecosystem.

ACKNOWLEDGEMENTS This research project was supported by funds from Universiti Sains Malaysia (grant no.

304/PBIOLOGI/6313037). The authors are grateful to Dr. Russell Shiel for helping with the identification of zooplankton.

REFERENCES Alhassan, E. H., Ofori-Danson, P. K. and Samman, J. (2015). Ecological impact of river impoundment on zooplankton. Zoology and Ecology. 25 (2), 136-142.

Czerniawski, R. and Pilecka-Rapacz M. (2011). Summer zooplankton in south rivers in relation to selective conditions. Central European Journal of Biology. 6(4), 659- 674.

Frutos, S. M., Poi de Neiff, A. S. and Neiff, G. (2009). Zooplankton abundance and species diversity in two lakes with different trophic states (Corrientes, Argentina). Acta Limnol. Bras. 21 (3), 367–375.

Ghani, A. A., Ali, R., Zakaria, N. A., Hasan, Z. A., Chang, C. K. and Ahamad, M. S. S. (2010). A temporal change study of the Muda River system over 22 years. International Journal of River basin Management. 8(1), 25- 37.

Gosselain, L.V., Viroux, L.P. and Descy, J. (1998). Can a community of small bodied grazers control phytoplankton in rivers? Freshwater Biology. 39(1), 9-24.

Idris, B. A. G. (1983). Freshwater zooplankton of Malaysia (Crustacea: Cladocera).

Universiti Pertanian Malaysia Serdang, Selangor, Malaysia.

Jack J. D. and Thorp J. H. (2002). Impacts of fish predation on an Ohio River zooplankton community. J. Plankton Res. 24, 119-127.

Jafari, N. and Alavi, S. (2010). Phytoplankton community in relation to physico-chemical characteristics of the Talar River, Iran. J. Appl. Sci. Environ. Manage. 14 (2), 51 – 56.

Jafari, N., Nabavi, S. M. and Akhavan, M. (2011). Ecological investigation of zooplankton abundance in the River Haraz, Northeast Iran: impact of environmental variables. Arch. Biol. Sci., Belgrade. 63 (3), 785-798.

Julien, P. Y., Ab. Ghani, A., Zakaria, N. A., Abdullah, R. and Chang, C. K. (2010). Case study: Flood mitigation of the Muda River, Malaysia. Journal of Hydraulic Engineering. 136, 25 - 161.

Mason, C. F. (1988). Biology of fresh water pollution. Longman Scientific and Technical, Harlow.

Melo, T. X., Lourenco, L. J. S. and Medeiros, E. S. F. (2014). Checklist of zooplankton from the upper Ipanema River (Pernambuco), an intermittent river in semi-arid Brazil. Check List. 10 (3), 524-528.

Paul, S. and Nandi, N. C. (2005). Assessment of health of Hugli River by benthos inhabiting Tolly‟s Nullah Outfall Region, Kolkata. In Fundamentals of Limnology. Kumar, A. (ed). pp 25-35. APH Publishing, New Delhi.

Pennak, R.W. (1989). Fresh-water invertebrates of the United States: protozoa

94 | P a g e to mollusca. 3rd ed. John Wiley & Sons, Inc., New York.

Rissik, D. and Suthers, I. (2009). The importance of plankton. In Plankton, a guide to their ecology and monitoring for water quality. 1st ed. Suthers, I. M. and Rissik, D. (Eds). pp 2-4. CSIRO Publishing, Melbourne.

Shiel, R. J. (1995) A guide to identification of rotifers, cladocerans and copepods from Australian Inland Waters. Cooperative Research Centre for Freshwater Ecology Identification Guide No. 3.

Murray Darling Freshwater Research Centre, Albury.

Wetzel, R.G. (1983). Limnology. 2nd ed. Saunders, Philadelphia.

Zar, J. H. (1999). Biostatistical analysis. Prentice Hall, Upper Saddle River, New Jersey.

95 | P a g e Effects of Excessive Light in Changing Life History of Aedes Aegypti Mosquito Wan Fatma Zuharah* a,b, Liew Yan Hui a, Nik Fadzly a,c a

School of Biological Sciences, Universiti Sains Malaysia, Minden, Malaysia Vector Control Research Unit, Universiti Sains Malaysia, Minden, Malaysia Centre of Marine & Coastal Studies (CEMACS), Universtiti Sains Malaysia, Minden, Malaysia Keywords : Aedes aegypti, Incandescent, LED, Light, Mosquito b

c

ABSTRACT Aedes aegypti, cosmopolitan species, is a vector that responsible for the spread of dengue fever and dengue hemorrhagic fever in Southeast Asia. Exposure to light use by modern human has caused changes in their life history. Here, we tested the effects of light exposure duartion on the life history of Ae. aegypti mosquito. Aedes aegypti mosquito eggs were exposed to three different durations of light exposure in the ratio of: (1) 1 light: 23 dark hours, (2) 6 light: 18 dark hours, and (3) 12 light: 12 dark hours. Two different types of light bulbs were chose for this study, (1) incandescent light bulb and (2) LED light bulb. Our results using Tukey test revealed no significant difference were found at P < 0.05 in life history aspects for (1) hatching rate, (2) survival rate and (3) development days between three different durations of light exposure. The sex ratio obtained was approximately 1: 1 ratio. In conclusion, we found that light does gave a little effect on affecting the development of Ae. aegypti life history, however the effect was found not significant. *wfatma@usm.my INTRODUCTION Aedes aegypti and Ae. albopictus are sympatric which are inclined to choose the same artificial containers to lay their eggs (Honorio et al. 2009). The structure of the septic tank in Malaysian houses that attach from house to house provided an ideal place for the mosquitoes to breed. The unpolluted water, which is stuck in the pocket or in the wall of the uncovered and broken septic tanks provided new breeding sites for Aedes mosquitoes (Nwoke et al. 1993, Barrera et al. 2008, MacKay et al. 2009) and was found that the Ae. aegypti is the more superior competent compared to Ae. albopictus. Both of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) have been known as vector species for dengue fever for a century.

Insectsâ&#x20AC;&#x; rate of activity such as roaming and finding food, resting posture may also be affected by the light (Bertholf, 1940). The reason why insect have such capability is because they possess special receptor which is called as photoreceptor allowing them to detect UV and colors from the light sources. Few studies in the aspect of behavioral or electrophysiological has been done to prove positive reaction of insect eye toward ultraviolet irradiation (Hamdorf et al. 1971, Meffert and Smola 1976). Beetles was found to have positive reaction and attracted to specific region of spectrum of light and different light intensities with wavelength range between 365 - 528 nm (violet-blue, blue, bluegreen), but only a very low percent show respond to longer wavelength ( yellow-orange, orange-red, infra- red) (Weiss et al. 1941). It is proven that, light plays a role as the switch or indicator controlling the daily activity of the insect. Light and temperature do cause an effect on the insectâ&#x20AC;&#x;s larvae to undergo diapauses, affecting their development period to reach adult stage and changing their normal behavior (Lutz 1932).

96 | P a g e Light often being use as a visual lure to attract nocturnal insect during night time to observe their abundance and occurrence in field study (Nowinszky 2003). Light is an electromagnetic radiation that is visible to human and it also refer as "visible light" that have wavelength in between 400 nm (Ultraviolet) to 700 nm (infrared). Incandescent light bulbs produce light by heating the filament at a fraction of radiation that falls into the visible light spectrum with electrical current until glowing occur. At higher temperature the light will shows color changes from red to white to blue. An improved version incandescent light bulb is with the addition of small amount of halogen such as bromine or iodine. However, incandescent light bulb is consider as a less efficient light bulb as it only able to convert less than 5% of the energy received into visible light, while other energy is released in heat energy (Keefe, 2007). Slowly incandescent light bulb is replaced with other more efficient light bulb such as fluorescent lamp, and light-emitting diode (LED) especially in the industrial lighting field (Schubert and Kim, 2005). Due to the advantages of LED which is durable, higher efficient of illumination, longer life-span compare to other type of lamp, it is widely used in all field such as industrial application, vehicle lighting, and even housing decoration lighting. Large number of insects being attracted to LED light including Trichoptera, Coleoptera, Hymenoptera, Hemiptera, and Diptera (Pawson and Bader 2014).

It cannot be denial that as urban expansion continues, white light LED lighting in industrial applications will also increase drastically and cause white light pollution in developing area and also higher chances of attracting flying insects.Therefore, our aim of study is to investigate the effects of lights (incandescent and LED) with the different exposure time on the hatching rate, survival rate and development of a dengue vector, Ae. aegypti mosquito.

EXPERIMENTAL A. Mosquito colony The laboratory strain of Aedes aegypti was obtained from Vector Control Researches Unit, Universiti Sains Malaysia (VCRU). Plastic containers measuring 16.5cm x 11.5cm x 7.0cm were filled up with 215ml of tap water and left for at least 24 hours to reduce the chlorine content which may affecting the hatching rate of the eggs.

B. Exposure of excessive light toward the mosquito population Two types of light bulbs were used in this study: (1) the light-emitting diode (LED) light and (2) the incandescent light bulb. A total of 100 eggs were placed into the plastic container containing 215ml of chlorine-free water. Containers was then exposed to three specific different duration of times for each type of light bulbs with the ratio of Light: Dark (L:D) of; (1) 12 hours : 12 hours which is the normal day times experience in the country, Malaysia, (2) 6 hours : 18 hours which similar to the period of time of light switch on for those rarely stay in home, and (3) 1 hour: 23 hours as the minimum light exposure as comparison. The exposure of light with the same duration was offered until the eggs were fully developed from the egg stage until matured adult stage in separated experiments. Larvae were feed with 0.5gm of larval food daily as the experiment processed. Throughout the experiment, at the interval of 24 hours, the data for the percentage of hatching rate, number of development days, the percentage of survivorship and the sex ratio of the adult mosquitoes were recorded.

C. Statistical analysis Data for hatching rate, survival rate and development days of Ae. aegypti mosquito after the exposure to the light were analyzed using two-way Analysis of variance (ANOVA) in IBM SPSS Statistic 22

97 | P a g e program for window 7. Log transformation of data was performed for hatching rate and survival rate to fulfill ANOVA assumption. Sex ratio of analysis is conducted by comparing the number of each group between male and female using T-test.

RESULTS AND DISCUSSION A. Hatching rate and survival rate after exposure to the lights.

The hatching rate of Ae. aegypti eggs in exposure to incandescent light rose with the increases duration of the light exposure, while the hatching rate of the eggs under LED light exposure decreases as the duration of light increases. However, no significant difference were found between the types of light (F=1.427, df=1, p=0.256) and duration of light exposure between LED and incandescent (F = 2.281, df = 2, P = 0.145, Table 1).

Whereas, survival rate of Ae. aegypti that reached adult stage was higher in incandescent light bulb compared to the one exposed under LED light bulb in all of the duration of light exposures. But, no significant differences was found in type of lights and duration of exposure (p>0.05, two way ANOVA, Table 1).

Table 1: Results of two-way ANOVA analysis on hatching rate and survival rate of Ae. aegypti after the exposure to incandescent and LED light bulbs at different duration of time.

Variables Mean square df F Sig. Hatching Rate

Survival Rate

Light (L)

0.028

1

1.422

0.256

Duration (D)

0.046

2

2.281

0.145

LxD

0.110

2

5.521

0.020

Error

0.020

12

Light (L)

0.032

1

3.435

0.089

Duration (D)

0.07

2

0.735

0.500

LxD

0.05

2

0.507

0.615

Error

0.009

12

B. Developments days after exposure to the lights The average development days required to reach adult stage was shorter in incandescent light compared to LED light bulb (Figure 1 & 2). In which with the ratio of light: darkness of 1: 23, the developments days required for the L1 larvae to reach adult stage in incandescent light bulb is 13.27 days, while in LED light bulb is 13.90 days. However, we found no significant differences among three different durations of light exposure in incandescent light at each stage of mosquito lifecycle (p>0.05), except during adult stage (F = 11.25, df = 2, P = 0.009, Figure 1). While, in LED light bulb we found there were no significant different during each development days at every stage of mosquito lifecycle between different light duration (Figure 2).

98 | P a g e

Figure 1: Comparison between different duration of light exposure using incandescent light bulb.

Figure 2: Comparison between different duration of light exposure using LED light bulb.

C. Sex allocation of adult mosquitoes after exposure to the lights Figure 3 showed that the entire male: female having approximate 1: 1 ratio in all of the condition.

There were no significant differences between sex allocation of male: female in all types of light and duration of exposure (p>0.05).

Figure 3: Sex allocation between male and female of Ae. aegypti after exposure to light.

Our study indicated that the hatching rate in incandescent light bulb condition is increasing as the duration of light exposure increases probably due to the heat released from the incandescent light bulb. Longer period of exposure of light from incandescent light bulb create a constant slightly higher temperature compare to normal room temperature which is around 27-35째C. In the case of LED light bulb, higher hatching rate was observed compared to the one in incandescent light bulb. But, hatching rate decreased when the duration of light exposure was in longer term at 12 hours. As heat is not produced from the LED light bulb, unlike incandescent light bulb which release most of it energy as heat (Keefe, 2007). Therefore, temperature of the eggs experience was less fluctuated compare to the one in incandescent light bulb which is closer and constant to room temperature of 27 to 28째C which is the optimum temperature that suitable for the egg hatching.

99 | P a g e The survival rate of the mosquito larvae throughout the process had similar trend for both incandescent light bulb and LED light bulb. Generally colony reared under incandescent light bulb has higher survival rate than those in LED light bulb. This may due to the different amount of larvae food intake. Mosquito larval are poikilothermic as their body temperature is affected by the temperature of the surrounding (Heinrich & Zoologists, 1981). Incandescent light bulb will produce more heat amount compare to LED light which does not produce heat. In response to heat from the incandescent light bulb will indirectly causing high metabolic rate on the larvae and consumption rate would be faster with more food is needed compared to those undergo LED light bulb condition.

The trends on the development rate of Ae. aegypti found against our original hypothesis. In which the shorten the duration of light exposure is, the development rate of the mosquito larvae will increase due to mosquito larvae will behave just like their adult stage where they tend to be more active during dark period and tend to eat more with faster development rate or it affecting the insect photoperiodism. However our result indicated that there were no significant differences on the development of larvae in each stage under influence of different duration of light exposure. However comparing between duration of 1: 23 and 12: 12, shorter light period required a longer time to reach adult stage compare to longer light period in both incandescent and LED condition. Unlike other experiment done by Dickson and Sanders (1945), the study showed that light actually play a roles in affecting the diapause of the Oriental fruit moth, Grapholita molesta during the larval stage where increasing in the light period will causes the larval undergo diapause and prolong the development periods to reach adult stage. In the study by Way and Hopkin (1950) involving temperature and photoperiod on Diataraxia oleracea during the larval stage, showed that at low temperature (10 to 20 °C) and short photoperiod (less than 4 hours) would induced the diapause of the larval while at high temperature (30 - 34°C) and long photoperiod (more than 14 hours) would prevent diapause of larval occur, however when rearing occurring in high temperature without any light, the diapause is prevented. This study by Way and Hopkin (1950) probably explained our study and development of Ae. aegypti required slightly more days at 1:23 compare to 12:12 light: dark ratio to reach certain stages. However these cases only be observed at the early stage of the larvae, and as closer to adult stage the pattern changed as the influence from the light does give less effect on the pupa to adult stage.

Most of the animals follow the approximate 1:1 Fisher‟s ratio of sex allocation and that include the Aedes aegypti in the experiment. From the experiment, even though the mosquito undergo different type of light bulb and even different type of duration, it does not cause any severe effect that would alter the sex of the mosquito skewed toward one side.

CONCLUSIONS In conclusion, exposure to LED light bulb had prolonged the development of Ae. aegypti mosquitoes‟ life cycle compared to those under influence of incandescent light bulb. Prolong of mosquito development time have a positive side which will offers longer time for controlling adult mosquito populations.

ACKNOWLEDGEMENTS The authors are grateful to the staff of School of Biological Sciences, Universiti Sains Malaysia and Vector Control Research Unit, Universiti Sains Malaysia for field assistance and mosquito culture.

100 | P a g e This project was supported by Fundamental Research Grant (FRGS: 203/PBIOLOGI/6711359) and Transdisiplinary Research Grant Scheme (TRGS: 203/PBIOLOGI/6765005) Ministry of Higher Education Malaysia and Universiti Sains Malaysia.

REFERENCES Honorio, N.A., Castro, M.G., de Barros, F.S.M., Magalhaes, M.D.A.F.M., and Sabroza, P.C. (2009) The spatial distribution of Aedes aegypti and Aedes albopictus in a transition zone, Rio de Janeiro, Brazil. Cad. Saude Publica, Rio de Janeiro 25(6), 1203-1214.

Nwoke, B.E., Nduka, F.O., Okereke, O.M., and Echighibe, O.C. (1993) Sustainable urban development and human health: septic tank as major breeding habitat of mosquito vectors of human diseases in South-Eastern Nigeria. Appl. Parasitol. 34(1), 1-10.

Barrera, R., Amador, M., Diaz, A., Smith, J., Munoz-Jordan, J.L., and Rosario, Y. (2008) Unusual productivity of Aedes aegypti in septic tanks and its implication for dengue control. Med Vet Entomol. 22, 62-69.

Mackay, A.J., Amador, M., Diaz, A., Smith, J., and Barrera, R. (2009) Dynamics of Aedes aegypti and Culex quenquefasciatus in septic tanks. J. Am. Mosq. Control Assoc. 25(4), 409-416.

Bertholf, L.M. (1940). Reactions to light in insects. Bios. 11(1), 39-43.

Hamdorf, K., Schwemer, J., and Gogala, M. (1971) Insect visual pigment sensitive to ultraviolet light. Nature. 231, 458-459.

Meffert, P. and Smola, U. (1976). Electrophysiological measurements of spectral sensitivity of central visual cells in eye of blowfly. Nature. 260 (5549):342 - 344.

Weiss, H.B., Soraci, F.A., and McCoy, E.E. (1941). Notes on the reactions of certain insects to different wave-lengths of light. J. NY Entomol. Soc. 49(1): 1- 20.

Lutz, F. E. (1932). Experiments with Orthoptera concerning diurnal rhythm. Am. Museum of Nat. History. No.550.

Nowinszky, L. (2004) Nocturnal illumination and night flying insects. App. Ecol. Environ. Res. 2, 17–52.

Keefe, T.J. (2007). The Nature of Light. http://www.ccri.edu/physics/keefe/light.htm Retrieved 200711-05 Schubert, E. F., and Kim, J. K. (2005). Solid-state light sources getting smart. Science. 308,1274– 1278.

Pawson, S.M., and Bader, M. K-F. (2014) LED lighting increase the ecological impact of light pollution irrespective of color temperature. Ecol. App. 24(7), 1561–1568 Way. M.J., and Hopkins B.A. (1950) The influence of photoperiod and temperature on the induction of diapause in Diataraxia oleracea L. (Lepidoptera). J. Experiment. Biol. 27, 365-376.

Dickson, R. C., and Sanders, E. (1945) Factors inducing diapause in the oriental fruit moth. J. Economic Entomol. 38, 605-606.

101 | P a g e Insecticide Residues on Poultry Manures: Comparing the Extraction Methods and Field Efficacy Test on Selected Insecticides in Managing Musca Domestica Population Song-Quan Ong, Abdul Hafiz Ab Majid*, Hamdan Ahmad and Fadhlina Hazwani binti Mohd Fadzlah Household & Structural Urban Entomology Laboratory, Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia, Email: abdhafiz@usm.my

In this study, bifenthrin, imidacloprid and fipronil were applied on the natural infest manures according to the manufacturer rate during a broiler breeding cycle. Two extraction methods were prior studied for their suitability and sensitivity in detecting the target compound and later, quantification and qualification of the insecticides in field condition were investigated. UAHHE modified from EPA 3550 method and SDIE were demonstrated and compared for their sensitivity in detecting the insecticides in poultry manures. The detection limits of the compounds were significantly higher when applied SDIE (bifenthrin-6.77ppm, fipronil-31.40ppm and imidacloprid-352.97ppm), compared to the UAHHE (bifenthrin-2.25ppm, fipronil-2.37ppm and imidacloprid-1.47ppm). In the field trial, three insecticides showed accumulation during the broiler breeding period and it is suggested that they were act as adulticides when applied on the poultry manures due to the significant correlation between the increment of insecticide residues to the reduction percentage of adult flies (<0.05).

Keywords Extraction method, Ultra-Performance Liquid Chromatography (UPLC), poultry manure, Musca domestica Introduction Insecticidal treatment on poultry manures is a common practice for controlling the population of the major pest, house fly, Musca domestica L (Diptera; Muscidae) in poultry farm.1) To assess the efficacy and safety of an insecticide, quantification of the insecticide residues on the treated substrates is essential to the evaluation.2) Yet the methods to determine the quantification of insecticide residues on the poultry manures are vague due to the limited reported detection methods. Although some standard organization such as Environment Protection Agency (EPA), America Standard Testing Methods (ASTM) and America Official Analytical Chemists (AOAC) propose some standard methods for study the insecticide residues, but these methods are focusing on soil, water, agricultural products or food,3-4) excluding poultry manures.

Qualification of insecticide residues in poultry manures is important in studying the efficacy of an insecticide. Department of Veterinary Services (DVS) Malaysia have reported some of the poultry farms in Malaysia were applied termicides to protect the wooden poultry houses and at the same time, controlling the house fly population. Nevertheless, the efficacy of the termicides toward the house fly is little been studied in Malaysia, although some of them have been used for controlling the adult fly.56)

Therefore, in this study, three insecticide; bifenthrin, imidacloprid and fipronil were prior studied using two extraction methods, ultrasonic assisted hexane-hexane extraction (UAHHE) that was modified from EPA 3550 method and solvent direct-immersion extraction (SDIE) for their sensitivity in extracting the residues in poultry manures and subsequently, quantified using Ultra-Performance Liquid Chromatography (UPLC). The greater sensitive extraction method was selected for the quantification and qualification investigation at the later field trial. The study aim to provide researchers and pest control manager a more sensitive technique in detecting the residues and their residues efficacy in controlling house fly on poultry farm Malaysia.

102 | P a g e Materials and Methods 1.0 Reagents The insecticides‟ selection were based on the information provided by the farmers in Perak, Malaysia that the insecticides were applied as insecticides to control house fly and termite in the poultry farms in the year of 2013.

High purity insecticides were used as standard for quantification comparison in Ultra-Performance Liquid Chromatography (UPLC). Bifenthrin (Fluka, 98.5%), fipronil (Fluka, 97.9%) and imidacloprid (Chem Service, 99.5%) were acquired from Sigma-Aldrich Malaysia whereas, the solvents: dichloromethane, acetone and hexane (Fisher Chemical, Malaysia) used for extraction were technical grade. For UPLC analysis, HPLC grade of acetonitrile (Fisher Chemical, Malaysia) and ultra-pure water from Milli-Q® purification system were used.

For the field trial, Maxxthor SC (Bifenthrin 10%, Ensystex Australasia Pty Ltd), Prothor SC (Imidaclopril 20%, Ensystex Australasia Pty Ltd) and Regent®50SC (Fipronil 5.0%, Bayer) were treated on the natural infected poultry manures according to their manufacturer recommended rate.

The manufacturer, content and the application rate were presented in Table 1.

2.0 Residues Study 2.1 Validation of the Insecticides Detection For the preparation of insecticide standards, high purity of chemicals was used for comparison. Five standards ranging in concentration from 50 to 500ppm were prepared by weighing the analytical grade of bifenthrin (Fluka, 98.5%), fipronil (Fluka, 97.9%) and imidacloprid (Chem Service, 99.5%) in a 25ml volumetric flask and acetonitrile (HPLC grade, Fisher Chemical, Malaysia) was added.

Later, the solutions were prior filtered by 0.20µm syringe filters (Acrodisc ®, Pall Corporation) before transferred to the glass vial for UPLC analysis.

2.2 Ultrasonic Assisted Hexane-Hexane Extraction (UAHHE) Extraction was conducted on the manures samples that treated with the insecticides (according to the manufacturer rate) on the poultry farm. Ultrasonic assisted hexane-hexane extraction (UAHHE) was a modified version of the EPA method 3550.7) The manure sample was first homogenized by an electrical stirrer and 5g of the wet sample was weighted in a universal bottle and 2g of anhydrous potassium sulfate was added to remove the moisture inside. The sample was mixed with a 10 mL of 3:7 (v/v) acetone/water solvent and sonicated for 10 minutes. Later, 5ml of hexane was added and the mixture was sonicated for another 10 minutes and vortex for 5 minutes. The mixture was allowed to separate into two layers by gravitation and 5 ml of the upper hexane layer was transferred into a clean 10mL centrifuge tube and 5ml of hexane was added. Then, the mixture was further sonicated for 10 minutes and centrifuged by the Hettich Universal 320 (Hettich Zentrifugen, UK) at 2000 rpm for 5 min. Afterwards, 5 ml of the upper layer was transferred into a new centrifuge tube and 5 ml of hexane was added and sonicated for 10 minutes. The upper layer removal, new hexane addition, sonication and then centrifugation were repeated three times before proceeded to the clean-up stage.

2.3 Solvent Direct-Immersion Extraction (SDIE) In contrast, solvent direct-immersion extraction (SDIE) was modified from CDFA (2011) and Hernandez et al. (2012).8-9) A 5 g of sample was weighted in a 100 ml beaker and 20 ml of solventacetone: water: specific solvent in the ratio of 1:1:2 of was added. The specific solvent was referred to

103 | P a g e the high solubility of active ingredient for the respective insecticide based on PubChem. 10) Imidacloprid was more soluble in dichloromethane (at 20ºC: dichloromethane 67 g/L), bifenthrin was highly soluble in hexane (at 20ºC, >600 g/L), and filpronil more soluble in acetone (at 20ºC, 545.9 g/L). The sample with the solvent were homogenized by a table shaker at 200 rpm for 2 hours and immersed for 24 hours. The sample was covered by aluminum sheet and a black-color plastic cover to prevent any photo-decay of the compound. The top solvent layer were removed to a clean 10mL centrifuge tube and subjected for centrifugation by Hettich Universal 320 (Hettich Zentrifugen, UK) at 2000 rpm for 5 min. The supernatant was preceded to the clean-up and concentrated stages.

2.4 Clean-Up and Concentrated Stages The clean-up stage was referred to the EPA 3660c.11) Solid-phase extraction (SPE) C-18 was preconditioned by 4 ml of methanol then 5ml of ultra-pure water and vacuumed until dryness. Five milliliters of extracted hexane from the UAHHE and supernatant from SDIE were loaded to the SPE, separately at the rate of 1drop/s and the residue were filtered through 0.20 µm syringe filter (Acrodisc®, Pall Corporation) before concentrated to dryness at 55 ± 5ºC in a ventilated oven. The dried remain was reconstituted in 1mL of acetonitrile in the amber glass vials for UPLC analysis.

2.5 Ultra-Performance Liquid Chromatography-UPLC Analysis Prior methods have been employed to measure residues of insecticide such as gas chromatographymass spectrometry (GC-MS) and high performance liquid chromatography (HPLC).12) However, GC is poor to determine some insecticides directly due to the weak volatility, polarity and thermal instability of the insecticides. In contrast to GC, LC is more effective and appropriate for the residual analysis of insecticides and in this study, ultra-performance liquid chromatography (UPLC) was applied for compound detection and quantify the insecticide. The compounds- imidacloprid, befenthrin and fipronil were analyzed using an ACQUITY UPLC™ WATER® system consisting of a PU-1580 pump coupled to an HG-1580-31 mixer and an photodiode array (PDA) detector with programmable excitation and emission wavelengths. Separation was achieved using an ACQUITY UPLC™ BEH C18 Column (1.7 µm x 2.1 mm x100mm). The premixed mobile phase used 20% water in methanol at a flow rate of 0.1 ml/min and the injection volume was 5 µL. The PDA detector was set with an excitation wavelength of 270 nm for Imidacloprid, 204 nm for bifentrin and 280 nm for fipronil. The initial mobile phase of water/ acetonitrile was 30:70 (v/v) for imidacloprid, 60:40 (v/v) for bifentrin, and 30:70 (v/v) for fipronil.13-14) 2.6 Quantitative Calculation The quantitative measurement for the insecticide‟s residue followed that of CDFA (2011)8): R=

(Spl H)(Std C)(Std V)(V) / (Std H)(Spl V)(Spl W)

Where R is the residues in part per million (ppm), Spl H is the sample peak height area, Std C is the standard concentration in ppm, Std V is the standard volume injected in µL, V is the volume after the injection in mL, Std H is the standard peak height area, Spl V is the sample volume injected in µL and Spl W is the sample weight in g.

3.0 Quantification Study and Field Trial of the Insecticides 3.1 Location The 12-week study was conducted from March to June 2014 on a broiler breeder poultry farm in an oil palm plantation in of Ayer Tawar, Perak, Malaysia, at the coordinates 4°21ꞌ20.48ꞌꞌN,

104 | P a g e 100°48ꞌ02.59ꞌꞌE. The farm occupies an estimated 10 hectares and contained 16 high-rise wooden poultry houses that measured approximately 60 meters long, 10 meters wide and 12 meters high.

Manure accumulated below the houses at depths of 1-1.8 meters and was removed after the breeding cycle (32-40 days after the introduction of one-day-old chicks).

3.2 Ethics Statement The poultry are commercially reared for the purpose of chicken meats. No testing was performed on these animals, only the insecticidal treatment of poultry manure; therefore, animal ethics are not applicable to this study.

3.3 Quantification Study of the Insecticide Residues Samples of 300-500g of manures were taken before each insecticidal treatment for residue study. The study of the insecticide residues were based on the significant sensitive extraction methods, which is the SDIE. Therefore, the samples of manures that were taken before each treatment in the testing period were extracted and cleaned-up as described in section of SDIE and Clean-Up and Concentrated Stages, respectively and detected by UPLC and the quality was calculated as the formula in section Calculation.

3.4 Insecticides Application The treatment was started on the tenth to twelfth day after the introduction of one-day-old broilers due to the removal of heat-retaining cloths and manures were begin to accumulate.

The investigation of quantification and qualification of the insecticides in field was evaluated by applying the insecticides, bifenthrin , imidacloprid and fipronil (Table 1.) on the naturally infested manures, which measured in 5 x 8 m2 at the manures accumulation site under the high-rise broiler poultry houses. Three insecticides were treated on three separated poultry house, respectively and the manufacturer-recommended dose was used for each application. The insecticides were dispersed using a polycarbonate hand sprayer (Mesto® Maxima, Germany). The sprayer was equipped with a 1.10 nm hollow cone nozzle that delivered a cone-shaped spray with low drift and was adjustable to a maximum pressure of 3 bars. The sprayer was also equipped with an 80 cm spray wand and 250 cm spiral hose that delivered the liquid at a rate of 4.5 liters per minute. A pressure indicator permitted the observation of the pressure in the spray tank.

Control for the house fly population usually executed as weekly based interval 1, 15) although it might also depends on the insecticides manufacturer recommendation. Nevertheless, the application interval for this study was fixed at 7 days; this is also a common treatment interval for the local poultry owner in controlling the house fly larva (personnel communication).

The control of this study consists of a poultry house that is not treated with any insecticides during the testing period. The treatment houses were aparted to the control house at least 100 meters (bifenthrin, imidacloprid and fipronil are aparted 500m, 180m and 800m, respectively to the control house) and oil palm trees that were located between the houses were served as boundaries to prevent major migration of the flies.

Testing plot was defined as a treatment area of 5 x 8 m2 of the manure surface where manure accumulation begins. The testing plot consisted of three separated area represented as three replicates.

The larval population density in the test plot was defined using infection areas (m2), which were measured using a 50 x 50 cm quadrate square with a 10 x 10 cm grid and five quadrates per testing plot were assessed. For adult population density measurements, a scudder grill 16) measuring 50 x 50 cm was used to sample the adult fly population. This method is the standard for measuring fly

105 | P a g e populations in many control programs because it neither repels nor attracts flies 1, 17) Five counts were obtained from the area near the testing plot where flies aggregated.

4.0 Statistical Analysis The concentrations between UAHHE and SDIE were compared their significance different using student t-test in SPSS 17.0 at α = 0.05.

For the efficacy of insecticides in field trial, the larval infection area and the adult fly scudder counts of the treatment houses at the end of the breeding period (4 th week) were compared to the control population using One–Way Analysis of Variance (ANOVA) in SPSS 17 the significant differences between means were determined by least significant difference (LSD) at p≤0.05.

The residues of the insecticides were direct-proportional to the reduction of adult flies by observation, therefore the correlation between the quantitative (residues) of insecticides and the adult fly population reduction (treatment population compared to the control population) in percentage was studied using Pearson Correlation at α = 0.05. The scudder grill count reduction percentage were prior transformed by arnsin- square root and subjected in SPSS 17.0 Pearson Correlation at α = 0.05.

Results All the result of residue were calculated in the part per million (ppm) for comparison and statistical analysis. Both SDIE and UAHHE methods have ensured the intimate contact between the manures and solvent, which is crucial for an extraction method.7) The result obtained in this study had demonstrated that SDIE extracted significantly higher residues of bifenthrin, imidacloprid and fipronil from poultry manures, in contrasted to UAHHE. The residues for both extractions were presented in Table 2. Also, as can be seen in the Figure 1 to 3, UAHHE showed lower residues detection compared to the SDIE. For the residue study, SDIE was selected as the extraction method. At the beginning of the study, sampling was conducted as the procedures of EPA (1992)18), which 5 places of the 300-500g manures were taken randomly to ensure homogeneously of the sample. In general, three insecticides demonstrated accumulation during the treatment period and the residues at the end of the treatment period showed increment compared to the beginning of treatment period. The residues in the manures for the 4 times treatment is presented in Figure 4, in which imidacloprid, bifenthrin and fipronil increased 39 folds, 0.24 folds and 1.05 folds, respectively at the end of the treatment period.

The effectiveness of the insecticides was represented by the comparison of population between the treatments and control houses at the end of the broiler breeding period (30-40 days), which was defined as the 4th week of the present study. For larvicidal effects of the insecticides, the larval density was represented by their infestation area as suggested by WHO (1986) 1). As can be seen in Figure 5, three insecticides demonstrated weak effect on the larva at end of the treatment period (4th week), in which imidaclopril and bifenthrin showed 31.82% and 33.33% increment, respectively while fipronil reduced only 10% of the infestattion area. The larval infestation area is detailed in Table 3.

As can be seen in Figure 6, Imidacloprid and bifenthrin have a fairly weak reduction percentage for the adult flies during the treatment period. Despite of these, fipronil, which is not a commonly used insecticide in controlling house fly program, showed as much as 51.51% reduction of adult flies compared to the control house. As the residue increase, the reduction percentage increased, therefore, the relationship between the residues and the reduction percentage (transformed by arcsine-square root) for each treatment were studied by Pearson correlation at significant level <0.05. In overall, the residues showed strongly positive correlation with the reduction percentage of adult flies (Table 3).

106 | P a g e Discussions Prior work has documented the methods for extracting certain insecticides from semi-solid sludge such as environment hazardous insecticides-imidacloprid that is suspected having harmful effects to honey bee19). These methods were proposed by recognized organizations such as EPA and ASTM but are focusing only on the extraction methods of soil and food sample. However, bio-waste such as manure was poorly been studied, even insecticidal application is common on the manures for controlling the house fly larval. In this study, SDIE provided more reliable detection limit compared to the single solvent system-UAHHE. The main factor for the higher detection limit by using SDIE could be due to the specific solvent system that based on the solubility of the insecticides, as suggested in King (1980)20), where a solvent selection should consist of high capacity to the target compound. Imidacloprid and fipronil are considerable polar insecticides (514 ppm/L and 1.9 mg/L in water, respectively) and therefore, they are more soluble in methanol and acetone, respecively.

Bifenthrin, a non-polar compound was more soluble in non-polar solvent such as hexane, isooctane and dichloromethane10). Although both methods were using hexane as the solvent for bifenthrin extraction, but SDIE was having significantly higher detection level (6.77ppm), compared to UAHHE (2.25ppmat p<0.05), this is suggested that SDIE consists of other advantages in inducing the compound to the solvent. Furthermore, elements in the extraction procedure are strongly affecting the result20). Ultrasonic in the UAHHE might destruct the compound as demonstrated by Amar et al.

(2014)21), imidacloprid was sensitive to sono-energy and this could influence the result. Prior studies had also suggest the potential of ultrasonic in destructing the structure of some analytes compound such as pesticides, herbicides and polychlorinated biphenyls (PCBs) 22-24) Previous study on the insecticide residues was focusing on the degradation period taken for the insecticides, either in photolysis or biodegradation25). Three of the insecticides were found accumulated during the 4 weeks application period. This could mainly due to their higher half-life time, which bifenthrin, imidacloprid and filpronil are having 65days, 38.9days and 125days, respectively.26-27) Other important factors that would also affect the degradation of the insecticides are microbes activities in the medium and the properties of the insecitides. 25, 28) Additionally, bifenthrin was also very low water solubility and high affinity as residue in soils.27) The effect of imidacloprid and bifenthrin on house fly was studied by numerous researches, but not as contact poison towards the larval on poultry manures. Thus, the inferior of imidacloprid in controlling the house fly larval might due to the insecticide killing mechanism, which is oral poisoning as demonstrated by numerous studies29) as a bait (for example QuickBayt速) for used against the adult fly.

The reasons of inefficiency of bifentrhin in controlling house fly larval might due to the common practice as the target stage for pyrethroid was adult stage.1) Additionally, although bifenthrin is declaimed to have a greater photostability30), but the research of Peck et al. (2013)31) had demonstrated the insecticidal effect of bifenthrin was greatly reduced after exposed to the sunlight for 3 weeks. The inference for the ineffectiveness of bifenthrin could due to the degradation of the compound after exposed to the sunlight. Whereas, fipronil showed minor reduction to the larva population, which reduced 9.09% of larva population compared to the control.

This is suggested the insecticides act more likely as adulticides on poultry manures. It is supported as the reduction percentage of adult flies was significantly correlated with the residue of the three insecticides. Even though the imidacloprid and bifenthrin reduced adult fly by 28.30% and 30.84%, respectively compared to the control house, nonetheless, fipronil reduced greatly (51.51%) of fly population compared to the control population.

The low reduction of imidacloprid and bifenthrin can be due to the dilution of dose because of the accumulation of manures. This is supported by situation of manures accumulation and rich microbes that will enhance the biodegradation process and causing lower residue efficacy. 25, 28) On the other

107 | P a g e hand, the slow-acting, fipronil was commonly applied as bait for the control of termite, is firstly studied for used against the house fly in Malaysia, although numerous attempts have had be done for controlling the adult flies.5-6) Conclusions This is concluded that SDIE was more sensitive extraction method for extracting insecticides in manures, due to of appropriate solvent system and protection for sono-sensitive compound compared to the UAHHE. In field trial, as the strong correlations between the residues and adult flies reduction suggested the tested insecticides act more as adulticide when applied on the poultry manures. Fipronil is a potential insecticides for controlling house fly in Malaysia, future attempts should be focused on the application mode (residue or bait).

Acknowledgements We thank to the laboratory assistant, Ms Anna from the Biochemistry Laboratory, School of Biological Sciences, and University Sciences Malaysia for supplying part of the regents and apparatus. The first author was financially supported by MyBrain15 program under the Department of Education, Malaysia. The project was granted under Research University Grant (RU) 1001 / PBIOLOGI / 811241 References 1) (WHO) World Health Organization. Vector Control Series: The Housefly Training and information Guide (Advance level) Danish Pest Infection Laboratory Lyngby, Denmark.(1986) 2)M Page, I. Michael, Haverty and E. Charles Richmond Insecticide Residues on Pine Bark: Influence of Trees, Sample Volume and Size on Variability Journal of Entomol. Sci. 24(2) 180-185 (1988) 3) AOAC Official Method 2007.01: Pesticide Residues in Foods by Acetonitrile Extraction 131and Partitioning with Magnesium Sulfate 4)ASTM E1527-13 2013 Standard Practice for Environmental Site Assessments: Phase I Environmental Site Assessment Process, ASTM International, West Conshohocken, PA, 2013, www.astm.org 5)JW Diclaro, C. Jeffrey, Hertz, M. Ryan, Welch, G. Philip, Koehler, and M. Roberto Pereira.

Integration of Fly Baits, Traps, and Cords to Kill House Flies (Diptera- Muscidae) and Reduce Annoyance J. Entomol. Sci. 47(1): 56-64 (2011) 6) HAA Khan, SA Shad, W Akram. Combination of Phagostimulant and Visual Lure as an Effective Tool in Designing House Fly Toxic Baits: A Laboratory Evaluation. PLoS ONE 8(9) 2013 7) (EPA) Environment Protection Agency U.S., "Method 3550 Interlaboratory Comparison Study: Methods for Volatile and Semi-Volatile Compounds," Environmental Monitoring Systems Laboratory, Office of Research and Development, Las Vegas, NV, EPA 600/4-84-027. (1984) 8)(CDFA) California Department of Food and Agriculture. Center for Analytical Chemistry Environmental Analysis. Determination of Bensulide and Imidacloprid in Surface Water EMON-SM05-023 (2011) 9) D Hernández, Moreno, Marcos Pérez, López, M. Prado Míguez, Francisco Soler,Begona Jiménez.

Porphyrin levels in excreta of rabbit as non-destructive biomarkers of diazinon exposure. Environ Toxicol. Phar. 34: 466–472 (2012) 10) PubChem Chemical and Physical Properties: Solubility http://pubchem.ncbi.nlm.nih.gov/

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11) (EPA) Environment Protection Agency U.S. EPA Method 3630c Silica Gel Cleanup 12)H. Obana, M. Okihashi, K. Akutsu, Y. Kitagawa, and S. Hori. Determination of neonicotinoidpesticide residues in vegetables and fruits with solid phase extraction and liquid chromatographymass spectrometry. J. Agric. Food Chem.51, 2501–2505 (2003) 13)S. Baskaran, R. Kookana S. &, R. Naidu. Degradation of bifenthrin, chlorpyrifos and imidacloprid in soil and bedding materials at termiticidal application rates. Pestic Sci 55:1222-1228 (1999) 14) Saran, Raj K., & Kamble, Shripat T. Concentration dependent degradation of three termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (isoptera: Rhinotermitidae). J. Eco. Entomo., 101(4): 1373-1383. (2008) 15)RE Williams.

Control of Poultry Pests Purdue Extension E-3-W http://extension.entm.purdue.edu/publications/E-3.pdf (2010) 16) H.I. Scudder. A new technique for sampling the density of housefly populations. Public Health Reports 62: 681-686. (1947) 17) LJ Bong and Z Jaal. Temporal changes in the abundance of Musca domestica Linn (Diptera: Muscidae) in poultry farms in Penang, Malaysia Tropical Biomedicine 26(2): 140–148 (2009) 18) (EPA) Environmental Protection Agency United States Preparation of Soil Sampling Protocols: Sampling Techniques and Strategies EPA/600/R-92/128 (1992) 19) JM Bonmatin, I Moineau, R Charvet, C Fleche, ME Colin, and ER Bengsch. A LC/APCIMS/MS Method for Analysis of Imidacloprid in Soils, in Plants, and in Pollens Anal. Chem., 75: 2027-2033 (2003) 20) CJ King. Seperation processes 2nd ed McGraw-Hill, New York p756-763 (1980) 21) L. Patil Amar, N. Patil Pankaj, R. Gogate Parag. Degradation of imidacloprid containing wastewaters using ultrasound based treatment strategies. Ultrason Sonochem. 21:1778–1786 (2014) 22) RMSM Mujeebur, P. Luigi, T. Ferruccio and M. Stanislav. Destruction Technologies For Polychlorinated Biphenyls (PCBs) International Centre for Science and High Technology United Nations Industrial Development Organization (ICS-UNIDO)( 2000) 23) H. Stacy, B. Alok and S. Steve. Pesticides and Herbicides Water Environ. Res. 74( 5): 1-28 (2002) 24) R Farooq, FK Lin, SF Shaukat, JJ Huang. Sonochemical degradation of organophosphorus pesticide in dilute aqueous solutions. J. Environ. Sci. 15:710. (2003) 25) BK Singh, A. Walker, JAW Morgan and DJ Wright. Effects of Soil pH on the Biodegradation of Chlorpyrifos and Isolation of a ChlorpyrifosDegrading Bacterium. Appl. Environ. Microbiol. 69(9): 5198-5206. (2003) 26) (CDPR) California Department Pesticides Regulation http://www.cdpr.ca.gov/ (2014) 27) (EPA) Environmental Protection Agency United States, Environmental Fate Assessment for the Synthetic Pyrethroids:, [Online]. Available at: http://www.epa.gov/oscpmont/sap/1999/february/pyreth. (1999) 28) STS. Gilani, M. Ageen, H. Shah and S. Raza. Chlorpyrifos Degradation in Soil And Its Effect On Soil Microorganisms J. Anim. Plant. Sc.i, 20(2): 99-102 (2010) 29) AT Nurita, A. Abu Hassan, , Nur Aida, H. & Norasmah Basar. Field evaluations of the granular fly bait, Quick Bayt® andthe paint-on fly bait, Agita® against synanthropic flies Tropical Biomedicine 25(2): 126–133 (2008) 30) LE Mokrey, KD Hoagland.. Acute toxicities of five synthetic pyrethroid insecticides to Daphnia magna and Ceriodaphnia dubia. Environ. Toxic Chem. 9:1045-1051. (1989) 31) GW Peck,.; Ferguson, J Holly.; LePage, T Jane.; Hebert, R. Vincent; O'Neal, Sally D.; and Walsh, Douglas B., "Evaluation of Sunlight-Exposed Pyrethroid-Treated Netting for the Control of Face Fly and Housefly (Diptera: Muscidae)" US Army Research. Paper 259. (2013).

109 | P a g e Table 1: List of the Tested Insecticides Product‟s name Active Ingredient (A.I.) and (Manufacturer) content Prothor (Ensystex Australasia Pty Ltd) Maxxthor (Ensystex Australasia Pty Ltd) Reagent®50SC (Bayer)

Imidaclopril 20% Inert 80 %

SC

Manufacturer Recommended Dose 50-100ml/10L

Bifenthrin 10% Inert 90 %

SC

125-250ml/10L

SC

0.625-75L/ha

Fipronil Inert

Formulation

5.0% 95.0%

Table 2. Comparison of Residues Detection (Means±S.E.) between UAHHE and SDIE Residue detection (ppm) UAHHE

SDIE

Bifenthrin

2.25±0.23

6.77±5.87*

Fipronil

2.37±0.03

31.40±17.40**

Imidacloprid 1.47±0.56 **significance level of α = 0.001 *significance level of α = 0.05 S.E. =Standard Error ppm part per million UAHHE Ultrasonic assisted hexane-hexane extraction SDIE solvent direct-immersion extraction

352.97±176.53**

Table 3. Quantification of the Insecticide Residues in Respond to the Larval Infestation Area and Scudder Grill (Means±S.E), and the Correlation between the Residues and the Scudder Grill Reduction.

Week# Residue Larval Scudder Grill Scudder Correlation between s Infestation area grill residues and Scudder (ppm) (cm2) reduction grill reduction (α = (%)** 0.05, 2-tailed ) st Control 1 300 ± 15.62 9.00 ± 0.45 2nd 375 ± 12.11 20.47 ± 0.86 3rd 408 ± 10.02 24.60 ± 1.55 th * 4 430 ± 21.89 28.73 ± 1.61a Imidacloprid

1st 2nd 3rd 4th

0.20 1.01 1.47 11.02

285 ± 11.25 350 ± 8.96 490 ± 18.25 435 ± 16.05*

9.18±2.41 18.40±1.08 21.20±3.70 22.04±4.50b

-2.01 10.11 13.82 23.30

r=0.981 Sig= 0.019

Bifenthrin

1st

0.10

255 ± 13.47

8.91±1.54

1.00

r=0.957

110 | P a g e

Fipronil

2nd 3rd 4th

1.23 1.91 2.13

360 ± 6.32 485 ± 17.22 440 ± 14.08*

15.52±0.93 17.43±2.60 19.97±1.23b

24.15 29.15 30.84

Sig=0.43

1st 2nd 3rd 4th

1.39 1.71 2.12 2.86

265 ± 7.56 325 ± 19.55 400 ± 22.11 400 ± 16.11*

9.11 ± 0.54 16.83 ± 0.70 18.87 ± 0.65 13.93 ± 0.74c

-1.22 17.78 23.29 51.51

r=0.968 Sig=0.32

#

The 4th week is defined as the end of the broiler breeding period, the larval and adult population were compared at the end of broiler breeding period. * n=5, Not significant at level of 0.05 Different alphabet indicates significant different (p≤0.05), n=5 ** The scudder grill reduction was the comparison of scudder counts between the treatment houses to the control house (no insecticide treatment) S.E. Standard Error ppm part per million

Figure 1: Bifenthrin: Top, Standard; Middle, UAHHE and Bottom, SDIE

111 | P a g e

Figure 2 Imidacloprid: Top, Standard; Middle, UAHHE and Bottom, SDIE

112 | P a g e

Figure 3 Fipronil: Top, Standard; Middle, UAHHE and Bottom, SDIE

113 | P a g e

Figure 4 Insecticidesâ&#x20AC;&#x; residue in response to the treatment period.

114 | P a g e

500 400 Control

300

Imidacloprid Bifenthrin

200

Fipronil

100 0 1st

2nd

3rd

4th

Week

Figure 5 Larval infestation areas in responds to the treatment period

35 30 25 Scudder Grill

Larval Infestation area (cm2)

600

20

Control

15

Imidacloprid Bifenthrin

10

Fipronil 5 0 1st

2nd

3rd

4th

Week

Figure 6 Scudder grill in responds to the treatment period

115 | P a g e A Study on Removal of Pb2+ Ion from Wastewaters by Using Water Cress (Enhydra Fluctuans Lour.)

Khin Sandar Thaw*, Aung Myint Htay** * Lecturer, Department of Chemistry, University of Yangon ** MSc Student, Department of Chemistry, University of Yangon

Abstract In this research, the role of water cress was investigated for the removal of toxic heavy metal Pb 2+ion from wastewaters of Industrial Zones. Wastewater samples were collected from the drains of paint factory I, paint factory II, battery factory I and battery factory II located at Industrial Zones and pH values were determined by pH meter. Water cress sample was collected from the shallow part of Inya Lake,Pyi Road, Yangon Region. Lead uptake was carried out using water cress (Enhydra fluctuans) with contact times. The element contents of wastewater before and after treatment with water cress were determined by AAS. It can be found that the highest removal percent of lead in wastewater is 87.03 % for 30 days. The physicochemical parameters such as pH, BOD, COD and DO of original wastewater and the wastewater treated with water cress were analyzed by the conventional methods.

After treatment with water cress, all physicochemical parameters were in the range of EPA standard.

The lead contents in water cress plants before and after 10,20,30 days treatment were determined by EDXRF.From the resultant data, relative abundance of lead 0.483 % in roots and 0.044 % in aerial parts of water cress were observed after 30-day adsorption.From the present study, it can be seen that water cress has a strong potential to up-take lead ion from the industrial wastewaters.

Introduction Water Cress(or) Kana-phaw (or) Enhydra fluctuans Water cress is a prostrate, spreading, annual herb. Stems are somewhat fleshy, 30 centimeters or more in length, branched, rooting at the lower nodes, and somewhat hairy. Leaves are stalk less, linearoblong, 3 to 5 centimeters in length, pointed or blunt at the tip, usually truncate at the base, and somewhat toothed at the margins.

Botanical characteristics of water cress Family Genus Species Botanical name English name

Myanmar name Plants part used

: : : : :

Asteraceae Enhydra fluctuans Enhydra fluctuans Lour.

Common enhydra, Buffalo spinach, Marsh herb, Water cress : Kana-phaw : Whole plants

Uses of water cress In Malaya, young parts are used as salad. Sometimes steamed before they are eaten. In the Philippines, leaves are pressed and applied to the skin as a cure for herpetic eruptions. The Malays use the young parts and bitter leaves as laxative. Leaves used for diseases of the skin and nervous system.

In Calcutta, fresh juice of leaves used as adjunct to tonic medicines; used for neuralgia and other

116 | P a g e nervous diseases. In Indian medicine, also used for various skin diseases and as a laxative. In Assam, India, plant juices used for skin diseases, nervous disorders and high blood pressure. Expressed juice of leaves used as demulcent in cases of gonorrhea; mixed with cow's or goat's milk. As a cooling agent, leaves are pounded and made into paste and applied cold to the head. Plant used for torpidity of the liver. Infusion is prepared the previous evening, boiled with rice and taken with mustard oil and salt. Used the tribal practitioners of the Marakh sect of the Garos for treatment of diabetes. In Ayurveda, used for liver disorders, skin and nervous disorders also as laxative. (Hinchman and Gatliff, 1998) Wastewater Wastewater is water that has been used and must be treated before it is released into another body of water, so that it does not cause further pollution of water sources. Wastewater comes from a variety of sources. Everything that you flush down your toilet or rinse down the drain is wastewater. Rainwater and run off, along with various pollutants, go down street gutters and eventually end up at a wastewater treatment facility. Wastewater can also come from agricultural and industrial sources.

Some wastewaters are more difficult to treat than others; for example, industrial wastewater can be difficult to treat, whereas domestic wastewater is relatively easy to treat (though it is increasingly difficult to treat domestic waste, due to increased amounts of pharmaceuticals and personal care products that are found in domestic wastewater).(Hammer, 1975) Industrial wastewater There are many types of wastewater such as domestic, commercial, industrial, agricultural and toxic hazardous etc. Wastewater contains numerous pathogenic or disease causing microorganisms that dwell in the human intestinal tract or that may be present in certain industrial waste. Wastewater also contains nutrients, which can stimulate the growth of aquatic plants and it may contain toxic compound . (Dunbabin and Bowmer, 1992) Toxicity of Lead Lead can affect the nervous system, gastrointestinal system, cardiovascular system, blood production, kidneys, and reproductive system. Lead poisoning has been recognized as a major public health risk, particularly in developing countries. Though various occupational and public health measures have been under taken in order to control lead exposure, cases of lead poisoning are still reported. Exposure to lead produces various deleterious effects on the hematopoietic, renal, reproductive and central nervous system, mainly through increased oxidative stress. These alterations play a prominent role in disease manifestations. Modulation of cellular thiols for protection against reactive oxygen species (ROS) has been used as a therapeutic strategy against lead poisoning. N-acetylcysteine, Îą-lipoic acid, vitamin E, quercetin and a few herbal extracts show prophylaxis against the majority of lead mediated injury in both in vitro and in vivo studies. This review provides a comprehensive account of recent updates describing health effects of lead exposure, relevant biomarkers and mechanisms involved in lead toxicity. It also updates the readers about recent advances in chelation therapy and newer therapeutic strategies, like nanoencapsulation, to treat lead induced toxic manifestations. (Chapman, 1996) Experimental Materials and Methods of Analysis The chemicals used to the experimental work were from British Drug House Chemical Ltd., England.

All specific chemicals used are described in details in each experimental section.

In all the investigations, the recommended and standard procedures of both conventional and modern techniques were employed. The experiment was carried out in the Physical Chemistry Research

117 | P a g e Laboratory (S 122) of the Department of Chemistry, University of Yangon and the spectroscopic analyses were studied at the Universities' Research Centre, University of Yangon.

Collection of Samples The water cress was collected from the Inya Lake which is located on Pyi Road, Hlaing Township, Yangon Region. Effluent samples were also collected from the drains of paint factories and battery factories from Industrial Zones. The water cress samples were washed with tap water and distilled water.

Determination of pH Materials Collected effluent samples from paint factory I, paint factory II , battery factory I and battery factory II Apparatus and Equipment pH meter and glassware Procedure The collected effluent sample ( 50 mL ) was placed in a 250 mL beaker and the pH of this effluent was measured by the recommended standard procedure in pH meter catalogue. The resultant pH data are described in Table 3.1.

Determination of COD by permanganate method Materials Effluent sample from battery factory I, potassium permanganate, potassium iodide, starch, sodium thiosulphate, sulphuric acid (sp. gr. 1.84, 95% reagent grade) Apparatus and Equipment Beaker, conical flask, volumetric flask, graduated pipette, burette, hot plate.

Procedure The water sample (50 mL) was placed in a conical flask. A 5 mL solution of 5 M potassium permanganate was added to the water sample and the flask was placed on the boiling water bath for 1 hr. Then the sample was cooled for 5 min. 0.5 M potassium iodide solution (5 mL) was added, followed by 10 mL of sulphuric acid solution (0.01 M). The solution was titrated with 1.0 M standard sodium thiosulpahte solution until a pale yellow color was obtained. Starch solution (1 mL) was added to the above solution to obtain a blue color. The titration was continued until the blue color disappeared completely. The blank determination was carried out using distilled water. The whole of the above procedure was repeated for another two times. The results are shown in Table 3.2.

Determination of BOD BOD was measured by incubating the sample at 20째C for five days.

Materials Effluent sample from battery factory I ,potassium hydrogen phosphate, urea, sodium hydroxide (pellets), sulphuric acid (reagent grade) Apparatus and Equipment Volumetric flask, graduated pipette, glass bottle, measuring cylinder, incubator

118 | P a g e Procedure Water sample was filled into glass bottles so that bubbling did not occur and initially dissolved oxygen (DO) content was determined by modified Winkler's method. A 10 mL solution of 0.05 % urea and the phosphate buffer solution were added to the bottles. The bottles were incubated at 20 ยบC for 5 days. After incubation, the oxygen concentration was measured by modified Winkler's method.

Finally, the 5 days biochemical oxygen demand was obtained from the difference between the initial DO content and DO of 5 days incubation (i.e., BOD) in mg L-1. The results are presented in Table 3.2.

Determination of DO DO of water sample was determined by modified Winkler's method.

Materials Effluent sample from battery factory I, potassium iodide, sodium hydroxide, sodium oxide, manganese (II) sulphate, sodium thiosulphate, sulphuric acid, potassium iodate Apparatus and Equipment Volumetric flask, narrow mouth glass bottle (BOD bottle), graduated pipette, burette Procedure Water sample was placed without bubbling in a BOD glass bottle. A solution (2 mL) of manganese (II) sulphate (0.05 M) and 2 mL solution of alkaline iodide were added to the sample and the bottle was stoppered. Then the bottle was inverted and rotated vigorously several times, and allowed to settle the brown precipitate, leaving a clear supernatant liquid. The precipitate was dissolved by adding 2 mL of concentrated sulphuric acid. The solution was put into a 250 mL conical flask and titrated immediately with standard sodium thiosulphate solution (0.1 M) until a pale straw color was obtained. Starch solution (2 mL) was added to the above solution to obtain a blue color. The titration was continued until blue color disappeared completely.

DO was calculated as described in Appendix-III and the results are shown in Table 3.2.

Study of the removal percents of Pb2+using water cress Materials Effluent sample from battery factory I, Water cress plant, Distilled water Apparatus and Equipment Plastic bowl, plastic bottles, balance Procedure The effluent (10.0 L) was placed in the plastic bowl. The water cress plants (100 + or -1 g) were placed in the prepared bowl. Whenever necessary, the solution level was adjusted with distilled water.

After 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days (different contact times), the solution, about 25 mL was taken out and stored in bottles. Then the amount of Pb2+ ion in the effluent was determined by AAS. The removal percents of Pb2+ion were calculated as described in the Appendix-IV. The results are shown in the Table 3.3.

119 | P a g e Energy Dispersive X-ray Fluorescence ( EDXRF ) Analysis of Water Cress after Adsorption of Lead Materials Dried water cress powder ( roots and aerial parts ) before and after adsorption of lead Apparatus and Equipment EDXRF spectrometer Procedure The procedure was done by the recommended standard instruction as reported in the catalogue. The resultant spectra are shown in Figure 3.2 and 3.3 and Table 3.4.

Determination of Physicochemical Properties of Effluent after Treatment with Water Cress The physicochemical properties (pH, COD, BOD and DO) of effluent samples after treatment with water cress were determined.

Materials The same as shown in Sections 2.3.2, 2.3.3 and 2.3.4.

Effluents (after treatment with water cress) Apparatus and Equipment The same as shown in Sections 2.3.2, 2.3.3 and 2.3.4 Procedure The determination of COD was carried out as the same procedure as described in Section 2.3.2, for BOD Section 2.3.3 and 2.3.4 for DO. The resultant data are described in Table 3.5.

Results And Discussion Physicochemical Properties of Collected Wastewater Samples Physicochemical properties and pH values of collected wastewater such as paint factory I, paint factory II, battery factory I and battery factory II located at Industrial Zones were determined before treatment of water cress plants. pH of wastewater of battery factory I located at Hlaing Thar Yar Industrial Zone showed that it is in the acceptable range to grow the water cress plants. Therefore, in this research, wastewater from battery factory I located at Hlaing Thar Yar Industrial Zone was chosen for the treatment with water cress for removal of lead ions. The resultant data are shown in Table 3.1. The qualities of wastewater samples were determined before the treatment with water cress.

The results of colour, pH, COD, BOD, and DO are shown in Table 3.2. The colour of wastewater was bluish green: its pH was 6.65.It was found that the effluents contaminated with 4.895 ppm of Pb2+ (according to AAS analysis ). In addition, COD, BOD and DO of effluents were 5.52, 5.7 and 6.5 ppm respectively. According to the AAS analysis, lead content in the effluents exceeded by EPA standard and the other parameters were also not in the range of EPA standard.

120 | P a g e Table 3.1

The pH Value of Collected Wastewater Samples from Industrial Zones

No 1 2 3 4

Industrial Zone Sample pH Hlaing Thar Yar Paint factory I 6.00 Hlaing Thar Yar Paint factory II 6.40 Hlaing Thar Yar Battery factory I 6.65 Shwe Pyi Thar Battery factory II 5.65 Table 3.2 The Physicochemical Parameters of Effluent from Battery Factory I Before treatment No. Parameter EPA* by water cress 1 colour bluish green no colour 2 pH 6.65 6.50-8.50 3 COD (ppm) 5.52 5 4 BOD (ppm ) 5.70 5 5 DO (ppm ) 6.50 4.00-6.00 6 Lead (ppm) 4.9 2 *EPA (2008) Classification of Selected Water Cress For this study, plant sample of the collected water cress (Kana-phaw in Myanmar name) was verified in the Department of Botany, University of Yangon. Based on the species of water cress, the collected plant sample is a semi- aquatic tropical plant grown as a leaf vegetable. It is found throughout the tropical and subtropical region of the world. The collected water cress samples were classified as Enhydra fluctuans Lour.

The Uptake of Lead from Effluent of Battery Factory I by Water Cress This experiment was carried out by growing water cress in the effluent at pH-6.65 to be reduced the concentration of Pb2+ in effluent. The measurement of Pb2+ concentration was taken of 3-days interval for 30 days. The results are shown in Table 3.3. From the resultant data, the maximum removal percent (87.03%) was found with increasing the time interval after 30 days.

From the resultant data of lead uptake of effluent by water cress, it can be used as natural sorbent for the removal of Pb2+ due to its high removal percent.

Day 0 3 6 9 12 15 18 21 24 27 30

The Removal Percent of Pb2+ Ion from Wastewater by AAS

Pb2+Content (ppm)

Removal Percent (%) 100

4.895 (original) 0 80 2.475 49.44 1.96 59.96 60 1.735 64.56 1.495 69.46 40 1.305 73.34 0.98 79.98 20 0.785 83.96 0 0.685 86.01 0 3 6 9 12 15 18 21 0.66 86.52 Contact time (day) 0.635 87.03 Figure 3.2 The removal percent of Pb2+ ion for different time intervals Removal percent (%)

Table 3.3

24

27

30

EDXRF Analysis of Water Cress after Adsorption of Pb2+ Water cress plants before and after adsorption of lead were dried at room temperature and analysed by EDXRF analysis for the presence of lead. The resultant spectra are shown in Figure 3.3 to 3.10. It can be seen that the absence of lead in the water cress plants before adsorption of lead. The observed

121 | P a g e spectra showed the presence of lead in water cress roots after 10, 20 and 30 days adsorption of lead (relative abundance of 0.261 %, 0.487 % and 0.483 % ) and in aerial parts of water cress after 10, 20 and 30 day adsorption of lead (relative abundance of 0.023 %, 0.027 % and 0.044 %). It was found that the equilibrium time for the removal of lead was 30 days in contact with roots and aerial parts of water cress with industrial wastewater. From the resultant data, the removal of lead from industrial wastewater by water cress was observed that roots of water cress was more effective than aerial parts of water cress.

Figure 3.6

EDXRF spectrum of water cress roots before and after 10-20-30-day adsorption

Figure 3.10 EDXRF spectrum of aerial parts of water cress before and after 10-20-30-day adsorption

Physicochemical Parameters of Effluent after Treatment with Water Cress Physicochemical parameters such as COD, BOD, DO and pH of effluent before treatment and after 30 days adsorption were investigated. The comparison of these parameters before and after treatment are presented in Table 3.5. All properties of COD, BOD and DO were in the range of EPA standard after treatment with water cress.

The resultant data are shown in Table 3.5 and Figure 3.7. It can be seen that water cress should be grown in the drains of Industrial Zones.

122 | P a g e

Table 3.4

Relative Abundance of Pb2+ Table 3.5 Comparison of Physicochemical Content in Roots and Aerial Parts Parameters of Effluents before and after of Water Cress before and after Treatment with Water Cress Adsorption by EDXRF

Days

Pb2+ content in Roots (%)

Pb2+ content in Aerial parts (%)

Parameters

Before treatment (ppm)

After treatment for 30 days (ppm)

0 10 20 30

0 0.261 0.487 0.483

0 0.023 0.027 0.044

pH COD BOD DO

6.65 5.52 5.7 6.5

6.5 2.94 3.6 5.5

Conclusion In this study, water cress (Enhydra fluctuans) samples were collected from the shallow part of Inya Lake, Pyi Road, Yangon Region. The wastewater samples were collected from the Industrial Zones .The removal percents of lead from wastewater were made by using water cress with contact times.

From the resultant data, the highest removal percent (87.03 %) of lead from industrial wastewater by water cress was observed at pH 6.50 and for contact time of 30 days. After 30 days, treated wastewater consists of BOD 3.60 ppm, COD 2.94 ppm and DO 5.50 ppm. The value of pH, BOD, COD and DO are in the range of EPA standard after treatment with water cress. From the EDXRF spectra, the relative abundance of lead were found 0.261%, 0.487 %, 0.483 % in water cress roots and 0.023 %, 0.027 %, 0.044 % in aerial parts of water cress after 10, 20 and 30 days. It was found that water cress roots were more adsorbed lead from wastewater than aerial parts of water cress. From the present study, it can be seen that water cress has a strong potential to uptake and accumulate the toxic heavy metals from industrial wastewater. Enhydra fluctuans Lour. can be used as natural sorbent for the removal of heavy metals from the industrial effluents economically. Water cress plants near the industrial zones have strong sorption capacity to accumulate all the heavy metals. Therefore, these plants near the industrial zones should not be consumed.

References Chapman, D. (1996). " Water Quality Assessment, A Guide to the Use of Biota Sediments and Water in Environmental Monitoring", London, 2nd Ed., Elfnspon, 8-91 Hinchman, R.R., M.C. Nergi and E.G., Gatliff. (1998). " Phytoremediation: Using Green Plants to Clean Up Contaminated Soil, Ground Water and Wastewater", Agr . Res .Magazine ,48 , 4-9 Win Ko Ko. (2011). "Determination of Pb2+ in Water Spinach (Indicator Plant) Location at Hlaing Thar Yar Industrial Zone". Yangon, MSc (Thesis), Department of Chemistry, University of Yangon, Myanmar Jakkis, S. (1981). "Lead Removal from Wet-Cell Battery Factory Wastewater by Chemical Treatment". Thailand, Pollut. Water Resour & Mana, Kasetsart University Dunbabin, J.S and K.H. Bowmer. (1992). " Potential Use of Constructed Wetlands for treatment of Industrial Wastewaters Containing Metals ". The Sci. the Total Env., 111,151-168 Hammer, M.J. (1975). " Water and Wastewater Technology". New York: John Willey & Sons, Inc , 9

123 | P a g e Nutritional Studies and Microbial Analyses of Fermented Flacourtia Rotendifolla (Na-ywe) Leaves Moe Moe Aye1, Kyaw Naing2 1.Lecturer, Dr., Department of Chemistry, University of Yangon 2.Pro-rector, Dr., University of Yangon

Abstract Fermented Na-ywe leaves is one kind of popular dessert in Pyay Township, Bago region, Myanmar. This food was investigated by both chemical and microbiological studies for the first time. The chemical studies involved the determination of nutritive constituents such as water, fat, pH, protein, ash, mineral elements, fibers and some vitamins. For hygienic value, pathogenic enteric bacteria i.e., total coliform count, Escherichia coli, Salmonella, Staphylococcus aureus and Vibrio parahaemolyticus were also determined by using their specific media.

Introduction Fermented Food in Myanmar Myanmar people consume various fermented food. These are from the sources of fish and shrimp as well as leaves, legumes, seeds and nuts.

Fish and shrimp products are: (i) Shrimp paste and sauce (ii) Fish paste and sauce (iii) Salted fish (iv) Pickled fish and prawn Leaves, legumes, seeds and nuts products are (i) (ii) (iii) (iv) (v) (vi)

Soybean paste and sauce Fermented defatted sesame press-cake Fermented bamboo shoot Fermented horsegram Fermented tea leaves Fermented Na-ywe leaves

Fermented Na-ywe Leaves

Figure 1.1 Na-ywe leaves

Family Specie Botanical name English name

: : : :

Flacourtiaceae fluctuans Flacourtia rotundifolia Meer.

Madagascar Plum

Myanmar name

: Na-ywe

124 | P a g e Sample Preparation

(a)

(b)

Figure 1.2 (a) Fermented Na-ywe leaves ample in the market (b) After peeling the cover sheets

Figure 1.3

Flow diagram for sample preparation of fermented Na-ywe leaves

Experimental Materials and Methods of Analysis In all the investigations, the recommended and standard procedures of both conventional and modern techniques were employed. The experiment was carried out in the Department of Chemistry, University of Yangon and also at the Universities' Research Centre, University of Yangon.

Collection of Samples Fermented Na-ywe leaves samples were purchased randomly from preserved food stores in Pyay Township, Bago Region and also from Hlaing Township, Yangon Region. The sample No.1 was prepared only for this research.

125 | P a g e Chemical Analysis The pH values were measured by using a pH meter. Water content was determined by oven-drying method. Fat content was determined by the Soxhlet extraction method. The relative compositions of some elements were determined by Energy Dispersive X-ray Fluorescence (EDXRF) spectrometry.

The trace contents of elements were determined by Atomic Absorption Spectrometry. Protein content and crude fibre content were determined by AOAC official method. The values of vitamins were determined by UV-Visible spectrometry.(Pearson,D.1976) Microbiological Analyses The determination of aerobic plate count, total coliform, E.coli and S.aureus were carried out by using the specific ready-made culture media system from 3MMicrobiology Products. The determination of V.P, salmonella and gram (+) bacteria were carried out by chemical tests. Identification of gram (-) bacteria was carried out by using API-20E media.(Prescott, L.K.,1999) Results And Discussion Nutritional Studies The pH values, water content, fat content, ash content, fibre content and protein content were as shown in Table 3.1.

Table 3.1 No. 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Some Nutritional values of Fermented Na-ywe Leaves Samples pH 4.7 4.8 4.7 4.9 4.6 4.5 5.0 4.7 4.9 4.6 4.8 4.7 5.0 4.8 4.6 4.9

Water content (%) 88.6 89.0 84.5 81.4 87.6 85.7 84.6 86.5 88.2 85.5 83.4 85.0 87.7 86.2 84.8 82.4

Fat content (%) 0.82 1.27 1.14 0.95 0.33 1.24 0.96 0.78 1.14 1.09 1.26 1.31 1.09 1.17 0.92 1.10

Ash content (%) 1.01 1.24 1.04 1.47 1.29 1.35 1.13 1.26 0.96 1.31 1.17 1.17 1.21 0.92 0.98 1.05

Fibre content (%) 7.42 7.14 7.69 11.33 10.52 10.46 9.62 10.10 8.46 10.98 11.40 11.32 8.65 8.73 9.38 9.40

Protein content (%) 9.87 8.81 7.68 9.81 8.12 9.37 9.12 8.63 9.56 9.39 7.94 8.38 9.75 9.00 9.50 9.12

The vitamin contents in Fermented Na-ywe Leaves Samples In general, vitamin contents increase during fermentation process. In this work, vitamin B1 (Thiamin), B2 (Riboflavin), B6 (Niacin) and vitamin C (Ascorbic acid) were determined by UV-Visible Spectrometry and the resultant values were as shown in Table3.2.

126 | P a g e Table 3.2 No. 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

The Vitamin contents of the Fermented Na-ywe Leaves Samples Thiamin ( g g-1) 12.9 10.5 12.8 11.9 12.5 10.9 11.2 11.6 12.9 12.8 12.5 11.0 12.7 10.9 11.3 12.2

Riboflavin ( g g-1) 0.55 0.39 0.73 0.61 0.42 0.53 0.55 0.36 0.50 0.47 0.58 0.62 0.57 0.49 0.55 0.68

Niacin ( g g-1) 7.2 6.9 7.1 7.3 6.9 7.0 6.8 6.5 7.1 7.3 7.3 6.7 7.5 7.2 6.7 7.1

Ascorbic acid ( g g-1) 29.1 29.5 31.5 31.3 30.3 29.4 30.7 29.3 30.5 29.7 30.2 29.1 29.7 29.0 29.6 29.2

Some Microbial Analyses of Fermented Na-ywe Leaves Samples The microbiological studies involved isolation and identification of microflora in each sample.

Moreover, for hygienic value, pathogenic enteric bacteria i.e., Escherichia coli, Salmonella, Staphlococcus aureus and Vibrio parahaemolyticus were also determined. All of these samples showed lower bacteria count than the reported values of fermented tea leaves samples. The resultant values were as shown in Table 3.3.

Table 3.3 No. 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

The Values of some Microflora in Fermented Na-ywe Leaves Samples Total Coliform (CFU g-1) 160 240 120 280 60 880 270 490 120 710 50 260 140 160 180 490

E.Coli (CFU g-1) ND ND ND 50 ND ND 20 20 ND ND ND 30 ND ND ND ND

S.aureus (CFU g-1) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Salmonella (CFU g-1) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

V.P (MPN g-1) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

127 | P a g e

Figure 3.1

Total coliform count and E.coli count by using ready-made culture medium of 3 M microbiology

Figure 3.3

Figure 3.2

S. aureus count by using ready-made culture medium of 3 M microbiology

EDXRF spectrum of the prepared sample

128 | P a g e Elemental Analysis of Fermented Na-ywe Leaves Samples by DXRF Elemental analysis was carried out for the determination of trace minerals, which are essential for human growth and metabolism but some are harmful. In this work, the relative compositions of elements were detected by EDXRF and then followed by AAS as described in Table 3.4 and figure.

Conclusion This research was the preliminary study of raw material, sample preparation process, food values, trace minerals and also the counts of some bacteria which are not only pathogenic but also the associates of fermentation. This food was found to be acidic pH range (4.5-5.0).This helps to preserve food. Their high water contents (81.4-89.0) was due to storage in water environment. Fat contents were not greater than 2.00%. The samples showed low range of protein content (7.9-9.8%) because it is a leafy product. The fibre contents were found to be (0.84-1.32%) on wet samples. Among the mineral elements, potassium and sodium were the most abundant constituents. Vitamin contents in these samples were not much different with other Myanmar fermented food. The samples contained a population of bacteria and fungi within the permissible range. E.coli was detected in four of the samples. It might be due to the contamination from environment or from hands of food handlers as well as from packing materials.

References Aung Myint, Oo Aung and Yee Yee Lwin (1996). " Neutural protease from Bacillus firmus of Myanmar Shrimp Sauce". J.Myanmar Chemical Society,3 , 1-4 FAO (1998). "Fermented Fruits and Vegetables", Food and Agricultural Organization of the United Nations, Rome. 32-35 FAO (2000). "Fermented grain legumes, seeds and nuts ", Food and Agricultural Organization of the United Nations, Rome.67 Pearson, D. (1976). "The Chemical Analysis of Food". J. and A.Chuchill Livingstone, New York.

370-380 Prescott, L. K. et.al (1999). " Microbiology ". McGraw-Hill Book Co., Inc. New York. 480-490

129 | P a g e

Information Technology, Electric

130 | P a g e Performance Evaluation for Simultaneous Perturbation Particle Swarm Optimization Shin Sempukuji *a, Hidetaka Ito a, Yutaka Maeda a a Graduate School of Science and Engineering, Kansai University.

Keywords: Optimization, PSO, Metaheuristic, Simultaneous perturbation method ABSTRACT Particle swarm optimization is one of the optimization techniques. Simultaneous perturbation optimization method is known as stochastic gradient method. Searching ability is improved by adding the gradient information to the ordinary PSO. We propose the PSO using the simultaneous perturbation optimization method. We have verified improvement of the proposed schemes that combine the PSO with simultaneous perturbation method using 50-dimensional evaluation functions. *k962343@kansai-u.ac.jp INTRODUCTION PSO (Particle Swarm Optimization) is one of optimization techniques. The PSO algorithm is modeled by the movement of swarm of birds or school of fish. PSO is a multi-point search. Individual search points were updated their positions sharing the individual best values and the group best value. This technique does not require direct calculation of derivatives of an objective function.

On the other hand, the simultaneous perturbation optimization is known as stochastic gradient method. The simultaneous perturbation optimization method is an extension of the usual difference approximation method to estimate a gradient by using a perturbation. It is possible for the method to estimate the slope without increasing the number of observations for the function by adding perturbations to all parameter elements simultaneously.

We propose new search methods by combining the PSO with the simultaneous perturbation. By using the proposed method, searching ability will be improve. Using some functions, we evaluated the proposed methods.

SIMULTANEOUS PERTURBATION PARTICLE SWARM OPTIMIZATION A. Particle Swarm Optimization  PSO contains three terms to update each search points. v t in right hand side of Equation (1) is an



inertia term. It denotes the same direction that has come until now. p t is a previous best value



(individual best value). The best value found by the all particles is nt (global best value).

Velocity vector and the position vector is given by the following equation.

      v t 1   v t  1  pt  x t   2 nt  x t     x t 1  x t  v t 1

(1) (2)

Here,  is convergence coefficient.  is attenuation coefficient. 1 ,2 is uniform random numbers.

131 | P a g e B. Simultaneous Perturbation method Simultaneous perturbation optimization method was proposed as an extension of the difference approximation. It is a stochastic gradient method. Algorithm of the simultaneous perturbation optimization method is as follows;

x t 1  x t  Δxt

(3)

xti 

(4)

f xt  ct   f xt  (i  1,2,n) c ti

Δxt is an estimator of partial derivative of the function f x  .  is gain coefficient.  Δx is gradient term. c t is perturbation vector.

C. Simultaneous Perturbation Particle Swarm Optimization Here, we describe six types of the proposed methods.

Scheme01

      v t 1  Δxt  v t  1  pt  x t   2 nt  x t 

(5)

It is a method obtained by adding the simultaneous perturbation optimization method to ordinary PSO. Scheme02

     v t 1  Δxt  1  pt  x t   2 nt  x t 

(6)

It is a method that replaced the inertia term of the PSO with the simultaneous perturbation optimization method.

Scheme03

    v t 1  Δxt  v t  2 nt  x t 

(7)

It is a method that replaced the term towards the PSO of self of optimum value with the simultaneous perturbation optimization method.

We used either the PSO or the simultaneous perturbation optimization method for search points in scheme 04, 05, 06.

Scheme04 uses the simultaneous perturbation optimization method only for minimum value in the search points. Other search points use the PSO. Scheme05 uses the simultaneous perturbation optimization method for minimum value and half of the search points. Other search points use the PSO. Scheme06 uses the simultaneous perturbation optimization method for half of the search points. Other search points use the PSO. EXPERIMENT CONDITIONS We set up the following conditions. ・Dimension of evaluation functions：50-dimension ・Total Search points：30 points

132 | P a g e ・PSO parameters：   1.0,   0.9 , 1 ,  2 are set differently for evaluation functions. ・Simultaneous Perturbation optimization parameters： Gain coefficient  is set differently for different schemes.

Perturbation c is set differently for different evaluation function. ・The initial values of the search points are randomly generated in the search area. ・Search area is set up differently for different evaluation functions.

TEST FUNCTIONS We use the following five evaluation functions to consider the performance of the proposed schemes.

These functions have different characteristic on shape. Therefore, it is convenient to evaluate ability of optimization techniques.

A. Rastrigin Function This function has many keen local minimums. n





f x   10n   xi2  10 cos2xi 

(8)

i 1

B. Rosenbrock Function The function has gentle slope leading to global minimum. n 1







f x    100 xi2  xi 1  xi  1 i 1

2

2



(9)

C. 2n-minima Function The function has flat bottom with some local minimums.

f x  



1 n 4  xi  16 xi2  5xi 2 i 1



(10)

133 | P a g e

D. Michalewicz Function This function contains keen global minimum and keen valleys. n



 

f x    sin xi  sin ixi2  i 1



20



(11)

E. Ackley Function The function has many shallow dips as local minimums.

 1 n 2 1 n  f ( x)  20 exp   0.2  xi   exp   cos2xi   20  exp 1 n i 1  n i 1   

(12)

RESULTS We obtained the results (see Table 1) by MATLAB for the proposed schemes. On average, we can see that efficiency of the proposed methods is better than the ordinary PSO. We were verified the six types of the proposed method by using five 50-dimensional evaluation functions. As a result, it was confirmed that at least one or more proposed schemes converge to optimal point of the five test functions. For some functions, PSO could not find the optimum value.

134 | P a g e Since different test functions have different characteristics, we could not find an almighty method.

However, some of the proposed method is efficient for some evaluation functions.

By combining the PSO with the simultaneous perturbation optimization method, it was confirmed that capability of PSO or the simultaneous perturbation method are improved.

CONCLUSIONS We proposed simultaneous perturbation particle swarm optimization methods that are combination of the ordinary PSO and the simultaneous perturbation method. We confirmed effectiveness of the proposed methods, compared with the PSO or the ordinary simultaneous perturbation optimization method.

Practical applications of the proposed methods and effectiveness of the methods in practical fields are also our future works.

Table 1 Comparison of the proposed methods for test functions Scheme Functions

Rastrigin

PSO

Scheme1

Scheme2

159.7516 (226.2487)

0.0143 (0.0553) 7.0E-08

0.0026 (0.0041) 3.0E-07

81.8899 (209.5687)

14.3459 (16.5568) 7.0E-10

29.1485 (30.5574) 2.0E-08

[-5 5]

Observations (million times) Best Average Gain coefficient

[-2 2]

Best Average Gain coefficient Perturbation

-minima (Styblinski-Tang)

Ackley

Scheme4

Scheme5

Scheme6

20

Perturbation

Rosenbrock

Scheme3

Search area Parameter

[-5 5]

[-30 30]

Michalewicz [-3.14 3.14]

0.0097 4.0700E-11 1.1676E-04 (0.0152) (2.1591) (2.0293E-04) 3.0E-08 4.0E-07 6.0E-08 -0.5～0.5 (-0.0001～0.0001 is excluded.)

11.1213 0.1085 0.0750 (12.5339) (0.4322) (7.2831) 4.0E-10 8.0E-09 1.0E-10 -0.01～0.01 (-0.0001～0.0001 is excluded.)

3.1593E-05 (5.6107E-05) 3.0E-08

0.0152 (0.4194) 2.0E-10

Best -1.4318E+03 -1.9563E+03 -1.9577E+03 -1.9352E+03 -1.8594E+03 -1.8734E+03 -1.8452E+03 Average (-1.2858E+03) (-1.8792E+03) (-1.9141E+03) (-1.8237E+03) (-1.6801E+03) (-1.6999E+03) (-1.6943E+03) Gain coefficient 4.0E-03 9.0E-03 4.0E-03 4.0E-03 6.0E-02 8.0E-08 -0.01～0.01 Perturbation (-0.0001～0.0001 is excluded.)

Best 8.0518 0.0100 0.0026 0.0197 3.2835E-05 0.0015 0.0436 Average (10.9022) (0.0125) (0.0031) (0.0235) (5.4950E-05) (0.0020) (5.2156E-04) Gain coefficient 1.0E-04 9.0E-05 9.0E-05 3.0E-04 4.0E-05 9.0E-06 -0.5～0.5 Perturbation (-0.0001～0.0001 is excluded.)

Best Average Gain coefficient Perturbation

-28.5801 (-23.1962)

-49.3800 (-48.6663) 9.0E-09

-49.5801 (-49.3459) 2.0E-07

-49.5680 -49.5539 -49.5594 (-49.4195) (-48.9840) (-49.3544) 1.0E-08 3.0E-08 1.0E-07 -0.00008～0.00008 (-0.000000001～0.000000001 is excluded.)

-49.5791 (-49.3982) 1.0E-07

REFERENCES Y. Maeda, H. Hirano and Y. Kanata (1995), A Learning Rule of Neural Networks Via Simultaneous Perturbation and Its Hardware Implementation, Neural Networks, vol.8, pp.251-259.

Charlie Vanaret, Jean-Baptiste Gotteland, Nicolas Durand, Jean-Marc Alliot (2014), Certified Global Minima for a Benchmark of Difficult Optimization Problems, Genetic and Evolutionary Computation Conference 2014.

S. Surjanovic, D.Bingham (2013), Virtual Library of Simulation Experiments: Test Functions and Datasets. Retrieved February 5, 2015, from http://www.sfu.ca/~ssurjano.

Riccardo Poli, James Kennedy, Tim Blackwell (2007) Particle swarm optimization An overview, Swarm Intell, pp.33-57.

135 | P a g e The Story of Chaotic Circuits and Systems at Sirindhorn International Institute of Technology,Thammasat University Buncha Munmuangsaen a, Banlue Srisuchinwong *,a a

School of Information, Computer, and Communication Technology, Sirindhorn International Institute of Technology, Thammasat University, Pathum-Thani, Thailand Keywords: Chaos, Jerk, Hyperjerk, Snap, Current-Tunable Circuits

ABSTRACT Since the first chaotic system has been identified in 1963 by Edward Lorenz, the design and development of chaotic circuits and systems over the subsequent years have been of increasing interest. In this review, two papers on chaotic systems and two papers on chaotic circuits are briefly reviewed as particular examples of chaotic circuits and systems discovered at Sirindhorn International Institute of Technology (SIIT), Thammasat University (TU). The first paper describes generalization of the simplest autonomous chaotic system, whereas the second describes elementary chaotic snap flows. The third describes four current-tunable chaotic circuits in a set of two diodereversible pairs, whereas the fourth describes a current-tunable chaotic jerk circuit. *banlue@siit.tu.ac.th INTRODUCTION In the early 1960s, a young MIT meteorologist Edward Lorenz (1963) accidentally discovered sensitive dependence on initial conditions while modeling atmospheric convection on a primitive digital computer leading to the discovery of the celebrated Lorenz equation in three first-order ordinary differential equations (ODEs). His results had little impacts until 1976 when Otto Rössler (1976) found another chaotic system with a single quadratic nonlinearity that is algebraically simpler than the Lorenz system. Many even simpler chaotic systems were systematically discovered by Sprott (1994, 2010) through an exhaustive computer search. One of the cases was then demonstrated in the explicit third-order form by Gottlieb (1996) which he called a „jerk function‟ since it involves a third derivative of displacement.

Sprott (1997) discovered the simplest autonomous chaotic jerk system. Generalization of such a simplest case was reported fourteen years later by Munmuangsaen et al (2011). The resulting chaotic jerk systems have been categorized as low dimensional systems. On the other hand, hyperjerk systems (Chlouverakis and Sprott, 2006), (Sprott, 2010) involve time derivatives of the jerk function. The dimensions of hyperjerk systems are apparently higher than the third-order jerk systems and therefore they are considered to be high dimensional systems. In particular, hyperjerk systems with a fourthorder derivative have been referred to as snap systems (Sprott, 2010). Some elementary chaotic snap flows, both conservative and dissipative flows, were studied at Sirindhorn International Institute of Technology (SIIT), Thammasat University (TU) and reported by (Munmuangsaen and Srisuchinwong, 2011).

The design and implementation of three-dimensional (3-D) autonomous chaotic circuits over the past three decades have been increasing due to a variety of applications in, for example, spacecraft trajectory control, stabilization of the intensity of a laser beam, noise radars and sonar (Kilias et al, 1995), synchronization (Carroll and Pecora, 1990) and secure communications (Cuomo et al, 1993),

136 | P a g e (Srisuchinwong and Munmuangsaen, 2011). Existing 3-D chaotic circuits may be separated into two types (Srisuchinwong and Nopchinda, 2013). Type-I involves chaotic circuits based on three firstorder ODEs, e.g. a well-known Chua‟s circuit (Matsumoto et al, 1985) and a chaotic circuit based on a Lambert-W function (Srisuchinwong and Amonchailertrat, 2013). However, such circuits are not especially simple and are relatively difficult to scale to arbitrary frequencies. Type-II involves chaotic jerk circuits described by a single third-order ODE. Circuits in Type-II have increasingly received great attention owning to the simplicity. Recently, current-tunable approaches have been reported for both Type-I (Srisuchinwong and Munmuangsaen, 2012) and Type-II (Srisuchinwong and Nopchinda, 2013), (Srisuchinwong and Treetanakorn, 2014).

In this paper, four particular examples of chaotic circuits and systems discovered at SIIT, TU are briefly reviewed. The first example summarizes the generalization of the simplest autonomous chaotic system (Munmuangsaen et al, 2011). The second summarizes the elementary chaotic snap flows (Munmuangsaen and Srisuchinwong, 2011). The third summarizes four current-tunable chaotic circuits in a set of two diode-reversible pairs (Srisuchinwong and Munmuangsaen, 2012). Finally, the fourth summarizes a current-tunable chaotic jerk circuit (Srisuchinwong and Nopchinda, 2013).

DISCOVERED CHAOTIC SYSTEMS A.

Generalization of the Simplest Autonomous Chaotic System

The algebraically simplest example of the dissipative chaotic system (Sprott, 1997) can be expressed as

x  ax  x   x2

(1)

which has only three terms in its jerk representation, or equivalently five terms in its dynamical system representation, with a single quadratic nonlinearity ( x 2 ) and a single parameter a. This raises a question of whether there are other simple chaotic systems of the form

x  x  x  f ( x)

(2)

where f ( x) is the nonlinear function required for chaos. To find chaotic solutions for each of the specified nonlinear function f ( x) , the procedure of a systematic numerical search developed in (Sprott, 1994, 1997, and 2010) was employed. By this method, many chaotic systems with different nonlinearities were found by Munmuangsaen et al (2011). Examples of the function f ( x) that can produce chaos are selected and listed in Table 1. The case of f ( x)   A exp( x) with A = 0.1 is of particular interest since it has the curious feature of having chaos in the limit of A  0 . The linear

x  x / A (so x  x / A , etc.) results in x  x  x   A2 exp( x / A) . This system in the limit of A  0 can be reproduced using a two-segment piecewise-linear approximation in which f ( x)   (1  x) for x  1 and zero otherwise, which produces chaos even in the limit of    . In this case, f ( x) has the characteristic of an ideal diode. It suggests that (2) can be transformation of

electronically implemented using a single diode as the nonlinear element. One example has been examined and reported in (Sprott, 2011).

137 | P a g e Table 1: Some functions f ( x) that produce chaos in equation (2).

Ca se

f ( x)

Ca se

f ( x)

GS 1

0.1exp( x)

GS 7

1/ ( x  2)2

GS 2

 exp( x  2)

GS 8

5 x  1  5 x

GS 9

0.4 /  x  1

1/  x  1

GS 3

B.

5.1cos( x  0.1)

GS 4

0.2 tan( x)

GS 10

GS 5

 sgn(1 4 x)

GS 11

GS 6

 x2  0.2 x3

GS 12

0.5

 4 sin( x  1)  2.2 x  cosh( x )  0.6 x

Elementary Chaotic Snap Flows

Early examples of chaotic snap flows have been demonstrated via an extensive numerical search with quadratic nonlinearities (Chlouverakis and Sprott, 2006), (Sprott, 2010). Through the use of the extensive numerical search at SIIT, Table 2 lists six examples of the resulting found chaotic snap flows (Munmuangsaen and Srisuchinwong, 2011). Four of them have demonstrated elegant simplicity of a single control parameter based on a single nonlinearity of either a quadratic, a piecewise-linear or an exponential type. Two others have demonstrated elegant simplicity of all unity coefficients based on either a single cubic nonlinearity or three cubic nonlinearities. The chaotic snap flow with a single cubic nonlinearity has only two terms. It can be noticed from Table 2 that three examples are conservative systems and three others are dissipative systems.

Table 2: Six elementary chaotic snap flows Ca

Equations

ses E CS1

x   Ax  x2  x

E CS2

x   Ax  x  x

E CS3

System types

Lyapunov exponent spectrums

Conservativ

0.03574, 0, 0, -0.03574

Conservativ

0.05537, 0, 0, -0.05537

x   x  Ax  x  ( x  1)

Dissipative

0.11107, 0, -0.43926, -0.67184

E CS4

x   x  Ax  x  exp( x)

Dissipative

0.16788, 0, -0.15074, -1.01714

E CS5

x   x3  x

Conservativ

0.07681, 0, 0, -0.07681

e e

e

138 | P a g e

E CS6

x   x  x  x3  x3  x 3

Dissipative

0.05351, 0, -0.20296, -0.85054

DISCOVERED CURRENT-TUNABLE CHAOTIC CIRCUITS

A.

Four Current-Tunable Chaotic Circuits in a Set of Two Diode-Reversible Pairs

Four current-tunable chaotic circuits in a set of two reversible pairs were discovered at SIIT (Srisuchinwong and Munmuangsaen, 2012). Each circuit consists of 6 minimum electronic components in the category of chaotic circuits that employ an op-amp. These components include a capacitor-inductor-capacitor (CLC) network, an op-amp (μA741), a diode and a current source, as shown in Fig. 1. Figures 1a and 1b show the first diode-reversible pair of the current-tunable chaotic circuits using a floating-diode technique. Figures 1c and 1d show the second diode-reversible pair of the current-tunable chaotic circuits using a virtually-grounded-diode technique. They are capable of current-tunable bifurcations in a current-mode approach. The dynamical representation of the equations that governs the behavior of the circuit can be found in (Srisuchinwong and Munmuangsaen, 2012).

Figure 1: Four current-tunable chaotic circuits (Courtesy of Srisuchinwong and Munmuangsaen, 2012).

Numerical results of trajectories on a normalized (X, Z) plane of the chaotic circuit shown in Fig. 1a are shown in Figs. 2a to 2d, whereas experimental results on a (VC2, VC1) plane are shown in Figs. 2e to 2h, exhibiting period-1, period-2, period-4 behaviors, and a chaotic attractor, respectively. Both results are well consistent. They can be disclosed through the use of either an adjustable current source I0 (LM334) or an adjustable potentiometer R0 as I0 = VSS/R0. A period-doubling route to chaos can be apparently observed. The chaotic attractors of each pair are found to be 180 rotated images of each other (not shown here).

139 | P a g e

a

b

c

d

e

f

g

h

Figure 2: Numerical and experimental results (Courtesy of Srisuchinwong and Munmuangsaen, 2012).

B. A Current-tunable Chaotic Jerk Circuit A current-tunable chaotic jerk circuit, as shown in Fig. 3, was discovered at SIIT (Srisuchinwong and Nopchinda, 2013). The normalized chaotic jerk equation is readily tunable by a current source I0 in the form of

X  a2 X  a1 X  a0 X  b1 sgn( X )  b2 I O

(3)

Figure 3: A current-tunable chaotic jerk circuit (Courtesy of Srisuchinwong and Nopchinda, 2013).

Numerical and experimental results are compared in Fig. 4. The current source I0 can be either an adjustable current source LM334, or a potentiometer R0 as I0 = VSS/R0. A conventional op-amp μA741 is used. Numerical solutions of trajectories on a ( X , X ) plane are shown in Fig. 4a to 4e, and on a ( X , X ) plane are shown in Fig. 4k to 4o using tunable I0. Oscilloscope traces on a (VC1, V1) plane are

respectively depicted in Fig. 4f to 4j. Continuously smooth transitions can be observed from a single right-half (Fig. 4f), a right-half unbalanced double-scroll (Fig. 4g), a fully balanced double-scroll (Fig. 4h), a left-half unbalanced double-scroll (Fig. 4i), and eventually, a single left-half (Fig. 4j) attractor.

Further details can be found in (Srisuchinwong and Nopchinda, 2013).

5

5

6

4 4

5

0

2 Xdot

0

Xdot

Xdot

Xdot

Xdot

2 0

0

-2

0

-2

-5

-4 -4 -2

0

a

2 X

4

6

-6 -4

-2

0

X

b

2

4

6

-5 -5

0 X

c

5

-6

-5 -4

-2

X

0

2

d

4

-6

-4

-2 X

0

2

140 | P a g e

e

g

f

4

5

0

X2dot

2 X2dot

X2dot

X2dot

10

0

5 X2dot

5

0

j

6

5

0

0

-2

-5 -10

i

h

-5

-2

0

2 X

4

6

k

-4

-4 -2

0

X

l

2

4

6

-6 -5

0 X

m

5

-5 -6

-5 -4

-2

n

X

0

2

4

-6

-4

-2 X

0

2

o

Figure 4: Chaotic trajectories of Fig. 3 using I0 (Courtesy of Srisuchinwong and Nopchinda, 2013).

CONCLUSIONS Four topics of simple chaotic systems and circuits discovered at Sirindhorn International Institute of Technology, Thammasat University have been briefly summarized as particular examples. The first topic has reviewed the generalization of the simplest 3-D dissipative chaotic system. The second topic has reviewed the elementary chaotic snap flows with different nonlinearities. Three of them are conservative systems and the three others are dissipative systems. The third topic has reviewed four current-tunable chaotic circuits in a set of two diode-reversible pairs with simple nonlinearities. The last topic has reviewed the current-tunable chaotic jerk circuit with continuously smooth transitions from one-scroll to double-scroll attractors.

REFERENCES Carroll, T. L. and Pecora. L.M. (1990) IEEE Trans Circuits Syst, 38, 453-456.

Chlouverakis, K.E. and Sprott, J.C. (2006) Chaos, Solitons & Fractals 28, 739–746.

Cuomo, K.M., Oppenheim, A.V. and Strogatz, S.H. (1993) IEEE Trans Circuit Syst, 40, 626-633.

Gottlieb, P.W. (1996) Am. J. Phys., 64, 525.

Kilias, T., Kelber, K., Mogel, A. and Schwarz, W. (1995) Int J Electron 79, 737-753.

Matsumoto, T., Chua, L.O. and Komuro, M. (1985) IEEE Trans. Circ. Syst., 33, 797–818.

Lorenz, E.N. (1963) J. Atmos. Sci., 20, 130–141.

Munmuangsaen, B. and Srisuchinwong, B. (2011) Chaos, Solitons & Fractals, 44 (11), 995-1003.

Munmuangsaen, B., Srisuchinwong, B. and Sprott, J.C. (2011) Phys. Lett. A, 375, 1445-1450.

Rössler, O.E. (1976) Phys. Lett. A, 57, 397–398.

Sprott, J.C. (1994) Phys. Rev. E, 50, R647-R650.

Sprott, J.C. (1997) Phys. Lett. A, 228, 271-274.

141 | P a g e Sprott, J.C. (2010). Elegant Chaos: Algebraically Simple Chaotic Flows, World Scientific, Singapore.

Sprott, J.C. (2011) IEEE Trans. Circuits Syst. II, Exp. Brief, 58, 240–243.

Srisuchinwong, B. and Amonchailertrat, N. (2013) J. Circuit Syst. Comp., 22 (8), art no. 1350075 (12 pages).

Srisuchinwong, B. and Munmuangsaen, B. (2011) WIPO, WO2011105972.

Srisuchinwong, B. and Munmuangsaen, B. (2012) Electron. Lett., 48 (17), 1051–1053.

Srisuchinwong, B. and Nopchinda, D. (2013) Electron. Lett., 49 (9), 587–589.

Srisuchinwong, B. and Treetanakorn, R. (2014) Electron. Lett., 50 (24), 1815–1817.

142 | P a g e A Paddy Moisture Measurement Using Hybrid Rectangular and Circular Waveguide Resonator Navapadol Kittiamornkula, Kamon Jirasereeamornkulb, Supaporn Kittisinc, and Kosin Chamnongthai*,b a School of Biomedical Engineering, Christian University of Thailand b Department of Electronics and Telecommunication Engineering, Kingâ&#x20AC;&#x;s Mongkut University of Technology Thonburi c Technology of Information System Management, Mahidol University Keywords: moisture measurement, microwave, resonator, paddy ABSTRACT This paper proposes a paddy moisture measurement using hybrid rectangular and circular waveguide resonator. This hybrid resonator is designed for material moisture measurement using microwave frequency. It can resonate the electromagnetic wave in microwave range specifically at 2.45 GHz which relates to material moisture. In the experiment, the anodizing aluminium is used as the material of hybrid resonator. Each connector of the hybrid prototype is SMA connector in order to transfer microwave signal from microwave generator to the hybrid resonator. Moreover, the experiment contains the moisture measurement of 10 paddy samples. To evaluate the experiment, the resonator result is compared with the basic moisture measurement which is dry-weight method.

The result shows 82.69% comparing with dry-weight method, but the processing time is extremely reduced, and it is the non-destructive method that the sample is not be destroyed in the measurement. *metalicaed@hotmail.com INTRODUCTION In agriculture countries, paddy has very importance for plant. If paddy has high quality, agriculture plant will increase highly. One of the importance factors of paddy quality is moisture. The moisture of plant paddy needs to be known in order to make plant efficiently.

The moisture measurement can be divided into 2 comprehensive techniques which are destructive technique and non-destructive technique. In destructive technique (Reeb James E. and Milota Michael R., 1999), the selected samples are fumed to measure their weight before and after fuming. Therefore, this technique takes very long time, and the selected sample has to be destroyed by heating.

In non-destructive granular moisture measurement, the resonator technique using microwave for determination of moisture and mass in single soybean seed was proposed. The simulation of microwave cavity resonance for measuring moisture and mass of a single grain was determined (W. Kraszewski and O. Nelson, 1994). For the design of microwave cavity for single grain characterization of mass and moisture, this paper presented a technique for circular resonant cavity with 11.5 GHz of microwave frequency. The simulation of mass, moisture and density of single grain was measured (E. Chaix et al., (1997). The microwave transmission technique for determination of wheat moisture using an artificial neural network (ANN) was presented. The ANN was used to measure density-independent moisture content using transmission coefficient measurement (Philip G. Bartley et al., 1997). In planar resonator sensor for moisture measurements, the planar ring resonator

143 | P a g e was presented for sensitive detector of the moisture content of dielectric material (Jerzy Skulski and Bogdan A. Galwas, 1998). A cylindrical resonating cavity in TM010 Mode for moisture measurement was proposed. This cylindrical model indicated the moisture content and the specific mass from the complex permittivity (Emmanuelle Bourdel et al., 2000). In a microwave attenuation measurement for grain moisture content using microwave at 10.5 GHz, the horn antennas was used to measure many kind of granular such as Korean short-grain rough rice, brown rice and barley (Ki-Bok Kim et al., 2002). For the principles of microwave moisture measurement in grain, techniques of moisture measurement, including electric moisture meter, were also discussed (Stuart O. Nelson et al., 2002).

For the non-destructive sensing technique of the physical properties of granular materials using microwave permittivity measurement, the dielectric of wheat, corn, and soybean were determined by measuring the scattering transmission coefficient or S21 in free space at frequencies between 2 to 13 GHz (Samir Trabelsi and Stuart O. Nelson, 2006). In dielectric resonator antenna (DRA) for wireless application, the description of a simple design on dielectric resonator antenna (DRA) using disk shape for wireless application was presented (M.S.M. Aras et al., 2008). In the analysis of a continuous fluidized-bed microwave rice kernel drying system, this paper proposed an analysis of a continuous fluidized-bed microwave rice kernel drying system with perpendicular slots of applicator on a concentric cylindrical cavity excited by perpendicular waveguides (C. Sangdao and M. Krairiksh, 2008). In a system of microwave cylindrical cavity resonator for granular material dielectric measurement using two waveguide transmitters, this paper presented the design of cylindrical cavity, and the distribution of electric field was demonstrated for 2.45 GHz (Kittiamornkul N. et al., 2010). In the effect to the distribution of electric field inside the resonator, this paper presents the distribution of electric field of sample which is solid and grain forms inside the designed cylindrical resonator, and the computer simulation was used to determine the electric field for each form of sample (Kittiamornkul N. et al., 2011).

This paper proposes the prototype of hybrid rectangular and circular waveguide resonator. The evaluation of the prototype of hybrid resonator is proved by comparing with basically destructive moisture measurement. The basic theory, simulation, experiment and conclusion are described in next section respectively.

EXPERIMENT According to microwave theory (Algie L. Lance, 1964), the design of hybrid rectangular and circular waveguide resonator was proposed by authors. The dimension of the hybrid resonator is shown by the following figure.

Fig. 1 Diagram of cylindrical resonator with two port waveguides (a= 7.674cm, b= 3.837cm, c=8.5135cm, d= 7.088cm, e=7.0865cm, f= 4.48cm, and g= 5cm) The experiment of the hybrid resonator was to show how this technique performs. The conducting specification of material used to construct the resonator is shown in following table.

144 | P a g e Table 1: Resonator Material Specification Part

Material

σ

Unit

Resonator

Anodizing aluminum

3.82×107

S/m

SMA Connector

Copper

5.96×107

S/m

The hybrid resonator was created by anodizing aluminum that has robust properties in moisture environment. The hybrid resonators is shown in Fig. 4. The portable microwave meter (J. Mearnchu, 2008). In processing, the microwave meter generates 2.45 GHz specifically.

The hybrid resonator was created by anodizing aluminum that has robust properties in moisture environment. The hybrid resonators is shown in Fig. 2 a.). The portable microwave meter (J. Mearnchu, 2008), which is supported by the Department of Telecommunication Engineering of KMITL, was used to measure the moisture content of sample. In processing, the microwave meter generates 2.45 GHz specifically.

In the experiment, 10 paddy samples was used in the experiment. The paddy sample fills in resonator fully, as shown in Fig. 2 b.). Then, the dielectric constant and dielectric loss factor are measured by the microwave meter, and they can calculate into moisture of the paddy sample.

a.)

b.)

Fig. 2 The hybrid resonator a.) The hybrid rectangular circular resonator b.) Paddy sample in the resonator The experiment setup of hybrid resonator with the sample inside is shown in Fig. 3

145 | P a g e

a.)

b.)

Fig. 3 The experiment setup a.) a.) The setup of port1 (circular waveguide as input) b.) The setup of port2 (rectangular waveguide as input) RESULTS AND DISCUSSION The relation between dielectric constant (r), dielectric loss factor (r) and the percentage moisture content (%MC) are demonstrated by the following equation (J. Mearnchu, 2008):

 r

%MC 

 r (0.3572   r   r) 0.0517

 0.1630 .............................................................................. (4)

The percent moisture content (%MC) obtained from the hybrid resonator was compared to that the %MC of the dry weight method which is the most trustworthy. A dry weight method is the ratio of sample weight before and after fuming. The results of this experiment used %MC of 10 paddy samples were shown in Table 2.

Table 2: The %MC of paddy samples Sample No. Dry-weight Port 1 Port 2 1

20.31

20.59

18.70

2

20.73

14.02

18.39

3

22.00

20.52

20.52

4

21.10

17.73

17.06

5

25.29

18.26

20.40

6

24.95

18.66

19.41

7

23.02

14.25

16.50

8

22.47

19.19

19.84

146 | P a g e 9

21.07

16.78

19.97

10

22.70

16.12

19.36

Average

22.36

17.61

19.01

According to Table 2, the total of %MC of hybrid resonator can be found by the average value of port1 and 2 which is 18.49%. Finally, the accuracy of the hybrid resonator is 82.69% comparing with dry-weight method which is a basic method.

In experiment, the hybrid resonator shows 82.69% comparing with dry-weight method. The result is close to basic method, and it is the non-destructive method. Moreover, the processing time is extremely reduced. For more reliability, more samples and different samples should be experimented.

CONCLUSIONS This paper proposes a paddy moisture measurement using hybrid rectangular and circular waveguide resonator. The hybrid resonator which is used in the experiment is constructed by anodized aluminum.

The hybrid rectangular and circular waveguide resonator is tested real environment in order to evaluate the efficiency comparing with basic moisture measurement which is the destructive method.

The experimental result shows 82.69% comparing with dry-weight method, but the processing time is extremely reduced, and it is the non-destructive method that the sample is not be destroyed in the moisture measurement.

REFERENCES Reeb James E. and Milota Michael R. (1999). Moisture content by the oven-dry method for industrial testing, 50th Western Dry Kiln Association Meeting, Portland, Oregon, USA., 66-74.

Kraszewski A.W. and Nelson S.O. (1994). Microwave Resonator Technique for Moisture Content and Mass Determination in Single Soybean Seeds, Transaction on Instrumentation and Measurement, Vol. 43, No. 3, 487 - 489.

Chaix E., Haigh A.D., Thompson F., Gibson A.A.P. and Campbell G. (1997). Microwave Cavity Design for Single Grain Mass and Moisture Characterization, Conference on High Frequency, Manchester, United Kingdom, 35 – 40.

Philip G. Bartley, Jr., Stuart O. Nelson, Ronald W. McClendon, and Samir Trabelsi, (1997).

Determining Moisture Content of Wheat with an Artificial Neural Network from Microwave Transmission Measurements, Transaction on Instrumentation and Measurement, Vol. 47, NO. 1, 123126.

Skulski J. and Galwas B.A. (1998). Planar Resonator Sensor for Moisture Measurements, 12th International Conference on Microwaves and Radar, Vol. 3, 692 – 695.

Bourdel E., Pasquet D., Denorme P. and Roussel A. (2000). Measurement of the Moisture Content with a Cylindrical Resonating Cavity in TM010 Mode, Transaction on Instrumentation and Measurement, Vol. 49, No. 5, 1023 – 1028.

Ki-Bok Kim, Jong-Heon Kim, Seung Seok Lee, Sang Ha Noh (2002). Measurement of Grain Moisture Content using Microwave Attenuation 10.5 GHz and Moisture Density, Transaction on Instrumentation and Measurement, Vol. 51, No. 1, 72 – 77.

147 | P a g e Nelson S.O., Trabelsi S., Kraszewski A.W. (2002). Principles of Microwave Moisture Measurement in Grain, 19th Conference on Instrumentation and Measurement Technology, Anchorage, AK, USA, Vol. 1, 99 – 102.

Trabelsi S. and Nelson S.O. (2006). Nondestructive Sensing of Physical Properties of Granular Materials by Microwave Permittivity Measurement, Transaction on Instrumentation and Measurement, Vol. 55, No. 3, 953 – 963.

Aras M.S.M., Rahim M.K.A., Asrokin A. and Aziz M.Z.A.A. (2008), Dielectric Resonator Antenna (DRA) for Wireless Application, International Conference on RF and Microwave, Kuala Lumpur, Malaysia, 454 – 458.

C. Sangdao and M. Krairiksh (2008), Analysis of a Continuous Fluidised-bed Microwave Rice Kernel Drying System, Transaction on Science and Technology, pp. 61-71.

Kittiamornkul N., Chamnongthai K., Jirasereeamornkul K., and Higuchi K. (2010). A System of Microwave Cylindrical Cavity Resonator for Granular Material Dielectric Measurement using Two Waveguide Transmitters, Conference on The Society of Instrument and Control Engineers, Taipei, Taiwan, 1888 – 1891.

Kittiamornkul N., Watcharakitchakorn O., Jirasereeamornkul K., Chamnongthai, K., Higuchi K. (2011). The study of electric field distribution of cylindrical cavity resonator with two waveguide transmitters for granular material dielectric measurement, Conference on The Society of Instrument and Control Engineers, Tokyo, Japan, 2433 – 2437.

Algie L. Lance (1964). Introduction to Microwave Theory and Measurements, McGraw-Hill, New York.

J. Mearnchu, T. Limpiti, D. Torrungrueng, P. Akkaraekthalin and M. Krairiksh (2008). A Portable Dielectric-Property Determination System using Coupled Antennas, MIKON International Conference, Poland, 884-887.

148 | P a g e Evaluation of Effect in Initial Setting of Tree Size for GP Kohei Matsumura*, Yoshiko Hanada**, Keiko Ono*** and Noriaki Muranaka** *Graduate School of Science and Engineering, Kansai University ** Faculty of Engineering Science, Kansai University ***Department of Electronics and Informatics, Ryukoku University

1.

Genetic Programming and dMSXF

Genetic programming (GP) is one of optimization methods that have been extended to handle a tree structure as a genotype of genetic algorithms (GAs). GP has been widely used for automatic programming. Which is described using tree structure, such as agent controls. To apply GP to tree structure optimization, it is important to design a crossover which inherits parentsâ&#x20AC;&#x; features to offspring effectively. In addition, crossovers should not cause a bloat where trees rapidly grow in early generation. Therefore many crossovers have been developed to consider the characteristics of tree structures. Among them, deterministic multi-step crossover fusion (dMSXF) [1] is a promising crossover to generate a wide variety of offspring that acwuire parentsâ&#x20AC;&#x; preferred characteristics with considering problem-specific features. dMSXF could effectively avoid the bloat however, it suppresses favorable growth of trees. Therefore the setting of the size in the initial population of the tree is very important. Might be difficult for GP to find optimal solutions if it start searching with small trees, in this paper, we evaluate the search performance of dMSXf with several settings of initial tree size, using symbolic regression problems. A symbolic regression problem is a problem to find an approximate function from an observed input output data set.

2.

Numerical Experiments

We verify effect of the configuration of size in the initial of trees on search performance of crossovers. dMSXF, size fair crossover(SFX) [2] and one point crossover (1x) are used in the experiments. SFX is the crossover method improving on 1x, and suppresses the growth of trees by limiting the size of the replacement subtree between the parents. An example function used for the verification is x4-x3+x2-x. The range of the variable x is [1, -1], and 21 sample points are placed at regular intervals in that this. Population size is 20, 50 and 100, and each run is terminated after 100 generations.

Each tree in the initial population is generated at random within the range Tinit. Here we examine 5 patterns of Tinit=[5,9], [5,15], [11,21], [15,25] and [21,31]. The number of offspring that are generated

Fig.1 Distribution of solutions

by one crossover is 12 or 24. Figure 1 illustrates the number of successes at 20 trials of three crossover methods. From this result, we can find that the size in the initial of tree at dMSXF has a major effect on the success rate. dMSXF cannot find optimal solutions with Tinit=[5,9]. This is because a bloat of trees is suppressed and trees cannot reach the tree size that expresses the function. For that reason, search performance of dMSXF at a small initial tree is inferior to another 2 methods. On the other hand, dMSXF shows very high search performance with a large initial tree set.

149 | P a g e

[1] Ikeda，K. and Kobayashi，S.:

Deterministic Multistep Crossover Fusion: A Handy Crossover

for GAs．Proc. of Parallel Problem Solving from Nature．PPSN VII．pp．162-171，2002． [2] W. B. Langdon, "Size Fair and Homologous Tree Crossovers for Tree Genetic Programming, Genetic Programming and Evolvable Machines, pp. 95--119, 2000

150 | P a g e Copyright Protection by Data Embedding to Image Katsuaki Kiwaki a*, Hidetaka Ito a, Yutaka Maeda a a

Graduate school of science and engineering, Kansai University Keywords : Copyright, Protection, Image data, Data embedding, Separating ABSTRACT Copyright protection is crucial issue in contemporary network society. Then, watermarking is key technology. For example, by replacing the noise component such as quantization error of digital information into the secret information, it is possible to embed the copyright information without being noticed by third party. In this work, we propose a technique which can embed a copyright image to target images using very simple procedure. Extracting the embedded image is also very easy. We examine validity of the proposed method. *k370672@kansai-u.ac.jp INTRODUCTION In todayâ&#x20AC;&#x;s network society, it is essential to protect the digital information in order for those who know little knowledge about network to use securely the internet. Through the internet, many images are copied and distributed. Most of these images do not have the copyright and the users make a judgment about copy and distribution. As one of the ways to protect these information, we propose a data embedding technique.

The data embedding is a technology which embeds a digital information into a different digital information without being noticed by other user. In this studying, a binary data is an embedding data.

We propose a method to mix an embedding data into an image and to separate the embedding image from the mixed image.

We embedded a binary copyright information into an image and separated the information from the mixed image. When the image is abused and outflowed illegally. By extracting copyright information, we obtained a legal basis from the extracted image.

EXPERIMENT A. Data embedding In this chapter, we will describe the data embedding technique used in this study. By mixing two images of the embedding and original images, we create a combined image. When we would like to check legitimacy of the image, we can separate the two images; copyright and original images from the mixed image. In this work, the original image is multilevel halftone color image, and the embedded image and binary data represented by black and white is used. The size of the original image was a vertical 100 pixels Ă&#x2014; horizontal 100 pixels.

We embedded an image S1 to an original image S2 to obtain a mixed image X. The mixing ratios of them are a1 and a2. In this case, images S1 and S2 have the size of m by n pixels. The mixing image xij is generated by the following Equation (1) using mixing ratio a1 and a2. Where, i and j denote row and column number of images, respectively.

151 | P a g e

xij  a1s1ij  a2 s2ij

(i=1,2,…,n j=1,2,…,m)

(1)

Mixing ratios a1, a2 are determined to satisfy the condition of equation (2).

a1  a2  1

(2)

In the separation process, S2 is unknown, and by using the mixed image X, and the mixing ratio a1, a2, we have to estimate the s1ij. The concept of embedded processing and separation processing is shown in Figure 1.

Figure 1 Embedding and separation processes B. Mixing ratio and embedding and separation performance To examine the performance of the data embedding and the separation, we embedded QR code shown in Figure 2 to three natural image shown in Figure 3 by changing the mixing ratio, and we examined accuracy of separated images and SN ratio. First, we examine influence for change of the mixture ratio. Next, we embed the QR code to the natural image and measured the SN ratio. Finally, we separated the QR code from the mixed image, and examine accuracy of the separated images. For different mixture ratio, we examines these feature.

RESULTS AND DISCUSSION In Figure 2, the embedded image can be clearly recognized. In this case, the separation accuracy is 100%. In Figure 3, it is difficult to recognize the embedded image. Then, the separation accuracy is 93.8%. Therefore, we can embed an image without being seen.

Figure 2 mixture ratio is 0.5

Figure 3 mixture ratio is 0.01

152 | P a g e CONCLUSIONS In this work, we proposed an electronic watermark technique. The proposed embedding and separation procedure is very simple. It is easy to utilize this technique for many uses such as copyright protection, information embedding and so on. For example, when the image is stolen by a third party, by separating the image of the copyright information, it is possible to make the image as legal basis. It is valid to use the proposed data embedding technique for copyright protection.

Effective embedding is important; relations between mixing ratio versus SN ratio, or mixing ratio versus extracting accuracy are serious issues for future consideration.

REFERENCES K.Matsui(1998), The basic of electronic watermark, Morikita Publishing.

S.Teduka (2011), The basic of the information technology, Kyoritsu Publishing.

Y. Seto (2007), The information security introduction, Japan Industrial Publishing.

K. Matsui, M. Iwakiri, The basic of the information hiding, Morikita Publishing.

R.G. Van Schyndel, A.Z. Tirkel, C.F. Osborne (1994), A digital watermark. Monash University.

153 | P a g e Optimization for Acoustic Characteristics of Wall Based on Three-degree-of-freedom Model Takuya Tanikado*, Yoshiko Hanada**, Masahiro Toyoda*** and Noriaki Muranaka** *Graduate School of Science and Engineering, Kansai University ** Faculty of Engineering Science, Kansai University ***Faculty of Environmental and Urban Engineering Departments, Kansai University E-mail: {hanada, muranaka}@ kansai-u.ac.jp

In acoustic designs, it is essential to choose the quality of materials which satisfies target frequency response of the acoustic absorptivity and the transmission loss for a wall boundary. Whole design cost is reduced by predicting these characteristics before developing a real model for experiments. In general approach, we evaluate acoustic behaviors of a wall by numerical simulating. Three-degree-offreedom model has been proposed to simultaneously consider frequency responses of both the acoustic absorptivity and the transmission loss, for a wall boundary. However, it is difficult to manually verify a large number of patterns of parameters under strict constraints. In this study, we formulated the parameter decision problem of three-degree-of-freedom model as an optimization problem. First, we define an objective function to find suitable parameter settings that simultaneously satisfies both the acoustic absorptivity and the transmission loss. Then, we adopt a genetic algorithm as an approximate solver in real time.

The following graphs are obtained by minimizing our proposed objective function. We present the detail of the objective function and other results of various cases at poster presentation.

transmission loss TL

acoustic absorptivity α 30

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154 | P a g e Noise Reduction in Thai Documents Using Sub-Cluster Labeling Nareeporn Piroonsup*, Sukree Sinthupinyo Department of Computer Engineering, Chulalongkorn University, Bangkok, Thailand Keywords : Noise Reduction, Thai Document, Semi-Supervised Learning ABSTRACT Labeling is a high time consuming process in several machine learning data preparation, especially in document analysis tasks. In this paper, we focus on a method which can help labeling in semisupervised learning for removing noise in Thai documents. In general, many researchers have applied the semi-supervised learning to problems having large data set because it can reduce human effort by clustering and automatic labeling. However, the general cluster-and-label method used in semi-supervised learning cannot distinguish noise from Thai characters well. Moreover several good tools for removing noise for other language documents always remove small characters in Thai. Hence, we propose a new approach that uses feature selection and sub-clustering algorithm to label components in clusters which comprises of mixed class user-labeled components. The results from the experiments show that our method can improve the overall accuracy of noise reduction in Thai documents.

INTRODUCTION The semi-supervised learning has been used with the problems in which there are too many data to label, especially in document analysis problem. In this paper, we use the semi-supervised learning to distinguish noise from characters in Thai documents. One major problem of the semi-supervised learning is how to label and use the unlabeled data effectively. Although automatically labeling the unlabeled data can help increase the number of training data, it can dramatically decrease the overall accuracy. This is because the newly labeled data may be incorrect and they become the noise in the training set. Nowadays the risk of using the unlabeled data is getting more interesting (Singh et al., 2009, Li and Zhou 2015).

RELATED WORKS Cluster-and-label is an efficient semi-supervised learning approach that has been used in various works especially in document analysis (Zeng et al. 2003, Zhang et al. 2015). The process is clustering all training data and labeling the unlabeled data in each cluster with a class of user labeled data in the cluster. Unfortunately, the clusters may consist of different-class labeled data. The traditional method to determine the class for labeling is a majority vote labeling (Demiriz et al. 1999). However, it may incorrectly labeled some unlabeled data and using this mislabeled data can degrade the accuracy of a classifier. The other approach avoids using mixed-cluster data (Dara et al. 2002) which discards many informative data for classification.

155 | P a g e PROPOSED METHOD In this paper, we propose the method using different feature sets to re-cluster data in the clusters containing different-class labeled data. We used the standard information gain to find good feature sets that can better re-cluster the data in such clusters. The whole algorithm is shown in Fig. 1.

EXPERIMENTAL A. Data Set We tested our method with 182 document images, in which there were 999,520 connected components, 137,444 characters and 862,076 noise components.

B.Results We compared our proposed method with the labeling by majority class. The accuracy of our method was 94.65% while the accuracy of the labeling by majority class was 41.31%.

Fig 1. The labeling algorithm using the selected feature set

CONCLUSIONS The proposed method can highly improve the accuracy in the semi-supervised learning method. We have tested with almost one million connected components and found that the improvement came from the more accurately label data in the mixed-cluster using the different feature set.

REFERENCES Singh et al. (2009). Unlabeled data: Now it helps, now it doesn’t. Advances in Neural Information Processing Systems 21, 1513–1520.

Li and Zhou (2015). Towards making unlabeled data never hurt. Pattern Analysis and Machine Intelligence, IEEE Transactions on 37 (1), 175–188.

Zeng et al. (2003). CBC: clustering based text classification requiring minimal labeled data. In Proceedings of the Third IEEE International Conference on Data Mining, 443–450 Zhang et al. (2015). TESC: An approach to text classification using semi-supervised clustering.

Knowledge-Based Systems 75, 152 – 160.

Demiriz et al. (1999). Semi-supervised clustering using genetic algorithms. In Proceedings of Artificial neural networks in Engineering, pp. 809–814.

Dara et al. (2002). Clustering unlabeled data with SOMs improves classification of labeled realworld data. In Proceedings of the 2002 International Joint Conference on Neural Networks.

156 | P a g e Personal Authentication System Using Pinna Related Transfer Function Tatsuya Miki a, Yoshinobu Kajikawa a a)

Faculty of Engineering Science, Kansai University Keywords : PRTF, biometrics, authentication, pinna, physiological trait, DNN ABSTRACT We propose a biometrics authentication method using acoustic features of pinna and ear canal. The proposed method is a personal authentication using acoustic transfer function called pinna related transfer function (PRTF). Hence, the proposed method can be realized with only a microphone and loudspeaker installed into smartphone without special equipment. We focus on the frequency characteristics of the PRTF for each ear model. In this paper, the PRTFs were measured using instrument that resembles a smart phone. In the proposed method, the frequency characteristics of the PRTF is used as input data and the classification between registrants and imposters are realized by deep neural network (DNN). Experimental results will indicate the feasibility of the PRTF to the biometrics authentication.

INTRODUCTION Recently, personal authentication technology with high accuracy has been required to improve the security and increase the spread of online trades. With this background, biometrics authentication technologies that use a physiological and/or behavioral trait have been studied at various research institutions. A physiological trait is an inherent feature to each human such as the fingerprint, iris, palm form, face, and ear form. On the other hand, a behavioral trait is a behavioral characteristic, such as voice-print, signature, and keystroke.

One of these authentication technologies focuses on the shape of pinna and utilizes the corresponding dynamic images [1]. This authentication technology aims to identify the person whose face is covered with cap and/or mask through security cameras.

In contrast, this paper focuses on the differences of the pinna related transfer function (PRTF), which is acoustic characteristic formed by pinna and ear canal, for each individual because the shape of ear is different among individuals as reported in some papers and medical books. Hence, we propose a biometrics authentication system using pinna related transfer function (PRTF) measured by smart phone with loudspeaker and microphone. The reference [5] proposed a personal authentication system using the PRTFs measured in headphone, earphone, and mobile phone. As a result, the use of mobile phone yields lower authentication rate than those of headphone and earphone. In this study, we suppose natural situations on the use of smart phones and measure pinna related transfer functions of ear model with a smart phone. In this paper, the classification between registrants and impostors is realized by deep neural network (DNN).

157 | P a g e

Figure 1 Overview of the conventional personal authentication system based on the PRTFs [5].

AUTHENTICATION USING PRTF The shape of the outer ear can be used for biometrics authentication [2]-[4]. In [5], the individual differences of the acoustic transfer functions were measured around the resonance frequency caused by the pinna and ear canal. Figure 1 shows the overview of the conventional work in [5]. The PRTFs were measured using a headphone, earphone, and mobile phone with a microphone. The impulse response of the ear hear(t) was measured by sending a noise signal n(t) into the pinna and ear canal.

The noise signal n(t) has flat frequency spectrum N(ω). Secondly, the impulse responses hear(t) is transformed to the frequency response H’ear(ω) using FFT. The PRTF is given by ( )

( ) . ( )

Next, the amplitude characteristics of Hear(ω) is obtained to find out the correlation between registrants and other queries. Finally, the person authentication is conducted based on the correlation.

As a result, the mobile phone yields lower authentication rate than the headphone and earphone. We think that high authentication rates of headphone and earphone are due to the covered outer ear or canal by headphone or earphone. However, it is unnatural that the user wears headphone or earphone for personal authentication. Thus, we focus on the personal authentication using a smart phone with a loudspeaker and microphone for more natural situations.

DEEP NEURAL NETWORK A DNN[6] is a feed-forward, artificial neural network that has more than one layer of hidden units between its inputs and its outputs. Each hidden unit, j typically uses the logistic function to map its total input from the layer below, xj , to the scalar state, yj that it sends to the layer above and given by.

where bj is the bias of unit j, i is an index over units in the layer below, and wij is the weight on a connection to unit j from unit i in the layer below. We first train a hidden layers using

158 | P a g e TABLE I. EXPERIMENTAL CONDITIONS Number of ear models Input signal Training sample Testing sample Learning rate Iteration(Autoencoder) Iteration(Back propagation)

4 Amplitude of PRTFs a a 0.01 500 40000

autoencoder that defines teaching signal to input signal in training data. The amplitude frequency characteristics of the PRTF are given as input data to the first hidden layer. The output signal from the first hidden layer is input to the second layer. This process is iterated by the last hidden layer. Finally, the weights in all layers are updated by using the back propagation algorithm.

EXPERIMENTAL RESULTS We measured some PRTFs to demonstrate the feasibility of person authentication using DNN. The measured PRTFs are given to the DNN to classify the registrants and impostors. Experimental conditions of authentication is shown in TABLE I. The classification between registrants and impostors is realized by deep neural network (DNN).

Fig. 2 An example of training results for the classification using DNN.

Fig. 3 An example of testing results for the classification using DNN.

One of training results under this condition is shown in Fig. 2. As a result, it is found that the classification is enough. One of testing results under this condition is shown in Fig. 3. As a result, it is found that the classification is not enough. This is because that the number of learning data is small.

CONCLUSIONS In this paper, we demonstrated the effectiveness of the proposed authentication system by using PRTF and DNN. We measured PRTFs and examined personal authentication using

159 | P a g e DNN. As a result, the proposed method distinguish the registrants and imposter in training phase, but the proposed method did not distinguish the registrants and imposter sufficiently due to small data set and insufficient learning in testing phase. In the future, we will measure much more number of PRTFs based on 3D camera and 3D printer technologies.

REFERENCES [1] N. Tashiro, M. Abe, T. Okamura, K. Shinohara. (2000). A study of individual identification using length of components on pinna using principal component analysis. vol. 24, no. 41, pp. 43–48.

ITE Technical Report.

[2] K. Soma, T. Watanabe, K. Sakai, O. Nakamura, Huang Zichong, H. Sai. (1997). Robustness of ear recognition against pose variation. vol. 110, no. 467, pp.67–72. IEICE Technical Report.

[3] K. Soma, T. Watanabe, K. Sakai, O. Nakamura, H. Sai. (2001). Ideal discriminant analysis for robust ear recognition. vol.25, no. 35, pp.25–30. ITE Technical Report.

[4] K. Shinohara. (2001). Individual authentication with pinna : Analysis of individual with pinna. vol. 25, no. 22, pp.25–30. ITE Technical Report.

[5] A. H. M. Akkermans, T. A. M. Kevenaar, D. W. E. Schobben. (2005). Acoustic ear recognition for person identification. pp.219–223. Fourth IEEE Workshop on Automatic Identification Advanced Technologies.

[6] G. Hinton, L. Deng, D. Yu, A Mohamed, N. Jaitly, A. Senior, V. Vanhoucke, P. Nguyen, T. S. G. Dahl, and B. Kingsbury. (2012). Deep neural networks for acoustic modeling in speech recognition. vol. 29, no. 6, pp.82–97. IEEE Signal Processing Magazine.

160 | P a g e

By Complex-valued Neural Network using a Simultaneous Perturbation Orbit Control of SCARA Robot Yoichiro Seki *a, , Hidetaka Ito a, Yutaka Maeda a a

Graduate School of Science and Engineering, Kansai University Keywords: complex-valued neural networks, simultaneous perturbation learning rule, inverse kinematics, orbit control, SCARA robot

ABSTRACT Robot technology has been used in various industries all over the world. However, the robot should have the intelligence to fulfill the further high functionality. Then, we focused on complex-valued neural networks. Artificial neural networks is a model of biological neural system, and has a high information processing ability. In addition, it is known that complex-valued neural networks are useful for the processing of translation and rotation in two dimensional plane.

In this work, we use complex-valued neural network as a controller of a robot system. Then, we have examined an effectiveness of the simultaneous perturbation learning rule for the robot control using the complex-valued neural network. We prepared a complex-valued neural network using the simultaneous perturbation learning rule, and we make the complex-valued neural network to learn the inverse kinematics of SCARA robot. By learning the inverse kinematics, orbit control of the SCARA robot can be realized by the complex-valued neural network. Then, we have set adjacent four target positions. We have carried out the orbit control for the four target positions by a simulation using MATLAB. As a result, we obtained an orbit that passes through the target positions. * k136353@kansai-u.ac.jp INTRODUCTION Currently, robot technology has been utilized in a wide variety such as industrial robots and home appliances. In particular, industrial robots are capable of performing the difficult task for humans. For example, robots perform the assembly of precision parts, transportation of materials, mass production of industrial products. Moreover, it is possible for robots to produce high-quality products.

However, the robot should have the intelligence to fulfil the further high functionality. Then, we focused on complex-valued neural networks. The robot can improve its own operation by using a complex-valued neural network. As a result, it is possible to perform precise control. Simultaneous perturbation learning rule can be carried out only using error information. Also, it is possible to respond flexibly to changes in the characteristics of a controlling object.

In this work, we considered an orbit control with four target positions in the control range of the robot arm.

EXPERIMENT A.

System configuration

161 | P a g e The system configuration is shown in Figure 1. Control subject is a SCARA robot. Controller is a complex-valued neural network.

Figure 1 System configuration First, the complex-valued neural network receives a teacher signal. It outputs angles corresponding to each axis of the robot arm. Result, SCARA robot arm moves. System compares the tip coordinates of the robot arm and teacher signal. To minimize the error, the system updates weights of the complexvalued neural network using the simultaneous perturbation learning rule.

B.

Structure of the complex neural network for learning the inverse kinematics

Figure 2 shows the structure of the complex-valued neural network.

Figure 2 Complex-valued neural network Figure 2 shows detailed configuration the complex-valued neural network used in this work.

Parameters in this neural network are all complex numbers. Real part of output of the network is xaxis component, the imaginary part is the y-axis component. Input has 5 nodes, an intermediate layer has 13 neurons, and output is 1 complex number. We have entered the tip coordinates of the robot arm and teacher signal (target position) in the neural network. In addition, we have feedback 1-3 sampling

162 | P a g e time delay of the angle of the robot arm. The reason for the feedback, we perform the dynamic orbit control. The neural network outputs the angle of the first-axis and the second-axis of the robot arm.

C.

Evaluation function

Weights of the complex-valued neural network are determined so as to minimize the error between the tip coordinates of the robot arm and the teacher signal. In this study, we compared the tip coordinates of the robot arm for each sampling time and target position of the robot arm. Summation of this square error is the evaluation function shown in equation (1).

J ď&#x20AC;˝ ď&#x192;Ľ{( xd ď&#x20AC;­ xa ) 2 ď&#x20AC;Ť ( yd ď&#x20AC;­ ya ) 2 }

(1)

Where, the target position of the robot arm is đ?&#x2018;Ľđ?&#x2018;&#x2018; and đ?&#x2018;Śđ?&#x2018;&#x2018; , the tip coordinates of the robot arm of each sampling time is đ?&#x2018;Ľ and đ?&#x2018;Ś . Until the value of the evaluation function is minimized, we run the simultaneous perturbation learning rule. And weights of the complex-valued neural network are modified.

D.

Procedure

In this study, we performed the orbit control of SCARA robot by the complex neural network with simultaneous perturbation learning rule. In the control of SCARA robot, it is necessary to carry out the learning of the neural network simultaneously.

The learning using the actual machine takes enormous time. Therefore, we have simulated the learning of the complex-valued neural network before the actual operation of the overall system. To do so, we can check the stability of operations in the simulation. Actual machine works with the weights obtained by the simulation.

RESULTS AND DISCUSSION

We have carried out the orbit control for the four target positions by a simulation using MATLAB. Figure 3 shows the learning result.

Figure 3 Complex-valued neural network

163 | P a g e Learning number is 5000 times, sampling time is 0.05 [sec], trial time is 2.5 [sec], learning coefficient of the complex-valued neural network is 0.003, and perturbation is 0.00001. The resulting orbit has generally passed the target positions. We have confirmed that the complex-valued neural network can learn the inverse kinematics of SCARA robot. Figure 4 shows changes of the evaluation function by the simulation. As the learning proceeds, the evaluation function approaches gradually to 0.

Therefore, we can see that the learning is stable.

Figure 4 Transition of the evaluation function in the simulation

CONCLUSIONS

We found that it is effective for the complex-valued neural network using the simultaneous perturbation learning rule to control and learn SCARA robot online.

As Future works, we plan the learning of the inverse kinematics by actual machine. We will check the same operation and simulation. Then, we will confirm the generalization and over-learning of the characteristics of the complex-valued neural network. Currently, we have recommended the research toward the realization of obstacle avoidance control.

REFERENCES

Y. Maeda, H. Hirano and Y. Kanata. (1995). A Learning Rule of Neural Networks Via Simultaneous Perturbation and Its Hardware Implementation, Neural Networks, vol.8, pp.251-259.

T. Nitta. (2000). The complex-valued neural network, Electrical Information and Communication Journal, Vol. 83, No.8, pp.612-615.

T. Nitta. (2009). Complex-valued neural networks, Information Science Publishing.

A. Hirose. (2012). Complex-valued neural networks, Springer.

164 | P a g e FPGA Implementation of Pulse Density Complex-valued Neural Network Using Simultaneous Perturbation Method Atsushi Tsumoto a, Hidetaka Ito,a, Yutaka Maeda a a

Graduate School of Science and Engineering, Kansai University Keywords: FPGA, Complex-valued neural network, Pulse density, Simultaneous perturbation, Hardware implementation ABSTRACT Using simultaneous perturbation learning rule of sign vector type, we designed a complex-valued neural network in VHDL based on numerical representation of pulse density. Circuit scale is economized. After that, we implemented the design by FPGA. We could realize FPGA complexvalued neural network with learning capability, and verify its performance for reduced and rotation problems.

INTRODUCTION Today, Neumann architecture computers are normally used in personal computers, smartphones and so on. These devices are operating in serial processing. It is difficult for them to operate in parallel essentially.

Accordingly, we made a FPGA complex-valued neural network (CNN) system in trial. By implementing neural network (NN) as FPGA, parallel processing, which is a feature of biological NN, is easily realized. Moreover high speed processing can be realized as well.

As learning rule, we adopt sign vector type of simultaneous perturbation (SP) learning rule. As numerical representation, we adopt pulse density system. We designed a complex-valued neural network by VHDL for FPGA. Sign vector type of SP learning rule can suppress the circuit scale in the hardware implementation. Pulse density system allow us to design a neuron model very easily by using AND elements and OR elements.

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6 ď&#x20AC;˝ 0.6 10 3 ď&#x20AC;˝ 0.3 10

165 | P a g e PULSE NEURON MODEL Pulse density representation First of all, we explain about pulse density representation used in this study. User determine specific interval with a certain maximum number of pulses. Then user obtained numerical value by dividing actual pulses number by maximum number.

For example, specific interval is defined as in Figure 1 and we determine that the maximum number is 10. If there are 6 pulses in the interval(see Figure 1 center), then this represents 0.6. If there are 3 pulses(see Figure 1 bottommost), then this is representing 0.3. This time, resolution is 0.1.

Multiplication operation in pulse density representation As in Figure 2, pulse input signal 1 is 0.6, and input signal 2 is 0.6, the result of the AND operation becomes approximately equivalent to 0.36(0.6 Ă&#x2014; 0.6). Result of AND operation; 0.4 represents approximately that of the multiplication.

Input1 Input2 AND result

Figure 2 operation

Multiplication.

Space addition operation in pulse density representation Two pulse density representation input values are added by passing through the OR element. When two adding input values are small each other(Figure 3), we can obtain approximate result. OR operation shows the linear addition characteristic.

On the other hand, when input values are large(Figure 4), OR operation does not result in linear addition, there will be some error. OR results saturates with value of one. We can say that OR result has characteristics of approximate sigmoid function.

As in Figure 3, pulse input signal 1 is 0.2, and input signal 2 is 0.4, the result of the OR operation becomes approximately equivalent to 0.6(0.2 ďź&#x2039; 0.4). Result of OR; 0.6 represents approximate addition.

Input1 Input2 OR result Figure 3 Space addition (low density).

166 | P a g e

Input1 Input2 OR result Figure 4 Space addition (high density) .

As in Figure 4, pulse input signal 1 is 0.6, and input signal 2 is 0.6, the result of the addition becomes 1.2(0.6+0.6). But OR result is saturated with ten pulses, that is, one. Now, OR result has become 0.8.

By utilizing a pulse density method, numerical representation can be express easily. In addition, the expression can be used to simplify the calculation in neuron model. Input and output function of the neuron model is represented by the Equation (1).

 m  y  f   wi xi     i 1 

(1)

x is the input, w is weight, θ is a threshold, the function f is a sigmoid function or a step function.

As explained, AND elements express multiplication of the weight and the input, OR element express the summation and approximate the sigmoid function. As a result, we can express neuron model only

by AND elements and OR elements (Figure 5). In this representation, we do not use multiplier circuit.

Thus, pulse neuron model can suppress logic elements resources.

In this study, we are handling a CNN. Features of the CNN are described in many references.

Figure 5 Pulse Neuron. Figure x shows the transition of the

167 | P a g e SIMULTANEOUS PERTURBATION (SP) OPTIMAIZATION METHOD Unlike difference approximation method, simultaneous perturbation (SP) optimization method is possible to correct parameters based only on twice observations of the evaluation function by adding perturbation to all elements simultaneously. In comparison with difference approximation method, SP learning rules can be used to reduce the computation time required for the parameter update. I show the optimization algorithm of this approach as follows.

Dxt,i =

J(x t + ct ) - J(x t ) ct,i

 xt,1        xt   xt,i       xt,n 

 ct,1        c t   ct,i       ct,n 

(i  1,･･･ , n)

(2)

 xt,1  ct,1        x t  c t   xt,i  ct,i       xt,n  ct,n 

The above equation Dx represents the estimated value of the partial derivative of function J with respect to xi. c t is perturbation vector, c i,t is the i-th element of the perturbation vector. The definition of the c t and c i,t is as follows; ■ c1 , c 2 , c 3 , ..., c n are independent of each other.

■ E ( c t ) = 0 E (·) shows the expected value.

■ c t,i ≠ 0 From Equation (2), we can see that the algorithm of SP learning rule is using only two function values of f (xt + ct) and f (xt - ct). Therefore, even if the number of dimensions of the parameter increases, SP learning rule can be obtained an estimate of the gradient vector from only two function values. For this reason, if the optimization problem has a high dimensional parameters, SP learning rule is effective as compared to a simple difference approximation.

Simultaneous perturbation learning rule of sign vector type In the simultaneous perturbation optimization method, we are using a uniform random numbers to generate the perturbation vector c t ordinarily. However, we think about the simultaneous perturbation optimization method using the Bernoulli distribution to generate the perturbation. Then, +c or -c is generated. The absolute value of the perturbation would be same. Only the sign of the perturbation is different. At this time, the procedure of simultaneous perturbation optimization method using a sign vector is replaced as follows.

168 | P a g e

Dxt,i =

J ( x t + cst ) - J ( x t )

(3)

cst,i

Where, st is sign vector, st,i represents the i-th element of

st with value of +1 or -1. c (> 0) is a

common perturbation for all elements.

In general, a random number generating circuit is complicated to realize in hardware. However, in SP learning rule of sign vector type, we change only sign of the perturbation. This method is possible to simplify the circuit design RESULTS We implemented the pulse density CNN for FPGA and verified whether CNN system is valid for the shrinking problem. We gave the following teacher signals to CNN(see Figure 6). Where, inputs are black circles, and the desired outputs are denoted by white circles.

Figure 6 Teaching signal.

Figure 7 Transition of evaluation function.

In this experiment, resolution of the pulse density system is a 12 bit, the system express the 0-1 in the 4096 stage. Our CNN contains 1, 10 and 1 neurons in input layer, intermediate layer and output layer, respectively. Moreover, c = 0.00757, Îą = 0.0002365 (= c / 32), and the initial weights are all 0. Total number of learning was fixed at 20 000 times. Figure 6 shows the transition of the evaluation function for the shrinking problem.

169 | P a g e CONCLUSION Since the operation verification of the complex neural network that the evaluation function is converged to every overlapping learning, we could confirm that the fabricated CNN system has learned the reduction problem. We found that pulse density type CNN using the simultaneous perturbation learning rule is valid to solve the reduction problem. As future works, improvement of the learning accuracy and shorting the learning time are important. Practical applications of the system are also interesting.

REFERENCES Y. Maeda, Y. Fukuda, T. Matsuoka, Pulse Density Recurrent Neural Network Systems with Learning Capability Using FPGA, WSEAS Transactions on Circuits and Systems, vol. 7, pp. 321330, 2008.

Y. Maeda, M. Wakamura, Simultaneous perturbation learning rule for recurrent neural networks and its FPGA implementation, IEEE Transactions on Neural Networks, vol. 16, pp. 1664-1672, 2005.

A. Hirose, Nature of complex number and complex-valued neural networks, Frontiers of Electrical and Electronic Engineering in China, p171-180, 2011.

170 | P a g e A Study on 3D Sound Field Analysis for Parametric Array Loudspeaker Hiroaki Bessho a, Yoshinobu Kajikawab a

Graduate School of Science and Engineering, Kansai University b Faculty of Engineering Science, Kansai University Keywords : Sound Field Analysis, Parametric array loudspeaker, KZK equation, IBFD method, CNFD method.

ABSTRACT In this paper, we examine a 3D sound field analysis for parametric array loudspeaker (PAL).

Nonlinear propagation of PAL can be analyzed by the KZK (Khokhlow-Zabolotskaya-Kuznetsov) equation. The KZK equation is the model equation for progressive wave beam that includes diffraction, absorption and nonlinearity of ultrasonic. This analysis utilizes an implicit backward finite difference (IBFD) method in nearfield (the field close source), and a Crank-Nicolson finite difference (CNFD) method in farfield to improve the computational efficiency of the spectral method for solving the KZK equation. In this paper, we examine the wave propagation in the room for unfocused sound beams.

INTRODUCTION Recently, with the spread of 3D imaging technique such as 3D TV or 3D movies, the sound reproduction giving realistic sensations has been desired, and then several kinds of sound field reproduction systems have been actively studied. The transaural systems are one of the sound reproduction systems using loudspeakers, but cross-talk paths occur due to the room transfer functions between each ear and each loudspeaker. This cross-talk paths can be compensated by the inverse filters for room transfer functions [1]. However, these filters require multiple channel processing, it consequently increases the computational complexity.

To solve this problem, sound reproduction system using parametric array loudspeakers (PALs) that have super directivity [2]-[4] are proposed. By the super directivity of PAL, the sound reproduction system that reduces the cross-talk paths can be implemented. Therefore, we examine the sound field analysis using KZK equation that is commonly used to analyze the nonlinear propagation of ultrasonic beams for sound reproduction system.

KZK EQUATION In this section, the KZK equation is explained. Ultrasonic waves that are used to reality is not plane wave but beam that has finite range in a transverse direction. If the propagation direction of sound beam is z-axis, the sound beam expands to and focuses on x-axis and y-axis by diffraction effects.

Three dimensional nonlinear model equation as Westervelt equation is given by

2 p 

1   2 p  2 p2 ( 1  )   , c02 2c02 t t 2  0 c04 t 2

(1)

where p is the sound pressure and c0 is the propagation velocity of waves. Focus on the beam that moves only +z direction, the delay time t′ = t – z/c0 is adopted to Eq. (1). Because waveform change on z-axis is more gradual than the waveform change on delay time, the following approximation can be used:

171 | P a g e

ď&#x201A;ś2 p 1 ď&#x201A;ś2 p 2 ď&#x201A;ś2 p ď&#x20AC;­ ď&#x201A;ť ď&#x20AC;­ , ď&#x201A;śz 2 c02 ď&#x201A;śt 2 c0 ď&#x201A;śt ď&#x201A;˘ď&#x201A;śz

(2)

Using the Eq. (2), we obtain

ď&#x201A;ś 2 p c0 ď&#x201A;ś 2 p 1 ď&#x201A;śp ď ¤ ď&#x201A;ś3 p ď ˘ ď&#x201A;śp 2 ď&#x20AC;˝ ( 2 ď&#x20AC;Ť )ď&#x20AC;Ť 3 ď&#x20AC;Ť , ď&#x201A;śt ď&#x201A;˘ď&#x201A;śz 2 ď&#x201A;śr r ď&#x201A;śr 2c0 ď&#x201A;śt ď&#x201A;˘ 3 2 ď ˛ 0 c03 ď&#x201A;śt 2

(3)

where r = â&#x2C6;&#x161; is the 2nd order laplacian on x and y plane. In the right-hand side in Eq. (3), the first term shows the diffraction, the second term shows the absorption and the third term shows the nonlinearity, respectively. Eq. (3) is KZK (Khokhlow-Zabolotskaya-Kuznetsov) equation and this is used to analyze the nonlinearity propagation of ultrasonic beam. In this paper, we use a time domain numerical algorithm which is developed on the basis of a form of Eq. (3):

ď&#x201A;śp c0 ď&#x20AC;˝ ď&#x201A;śz 2

ď&#x201A;ś 2 p 1 ď&#x201A;śp ď ¤ ď&#x201A;ś2 p ď ˘ ď&#x201A;śp 2 ď&#x201A;˘ ď&#x201A;˘ ď&#x192;˛ď&#x20AC;­ď&#x201A;Ľ ( ď&#x201A;śr 2 ď&#x20AC;Ť r ď&#x201A;śr )dt ď&#x20AC;Ť 2c03 ď&#x201A;śtď&#x201A;˘2 ď&#x20AC;Ť 2ď ˛0c03 ď&#x201A;śtď&#x201A;˘ . tď&#x201A;˘

(4)

NUMERICAL ANALYSIS OF KZK EQUATION In this section, we investigate the propagation of sound beam to use KZK equation. Figure 1 shows the flowchart of numerical analysis. As shown in the Figure 1, the equation is solved sequentially, at each range step, as follows: first the diffraction term, then the absorption term, and finally the nonlinear term are integrated.

A. Numerical algorithm The following variable transformations are applied for Eq. (4):

ď ł ď&#x20AC;˝ z z0 ,

ď ˛ď&#x20AC;˝

(5)

r/a , 1ď&#x20AC;Ťď ł

ď ´ ď&#x20AC;˝ ď ˇ0 t ď&#x201A;˘ ď&#x20AC;­

(6)

(r / a) 2 , 1ď&#x20AC;Ťď ł

(7)

P ď&#x20AC;˝ (1 ď&#x20AC;Ť ď ł ) p p0 ,

(8)

where a is the characteristic radius of the source, Ď&#x192; is a dimensionless range coordinate in terms of the Rayleigh distance z0 = Ď&#x2030;0a2/2c0 at the characteristic angular frequency Ď&#x2030;0, Ď is the dimensionless transverse coordinate, Ď&#x201E; is a dimensionless retarded time, and p0 is a characteristic source pressure amplitude.

Substitution of Eq. (5)-(8) into (4) yields

ď&#x201A;śp 1 ď&#x20AC;˝ ď&#x201A;śď ł 4(1 ď&#x20AC;Ť ď ł ) 2

ď ´

ď&#x192;˛

ď&#x20AC;­ď&#x201A;Ľ

(

ď&#x201A;ś 2 p 1 ď&#x201A;śP ď&#x201A;ś2P NP ď&#x201A;śP ď&#x201A;˘ ď&#x20AC;Ť ) d ď ´ ď&#x20AC;Ť A ď&#x20AC;Ť , 2 2 ď ˛ ď&#x201A;śď ˛ (1 ď&#x20AC;Ť ď ł ) ď&#x201A;śď ´ ď&#x201A;śď ˛ ď&#x201A;śď ´

(9)

where A = Îą0z0 is the absorption coefficient, and N = z0 /đ?&#x2018;§ is the nonlinearity coefficient.

172 | P a g e The effects of diffraction, absorption, and nonlinearity are taken into account by the Eq.(10)-(12)

p 1   4(1   ) 2







p 2P A 2,   p NP P  .  (1   ) 

(

 2 p 1 P  )d ,  2  

(10)

(11)

(12)

The Rayleigh distance z0 is a rough indication for the boundary between the nearfield and farfield. For σ < 0.1, the observation point is in nearfield, and for σ >0.1, the observation point is in farfield. For Eq. (10), the discontinuity in the step function produces numerical fluctuations in the field near the source. In this nearfield region, we use an implicit backward finite difference (IBFD) method, which is effective in damping the fluctuations. In the farfield region, we use a Cranc-Nicolson finite difference (CNFD) method, which permits larger stepsize. We combine IBFD and CNFD methods to improve the computational efficiency of the spectral method for solving the KZK equation.

B. Computational analysis for unfocused sound beams We analyze wave propagation of sound beams. In this paper, we explain only the flow of the mode of analysis due to the space limitations.

1) Input the required information such as an input signal, environmental parameters, and computational grids.

2) Analyze diffraction, absorption, and nonlinearity respectively using IBFD method.

3) Analyze diffraction, absorption, and nonlinearity respectively using CNFD method.

4) Output sound pressures at observation points or regions.

CONCLUSION In this paper, we examined the sound field analysis for parametric array loudspeaker and explained the propagation of sound beam using KZK equation by combining IBFD and CNFD methods. In the future, we will analyze the wave propagation for parametric array loudspeakers in an actual room.

REFERENCES [1] A. Kaminuma, S. Ise, K. Shikano, “A method of an inverse filter design for multi-channel sound reproduction system using minimum-norm- solution in frequency domain, ”J. Acoust. Soc. Jpn., vol.

57, no. 3, pp. 175–183, Mar. 2001.

[2] S. Sakai, T. Kamakura, “Technologies and applications of high directivity loudspeaker,” Jour. Jpn. Soc. Mech. Eng., vol. 1074, no. 111, pp. 432-436, May 2008.

[3] W.-S. Gan, E.-L. Tan, S. M Kuo, “Audio projection: directional sound and its applications in immersive communication,” IEEE Signal Processing Magazine, vol. 28, no. 1, pp. 43–57, Sep. 2011.

[4] W.-S. Gan, E.-L. Tan, C.-H Chen, “Spatial sound reproduction using conventional and parametric loudspeakers,” Proc. of APSIPA ASC 2012, Holly Wood, USA, Dec. 2012.

173 | P a g e [5] Y.-S Lee, â&#x20AC;&#x153;Numerical solution of the KZK equation for pulsed finite amplitude sound beams in thermoviscous fluids,â&#x20AC;? The University of Texas at Austin, http://people.bu.edu/robinc/kzk/YSLeePhD1993.pdf, Dec. 1993.

Fig. 1 Computational procedure in terms of the axial coodinate z

174 | P a g e Multi-channel Feedforward ANC System Using Microphone Arrays for Noise Source Separation Satoshi Kinoshita *,a, Yoshinobu Kajikawa a a

Faculty of Engineering Science, Kansai University, Osaka, Japan Keywords : Active noise control, Multi-channel ANC system, Sound source separation, Generalized sidelobe canceller

ABSTRACT Multi-channel feedforward active noise control (ANC) system can reduce various noise such as the broad-band noise by arranging the reference microphones close to noise sources. However, the performance of ANC system deteriorates when the reference microphones cannot be arranged close to noise sources because each microphone catches undesired noise in addition to desired noise. To solve this problem, we propose a multi-channel feedforward ANC system combined with sound source separation. This system uses microphone arrays as reference microphones and the separated signals is used as the reference signals of the ANC system. In this paper, we examine the effectiveness of the proposed system through some simulations. *k538866@kansai-u.ac.jp INTRODUCTION Feedforward active noise control (ANC) [1]â&#x20AC;&#x201C;[4] system can reduce various noise such as the broadband noise by arranging the reference microphones close to noise sources [5]. However, it is difficult to control of noises when the reference microphones cannot be arranged close to the noise sources due to various noises generated from the various noise sources or noise source movements. To solve this problem, Hase et al. [6] proposed an ANC system with noise source localization. In this system, some microphones are arranged around the noise control point and the nearest microphone to noise source is used as the reference microphone to reduce the noise. However, the performance of ANC system deteriorates when noises are generated from different noise sources and the multiple microphones are used because each microphone catches undesired noise in addition to desired noise.

Therefore, we propose a multi-channel feedforward ANC system combined with sound source separation in this paper. This system uses microphone arrays as reference microphones to separate noises and the separated signals are used as the reference signals of the ANC system. In this paper, we examine the effectiveness of the proposed system using generalized sidelobe canceller (GSC) [7] as noise source separation method through some simulations.

SOUND SOURCE SEPARATION BY MICROPHONE ARRAY Microphone array can obtain spatial information of the sound such as time difference of arrival and amplitude difference. Beamforming is the basis of the array signal processing and can be applied to sound source separation by directionality control. In this chapter, we explain the generalized sidelobe canceller (GSC).

A. Generalized sidelobe canceller (GSC)

175 | P a g e GSC is one of the adaptive beamformer composed of multiple channel fixed filters and adaptive filters. The block diagram of GSC is shown in Figure 1. In the GSC, the processing flow is as follows.

First, the observed signals of each microphone z m (n) are compensated for propagation delay by using the fixed filters w c,m (n). Next, those output signals are branched to the upper and the lower sections.

In the upper section, the output signals of the fixed filters are added together to emphasize the desired signal. On the other hand, in the lower section, the difference between adjacent output signals of the fixed filters is calculated. In consequence, the input signals of the adaptive filters Z b,m (n) contain only noises rather than the desired signal. The output signal of the GSC x(n) is obtained by subtracting the total output signal of the adaptive filters y a(n) from the sum of the output signals of the fixed filters yc(n) . Hence, GSC has higher noise reduction performance compared with the fixed beamformer. In this paper, we utilize the normalized least mean square (NLMS) algorithm as the updating algorithm of the adaptive filters. The updating equation of the adaptive filters is given by

where wa,m n is the filter coefficient vector of the m th adaptive filter, z b,m n signal vector of the m parameter.

is the input

MULTI-CHANNEL FEEDFORWARD ANC SYSTEM USING MICROPHONE ARRAYS FOR NOISE SOURCE SEPARATION Block diagram of a Case(2,1,1) ANC system using microphone arrays for noise source separation is shown in Figure 2. The Case(2,1,1) ANC system represents a multi-channel feedforward ANC system that uses two reference microphones, one secondary source, and one error microphone. The proposed system uses microphone arrays as reference microphones to separate noises and the separated signals are used as the reference signals of the ANC system. In this paper, we utilize the filtered-x NLMS (FXNLMS) algorithm as

Figure 1: Block diagram of GSC using three microphones

176 | P a g e

Figure 2: Block diagram of a Case(2,1,1) ANC system using microphone arrays for noise source separation. the updating algorithm of the noise control filters. The updating equation of the noise control filters is given by

where j is the index number of the reference microphone array, w j n is the filter coefficient vector of the j th noise control filter, x j n is the j th filtered reference signal vector, e n

SIMULATION RESULTS In this chapter, we demonstrate the effectiveness of the proposed Case(2,1,1) ANC system using the GSC as noise source separation method. We compare the noise reduction performance and the noise separation performance of the two ANC systems, without the noise source separation and with the noise source separation using the GSC in this simulation. Figure 3 shows the arrangement of noise sources, reference microphone arrays, an error microphone, and a secondary source in the simulation.

In Figure 3, the mic. array 1 suppresses the noise signal of the noise source 2 and the mic. array 2 suppresses the noise signal of the noise source 1. Table 1 shows the simulation conditions. The fixed filters of the GSC were created by using the acoustic path models from each noise source to each microphone. Each step size parameter was set to optimal values based on some preliminary simulation results. Noise reduction performance is evaluated by the “Reduction” defined by

Where d 1(n) and d 2(n) are the noise signals at the error microphone, and e(n) is the error signal.

Noise separation performance is evaluated by the “Magnitude-squared coherence (MSC)” defined by

177 | P a g e Where Pxy (f) is the cross power spectral density between two signals, and Pxx (f) and Pyy (f) are the power spectral densities of each signal. The MSC indicates the degree of coincidence of the two signals at each frequency.

Figure 4 shows the comparison of the noise reduction performance in each ANC system. From Figure 4, we can confirm that the noise reduction performance of the ANC system with the noise source separation is higher than that of the ANC system without the noise source separation.

Figure 5 shows the comparison of the noise separation performance in each ANC system. Figure 5 (a) shows the MSC between the noise signal of the noise source 2 and the observed signal in the mic. 10, and Figure 5 (b) shows the MSC between the noise signal of the noise source 2 and the output signal of the GSC in the mic. array 2. From Figure 5, we can confirm that the noise separation performance of the output signal of the GSC is overall higher than that of the observed signal without the noise source separation.

From these results, the proposed Case(2,1,1) ANC system can realize superior noise reduction performance through the use of noise source separation and the sound source separation is possible to separate the noises.

Figure 3: Arrangement of noise sources, reference microphone arrays, an error microphone, and a secondary source in the simulation.

CONCLUSIONS In this paper, we have proposed the Case(2,1,1) ANC system using microphone arrays for noise source separation and confirmed the effectiveness of the proposed ANC system. In the future, we will examine the noise reduction performance of the ANC system combined with noise source separation based on the noise source localization.

178 | P a g e

Figure 4: Comparison of the noise reduction performance in each ANC system.

Figure 5: Comparison of the noise separation performance between the noise signal of the noise source 2 and each signal.

REFERENCES [1] P. A. Nelson and S. J. Elliott, Active control of sound, Academic Press, London, 1992.

[2] S. J. Elliott and P. A. Nelson, “Active noise control,” IEEE Sig. Process. Mag., vol. 10, no. 4, pp.

12–35, Oct. 1993.

[3] S. M. Kuo and D. R. Morgan, Active noise control systems, John Wiley & Sons, New York, 1996.

[4] S. M. Kuo and D. R. Morgan, “Active noise control: a tutorial review,” Proc. of the IEEE, vol.

87, no. 6, pp. 943–973, Jun. 1999.

[5] Y. Kajikawa, W. S. Gan and S. M. Kuo, “Recent advances on active noise control: open issues and innovative applications,” APSIPA Trans. Sig. Inf. Process., vol. 1, pp. 1–21, Aug. 2012.

[6] S. Hase, Y. Kajikawa, “Multi-channel ANC system using optimized reference microphones based on time difference of arrival,” EUSIPCO2015, Nice, France, Sep. 2015. (to be published) [7] L. J. Griffiths and C. W. Jim, “An alternative approach to linearly constrained adaptive beamforming,” IEEE Trans. Antennas Propagation, vol. AP-30, pp. 27–34. Jan. 1982.

179 | P a g e A Quantitative Evaluation of Fatigue Based on Brain Activity Measurement Kazuma Musha*, Yoshiko Hanada**, Noriaki Muranaka** *Graduate School of Science and Engineering, Kansai University ** Faculty of Engineering Science, Kansai University This study discusses a quantitative evaluation method for fatigue through a measurement of brain activities. Consciousness about fatigue which one gets by work varies among different individuals.

Insensible fatigue has possibilities to cause that a serious accident. That could be avoided if one becomes aware of fatigue by a quantitative evaluation method and manages oneself. We suppose that fatigue appears in trend of brain activities. Near-infrared spectroscopy (NIRS) is one of approaches for measurement of brain activities, which evaluates them by measuring an amount of oxyhemogrobin. We analyze difference between a fatigued condition and the ordinary in change in amount of oxyhemogrobin. NIRS requires a simple task in order to understand the change in oxyhemogrobin, and it is very important to choose a suitable one. Here we evaluate validity of four tasks; Trail Making Test, Verbal Fluency Task, Stroop Color-Word Test and Calculation Task. In the experiments, we measure brain activity of examinees who have got tired after driving a car.

The measurement procedure of NIRS is divided into three phases of Rest time, Task time and Pre time. Pre Time is a phase to fix a standard position of the brain blood flow. Task Time is a main phase to let an examinee perform a task, and to examine the brain activity. Rest Time is a phase for the break. We repeat Task time and Rest time several times. We set, respectively, Pre time to 5 seconds, Task time to 20 seconds and Rest time to 40 seconds. In addition, we suppose about what kind of influence fatigue has on brain activities. Figure 1 and 2 show the brain activities measured by a certain channel of an examinee before and after the driving, respectively. The area between two vertical lines shows the change in Task time. From these figures, we can see that the change is distorted after the driving while Fig.1 shows gently sloping change. This trend is also observed in other examinee. We presume that is cause of fatigue that the examinee got, and evaluate a fatigue degree from the distortion. We would show other results and a formulation of the fatigue degree in the conference room.

Fig 1. Brain activity before the driving

Fig 2. Brain activity after the driving

180 | P a g e Measurement of Brain Activity by Intellectual Function under Learning Through NIRS Naoki Shiroyama *, Yoshiko Hanada**, and Noriaki Muranaka** *Graduate School of Science and Engineering, Kansai University ** Faculty of Engineering Science, Kansai University As the way to understand a brain function, functional neuroimaging is used to map brain activities into an image. Development in this technology enables us to comprehend an aspect of activities of the cerebral cortex, and then brain research has been applied to an education field. The cerebral cortex controls our intellectual functions, e.g., thought, calculation and reasoning. Therefore, it is reasonable to evaluate learning effects in education field from cerebrumâ&#x20AC;&#x;s activities. By clarifying a mechanism of brain activities under learning, improvement in learning method would be expected. The purpose of our study is to identify activated regions in the cerebral cortex under learning.

In this study, we measure brain activities of examinees who handle a task which emulates actual learning, by using Near-infrared spectroscopy (NIRS). NIRS is one of equipment for functional neuroimaging, which measures hemoglobin concentration in the brain. It is little-affected from body motions, so that examinees need not keep a fixed posture. The measuring points are both the temporal lobe and its neighboring regions. We adopt two pencil-and-paper tests. These tests evaluate both the calculation function and the linguistic function which associate with learning. In order to measure brain activities while exercising the calculation function, we conduct the multiplication test which consists of two digits times one digit. In order to measure brain activities while exercising the linguistic function, we conduct the test where an examinee rearranges four words to make sentences.

Through the experiment, to verify brain activities, we examine correlation between amount of change in hemoglobin and scores of the tests.

The result of measurement showed that both functions activate Broca's area in the frontal lobe of the cerebral cortex. In the examination for the calculation function, examinees who get a good score do not activate the area much, compared to examinees who get a bad score. On the other hand, for the linguistic function, examinees who get a good score activate preferentially the left brain.

181 | P a g e

Civil Engineering

182 | P a g e Slump and Compressive Strength of Concrete made with Wet Fly Ash Warangkana Saengsoy *,a, Thuy Bich Thi Nguyen b, Somnuk Tangtermsirikul b a

Construction and Maintenance Technology Research Center (CONTEC), School of Civil Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University b School of Civil Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University Keywords: Wet fly ash, Moisture content, Slump, Compressive strength, Double mixing method ABSTRACT This study is aimed to investigate feasibility of using wet fly ash as a cement replacing material in concrete. Effect of moisture content and exposure period of wet fly ash on basic properties of concrete are studied. Slump and compressive strength of concrete made with the wet fly ash are investigated and compared with those made with dry fly ash. Wet fly ashes prepared in laboratory are used in the investigation. Moisture contents of the wet fly ashes are 25%, 45%, and 65%. The wet exposure periods of fly ash are 1 month, and 3 months. The test results revealed that slump and compressive strength of concrete made with wet fly ash are slightly improved. The effect of different moisture contents of the wet fly ash can be explained by the concept of double mixing method. The micro hardness test results showed that ITZ of the concrete made with wet fly ash is improved due to the effect of double mixing method. On the other hand, exposure period of the wet fly ash has no effect on the slump and compressive strength of concrete since the chemical composition of the wet fly ash is unchanged. *warangkana@siit.tu.ac.th INTRODUCTION Fly ash is one of the by-products derived from the coal combustion. In Thailand, almost 3 million tons of fly ash is produced annually. In the past, almost all produced fly ash had been used in concrete industry. However, it is estimated that about 0.6 to 0.7 million tons of fly ash, which is in excess of the demand, is being dumped without effective use during recent years. Some un-used fly ashes are water-sprayed to have moisture content about 15% to 20% before being dumped near the power plants. This produces large stocks of wet fly ash which are needed to be more properly managed. Use of wet fly ash as a cement replacing material in concrete seems to be possible in practice [1, 2], especially for the low CaO fly ash. The high CaO fly ash, since partially cementitious, is not possible to be reused as a pozzolan if wetted. Additional drying and grinding processes are required for wet high CaO fly ash [3], which also make it not cost effective. Therefore, the possibility of using wet fly ash in concrete is studied for the low CaO fly ash. Since moisture content of the dumped wet fly ash can vary depending upon the exposing environment, effect of the moisture content and exposure period of wet fly ash on basic properties of concrete are studied. Slump and compressive strength of concrete made with the wet fly ash are investigated and compared with those made with dry fly ash.

Wet fly ashes prepared in laboratory are used in the investigation.

183 | P a g e EXPERIMENTAL PROGRAM The aim of this study is to investigate the effect of moisture content and exposure period of wet fly ash on slump and compressive strength of concrete. The basic properties of wet fly ash including chemical and physical properties were also tested.

Two types of low CaO fly ash (FA-HM and FA-HS) produced from different blended-coal ratios and coal sources were used. FA-HM is fly ash produced from a blended Hunter Valley and Melawan. FAHS is fly ash produced from a blended Hunter Valley and Spring Creek. Wet fly ashes were prepared in the laboratory by adding water into the dry fly ashes to achieve moisture contents of 25%, 45%, and 65%. Wet fly ashes with moisture contents of 25%, 45%, and 65%, prepared from FA-HM, are named as FA-HM-25, FA-HM-45, and FA-HM-65, respectively. While those prepared from FA-HS are FA-HS-25, FA-HS-45, FA-HS-45, and FA-HS-65, respectively. The wet fly ashes were kept in those conditions for 1 month, and 3 months before being tested in order to investigate the effect of wet exposure period. It is noted that the study on properties of wet fly ash and effect of moisture content of wet fly ash were conducted based on wet fly ash with exposure period of 3 months. Slump and compressive strength of fly ash concrete with water to binder ratios (w/b) of 0.45 and 0.55 were measured. The replacement ratios of total binder by fly ash were 0, 0.3 and 0.5.

RESULTS AND DISCUSSION

A. Basic Properties of Fly Ash Specific gravity of dry and wet fly ashes is shown in Table 1. The specific gravity of the wet fly ash is similar to the originally dry fly ash. The specific gravity of the wet fly ash is unaffected by different moisture contents. Blaineâ&#x20AC;&#x2122;s fineness values and median particle sizes of FA-HM and FA-HS are comparable as shown in Table 2. The particle size of the fly ash is slightly larger than that of OPC. Chemical composition of dry and wet fly ashes is given in Table 3. It can be seen that the chemical composition of the wet fly ash is unchanged when compared to that of the originally dry fly ash. The chemical composition of the wet fly ash with different moisture contents is also similar. All fly ash samples have low CaO content, which is about 2%. The sum of three main oxides (SiO 2, Al2O3, and Fe2O3) is about 86%. All fly ashes have quite high loss on ignition but low SO3 content.

Table 1 Specific gravity of OPC, dry fly ash, and wet fly ash Sampl

OPC

e Specif ic gravity

3.11

FAHM

FAHM-25

FAHM-45

FAHM-65

2.11

2.12

2.15

2.14

FA-

FAHS-25

FAHS-45

FAHS-65

2.1

2.1

2.11

2.09

HS

Table 2 Blaine fineness and median particle size of OPC and dry fly ashes Sample Blaine fineness (cm2/g) Median particle size (Îźm)

OPC 3,660

FA-HM 3,400

FA-HS 3,350

18.22

21.76

22.61

184 | P a g e Table 3 Chemical composition of dry and wet fly ashes FAHM

FAHM-25

FAHM-45

FAHM-65

FA-

FAHS-25

HS

FAHS-45

FAHS-65

SiO2

61.09

61.39

61.26

61.3

63.53

63.26

63.84

62.89

Al2O3

20.35

19.99

20.11

20.06

20.81

21.14

20.26

20.75

Fe2O3

5.20

5.32

5.19

5.14

4.06

4.04

4.07

4.12

CaO

2.32

1.75

1.83

1.90

1.84

1.85

1.92

1.92

MgO

1.35

1.36

1.35

1.37

1.00

0.92

0.96

0.98

SO3

0.28

0.28

0.21

0.25

0.15

0.21

0.20

0.17

Na2O

0.79

1.16

1.33

1.03

0.95

0.87

1.13

1.15

K2O

1.36

1.32

1.4

1.41

1.23

1.20

1.22

1.29

TiO2

0.98

1.01

0.93

0.99

1.05

1.03

1.02

1.05

P2O5

0.23

0.30

0.32

0.30

0.29

0.40

0.31

0.35

SrO

0.09

0.08

0.08

0.08

0.12

0.12

0.11

0.12

ZrO2

0.07

0.07

0.06

0.07

0.08

0.07

0.07

0.08

Free CaO

0.03

0.02

0.02

0.04

0.06

0.06

0.04

0.07

LOI

5.68

5.81

5.82

5.94

4.55

4.81

4.84

4.99

B. Slump of Concrete Slump of fly ash concrete with water to binder ratios (w/b) of 0.45 and 0.55 were measured. The replacement ratios of total binder by fly ash were 0, 0.3 and 0.5. Slump of concrete made with and without fly ashes is shown in Fig. 1. It can be seen that the slump of fresh concrete containing dry fly ash is lower than that of OPC concrete. In case of low water to binder ratio (w/b = 0.45), the slump of concretes with and without fly ash is almost similar. In the case of high water to binder ratio (w/b = 0.55), effect of moisture content of wet fly ash on the slump is very clear that wet fly ash can improve the slump of concrete when compared with dry fly ash.

12

12

10 8

8

8.5

9

7.5

6 4

4

3.5

4

4

4

2

10 8

8

7.5 6.5 5.5

6 4

6.5

4 2.5

3

3

3

2

0

0

w/b=0.45 OPC

Initial slump (cm)

Initial slump (cm)

10.5

FA-HM

FA-HM-WL-25

w/b=0.55 FA-HM-WL-45

FA-HM-WL-65

(a) 30% replacement of fly ash

w/b=0.45 OPC

FA-HM

FA-HM-WL-25

w/b=0.55 FA-HM-WL-45

FA-HM-WL-65

(b) 50% replacement of fly ash

Fig. 1 Slump of concrete

185 | P a g e

600

500

Compressive strength (kgf/cm2)

Compressive strength (kgf/cm2)

C. Compressive Strength of Concrete Compressive strength of fly ash concrete with water to binder ratios (w/b) of 0.45 and 0.55 was measured. The replacement ratios of total binder by fly ash were 0, 0.3 and 0.5. Fig. 2 and Fig. 3 illustrate compressive strength development of concrete with and without fly ash. Compressive strength of concrete with fly ashes is lower than that of concrete with cement only. Although the concretes with fly ash have lower strength development at early age, their strength development in long term (91 days) is almost equal to that of the OPC concrete due to pozzolanic reaction of the fly ash at later age. Compressive strength of concrete made with wet fly ash is slightly improved when compared with that of dry fly ash concrete (except FA-HM with w/b = 0.55 and 30% replacement of fly ash at 3 days).

Effect of wet exposure period of wet fly ash on compressive strength of concrete at 28, and 91 days is shown in Fig. 4. The ratios between compressive strength of concrete with wet fly ash at 3 months of exposure period and that at 1 month of exposure period are almost equal to 1. Compressive strength of concrete made with wet fly ash is considered unaffected by the exposure period. This is because chemical composition of the wet fly ash is unchanged.

500 400 300

200 100

400

300

200

100

0

0 3 days

7 days

28 days

3 days

91 days

OPC

FA-HM-30

FA-HM-WL-25-30

FA-HM-WL-45-30

7 days

28 days

91 days

Age of samples (days)

Age of samples (days) FA-HM-WL-65-30

OPC

(a) 30% replacement of fly ash

FA-HM-50

FA-HM-WL-25-50

FA-HM-WL-45-50

FA-HM-WL-65-50

(b) 50% replacement of fly ash

500

Compressive strength (kgf/cm2)

Compressive strength (kgf/cm2)

Fig. 2 Compressive strength of concrete with w/b = 0.45

400

300

200

100

500

400

300

200

100

0

0 3 days

7 days

28 days

91 days

3 days

Age of samples (days) OPC

FA-HM-30

FA-HM-WL-25-30

FA-HM-WL-45-30

7 days

28 days

91 days

Age of samples (days) FA-HM-WL-65-30

(a) 30% replacement of fly ash

OPC

FA-HM-50

FA-HM-WL-25-50

FA-HM-WL-45-50

FA-HM-WL-65-50

(b) 50% replacement of fly ash

Fig. 3 Compressive strength of concrete with w/b = 0.55

186 | P a g e 1.2

1 0.8 0.6 0.4 0.2

0 w45r30

w45r50

w55r30

w55r50

Age of samples (days) FAHM

FAHM25

FAHM45

Ratio of compressive strength of concrete made with fly ash at 3 months to 1 months (%)

Ratio of compressive strength of concrete made with fly ash at 3 months to 1 months (%)

1.2

1 0.8 0.6 0.4 0.2

0

FAHM65

w45r30

FAHM

(a) 28 days

w45r50

w55r30

w55r50

Age of samples (days) FAHM25

FAHM45

FAHM65

(b) 91 days

Fig. 4 Effect of wet exposure period of wet fly ash on compressive strength of concrete D. Effect of Double Mixing Method on Micro Hardness of Concrete In the double mixing method, the charging of water is divided into two stages [4]. It was found in the literature that the improvement in strength of recycled aggregate concrete can be achieved by using the double mixing method. The content of the first charge of water also has an effect on the properties of concrete [4]. When wet fly ash is used in the mixture, preexistence of water in the wet fly ash can be considered as the first charge of water in the double mixing method. Therefore, moisture content of the wet fly ash is supposed to have effects on the properties of concrete as well. The double mixing method is known to improve the interfacial transition zone (ITZ) of normal aggregate concrete [4]. In this study, Vickers micro hardness test on the ITZ of concrete made with dry and wet fly ash was conducted. Concrete samples with water to binder ratio of 0.55 and 30% replacement of fly ash were used. The Vickers micro hardness was measured within the distance of 20-250 µm from the aggregate surface. The average value of Vickers micro hardness measured at 50±30 µm distance from the surface of sand particles is shown in Table 4. The profile of hardness values at different distance from sand particles is shown in Fig. 4. The hardness values near sand particle of concretes made with wet fly ash are higher than that of the concrete made with dry fly ash. The hardness values increase with an increase in moisture content of the wet fly ash. The hardness values of the paste near sand particles of concrete made with dry fly ash are lower when compared to the values measured far from the sand particles, which is a typical behavior for normal aggregate due to the effect of interfacial transition zone (ITZ). On the other hand, the hardness values of concrete made with wet fly ash decrease as the distance from the sand particles increases. However, the hardness values measured far from the sand particles of concrete made with the wet fly ash are only slightly lower than that of dry fly ash. This can be confirmed that ITZ of the concrete made with wet fly ash is improved due to the effect of double mixing method. The improved ITZ increases compressive strength of the concrete made with wet fly ash.

Table 4 Average values of Vickers micro hardness near sand particles of concrete made with dry and wet fly ashes Samples

FA-HM

FA-HM-25

FA-HM-45

FA-HM-65

Vickers micro hardness

43.58

48.41

56.13

63.43

187 | P a g e

100

Hardness Vickers

80 60

40 20 0 0

50

100

150

200

250

Distance (µm) CFAHM

CFAHM25

CFAHM45

CFAHM65

Fig. 4 Profile of hardness values from polynomial fit at different distance from sand particles of concrete with dry and wet fly ashes CONCLUSIONS Effect of moisture content and exposure period of wet fly ash on basic properties of concrete were investigated. From the test results, it can be concluded that chemical composition of the wet fly ash is unchanged when compared to that of the originally dry fly ash. Slump and compressive strength of concrete made with wet fly ash are slightly improved. Preexistence of water in the wet fly ash (moisture content) is considered as the first charge of water in the double mixing method which leads to improvement of the ITZ of concrete made with the wet fly ash. Compressive strength of the concretes made with wet fly ash are considered unaffected by the exposure period of the wet fly ash.

ACKNOWLEDGEMENTS The authors would like to acknowledge the research support from BLCP Power Co., Ltd. and the Center of Excellence in Material Science, Construction and Maintenance Technology, Thammasat University.

REFERENCES [1] Haldive S.A., Kambekar A. R., ‘Experimental study on combined effect of fly ash and pond ash on strength and durability of concrete’, International Journal of Scientific & Engineering Research Volume, Issue 5, May-2013.

[2] Andrew B., & Aceves P.G., ‘The use of Non-Commercial Fly ash in Roller compacted concrete structures’, World of Coal Ash (WOCA) conference, Denver, CO, USA May 9-12, 2011,.

[3] Raungrut Cheeratot & Chai Jaturapitakkul, ‘A study of disposing fly ash from landfill to replace Portland cement’, Accepted 2 February, 2004.

[4] Nobuaki Otsuki, M.ASCE, Shin-ichi Miyazato, and Wanchai Yodsudjai, ‘Influence of Recycled Aggregate on Interfacial transition Zone, Strength, Chloride Penetration and Carbonation of Concrete’, Journal of materials in civil engineering, 15, 2003, 443-451.

188 | P a g e Effect of High Free Lime and High Sulfur Trioxide Contents on Volume Change of Cement-FlyAsh Mixtures Parnthep Julnipitawong*,a, Adnan Nawaz b, Pitisan Krammart c, Somnuk Tangtermsirikuld a

Construction and Maintenance Technology Research Center, School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b School of Engineering and Technology, Asian Institute of Technology c Department of Civil Engineering, Rajamangla University of Technology d School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University Keywords Free lime, Autoclave expansion, Alkali aggregate reaction, Sulfate resistance ABSTRACT This study highlights the effect of high free lime and high SO3 in fly ash on durability properties especially volume change properties of mixtures containing fly ash with different free lime and SO3 contents. Three types of fly ashes were studied, and free lime was added to increase free lime contents of fly ash up to 10% for each type of fly ash. The autoclave expansion, alkali aggregate reaction, and sulfate resistance tests of fly ash mixtures were investigated. Experimental results showed that in cases of mixtures with 20% fly ash replacement and free lime content up to 10%, and, mixtures with 40% fly ash replacement and free lime content up to 7%, the autoclave expansion were within the specified limit of ASTM C618. Fly ash mixtures with high free lime led to higher expansion in cases of alkali-aggregate reaction and sulfate resistance tests. In the sulfate resistance test, fly ash mixtures tend to expand more than cement-only mixtures, especially in the case of fly ashes with very high SO3 content. Test results also showed that it is possible to utilize 20% of tested fly ash in a mixture as binder, with SO3 content less than 5% and free lime content up to 10% while not causing volume instability problems in this study. *parnthep@siit.tu.ac.th INTRODUCTION The Mae Moh power plant of the Electricity Generating Authority of Thailand is one of the main production sources of fly ash, contributing around 90% of total national production. Kaewmanee et al.

(2013) fly ash obtained from this source has shown recently increased content of free lime, which may result in undesired expansion and volume instability when used in concrete as a cement replacement.

Therefore, the durability issues and timedependent properties of concrete containing fly ash with high free lime content should be considered. In ASTM C618 and many other standards, there is no limit specified for free lime content of fly ash. However, EN-450 specifies the free lime content to 1%, or up to 2.5% if the autoclave expansion is still within the established limits. Kaewmanee et al. (2013) conducted a study on expansion properties of fly ash mixes, with various free lime contents and reported a set of experimental results to clarify the effect of free lime content on fly ash mixes.

Results showed that normal consistency and water demand were not much affected by free lime, and free lime content up to 4.51% had only slight effect on chemical properties of cement-fly ash mixtures. The autoclave expansion results of the tested mixtures with 4.51% free lime content in fly ash were still within the limit according to ASTM C618. The expansion

189 | P a g e due to alkali-aggregate reaction of mixtures with fly ash having 4.51% free lime contents was increased but the expansion was less than that of cement-only mixture. However, fly ash obtained from the Mae Moh power plant has not only shown increased free lime content, but also tendency of SO3 content, which may lead to long term expansion and volume instability. Therefore, the objective of this study is to investigate the effect of high free lime and high SO3 contents of fly ashes on expansion properties of cement-fly ash mixtures.

EXPERIMETAL PROGRAMS A. Materials In this study, three types of fly ashes F(A), F(B) and F(C) and an ordinary Portland cement (OPC) type I, were used. F(A), F(B), and F(C) were obtained from the Mae Moh power generating plant.

Tables 1 and 2 show the physical properties and chemical compositions of cement and fly ashes.

Based on of TIS 2135, F(A), F(B) and F(C) were classified as Class 2b which are high CaO fly ashes.

The amount of SO3 in F(B) and F(C) is higher than limit of 5%, as specified by TIS 2135. Free lime was added in F(A), F(B) and F(C) to prepare fly ashes having total free lime contents of 5%, 7% and 10%, respectively. In this study, river sand complied with ASTM C33 with a specific gravity of 2.60 was used as the fine aggregate.

B. Experiments The volume change properties of cement-fly ash mixtures were determined from autoclave expansion, expansion due to alkali aggregate reaction and sodium sulfate expansion. The autoclave expansion test was tested according to ASTM C151. Test Mixtures containing 20% and 40% of each type of fly ash were used to investigate the autoclave expansion. The expansion due to alkali aggregate reaction was conducted as per ASTM C1260. Mortar bar specimens (2.5 x 2.5 x 28.5 cm) were prepared with crushed reactive gravel, graded as per ASTM C1260. Test mixtures containing 20% of each type of fly ash were prepared. Sand to binder ratio of 2.25 and water to binder ratio of 0.47 were used for all test mixtures. Mortar bars were submerged in a sodium hydroxide (NaOH) solution under temperature of 80 ¹ 2 °C. The expansion due to alkali aggregate reaction was recorded for 14 days. The 2.5 x 2.5 x 28.5 cm mortar bar specimens were prepared in case of sulfate resistance test. Test mixtures containing 20% and 40% of each type of fly ash were prepared. Sand to binder ratio and water to binder ratio were controlled at 2.75 and 0.55, respectively for all mixtures. After 24 hours of mixing, test samples were cured in water for 28 days and then immersed in a sodium sulfate solution for the sulfate expansion test.

190 | P a g e RESULTS AND DISCUSSIONS A. Scanning electron microscope (SEM) images Fig. 1 shows SEM images of fly ash. It can be seen that the particles of the Mae Moh fly ash are round or spherical shape while irregular shape of free lime particle dispersing around fly ash particles can be seen

Fig. 1 SEM image of fly ash B. Autoclave expansion Fig. 2 shows that F(B) mixtures showed highest autoclave expansion as compared to other fly ash mixtures. This is because of the highest free lime content of 3.93% in F(B), as compared to 3.03% in F(C) and 1.71% in F(A). Similarly, F(C) mixtures showed higher expansion as compared to F(A) mixtures. The cement-only mixtures showed shrinkage as compared to the expansion of fly ash mixtures. Although, the autoclave expansion of fly ash mixtures is higher than that of the cement-only mixture, the expansion values are still within the specified limit of 0.8%, as specified in ASTM C618, even in the cases of fly ashes with high calcium oxide and high free lime contents. Figs. 2b-d show that the mixtures containing fly ashes F(A), F(B), and F(C) with added free lime had a higher degree of expansion with an increase of free lime content. Although the expansion results of mixtures with added free lime are higher than the cement-only mix, they are within the ASTM C618 limit except in the cases of mixtures containing 40% of F(A10), F(B10), and F(C10) fly ashes.

Fig. 2 Autoclave expansion of mixtures

191 | P a g e C. Alkali-aggregate reaction It was revealed that expansion of F(A), F(B), F(C) mixtures were lower than cement-only mixture as shown in Fig. 3a. The mixtures containing F(B) and F(C) showed higher expansion as compared to F(A) mixtures because of higher free lime and calcium oxide contents. The addition of free lime resulted in increased expansion as shown in Fig. 3b. This may be because of the additional alkalinity in the form of calcium hydroxide provided by the free lime. All mixtures showed smaller expansion than the cement-only mixture except F(C10) mixture case. It can be postulated that calcium hydroxide is consumed in the pozzolanic reaction, therefore, the alkalinity level of the fly ash mixtures reduced, resulting in lower expansion as compared to the cement-only mixture.

D. Sulfate resistance Figs. 4a-d shows the expansion results of mixtures submerged in sodium sulfate solution up to 224 days. Fig. 4a demonstrates that cement-only mixtures showed higher expansion than fly ash mixtures.

From Figs. 4b and 4c, expansion of F(A) and F(B) mixtures, with added free lime and 20% fly ash replacement, was lower than the cement-only mixtures. In the case of F(C) mixtures, only mixture containing 20% of F(C) fly ash with 5% free lime content showed lower expansion than cement-only mixture (Fig. 4d). It can be concluded that addition of free lime in fly ashes resulted in higher expansion of the mixtures as shown in Figs. 4b-d.

(a) Primary fly ashes

(b) Fly ashes with added free lime

Fig. 3 Expansion due to alkali-aggregate reaction

192 | P a g e

Fig. 4 Expansion of mortar bars submerged in Na2SO4 solution after 224 days.

CONCLUSIONS For this study conclusions can be drawn as follows: Autoclave expansion increases with the addition of free lime in fly ash mixtures. Mixtures with 20% fly ash replacement and free lime content up to 10% showed autoclave expansion within the specified limit of ASTM C618. In the case of 40% fly ash replacement, fly ash mixtures with 7% free lime content, gave autoclave expansion within the specified limit of ASTM C618.

Addition of free lime content increases the expansion due to alkali-aggregate reaction. Mixture containing 20% of fly ash with 7% free lime content gave lower expansion than the cement-only mixtures.

In case of sodium sulfate resistance test, the mixture containing 20% fly ash replacement (FA(10)) with 10% free lime and 4.26% SO3 contents had expansion less than that of cement-only mixture.

ACKNOWLEDGEMENT The authors would like to thank the Electricity Generating Authority of Thailand for providing research support and fly ash samples for this study. This research is also supported by the Center of Excellence in Material Science, Construction and Maintenance Technology, Thammasat University,

193 | P a g e the Higher Education Research Promotion and National Research University Project, the Office of the Higher Education Commission of Thailand.

REFERENCES Annual Book of ASTM Standards. (2003). ASTM C618 standard specification for fly ash and raw or calcined natural pozzolan for use as a mineral admixture in Portland Cement Concrete; European Standard. (1994). EN 450 fly ash for concrete â&#x20AC;&#x201C; definitions, requirement and quality control, Brussels.

Kaewmanee K., Krammart P., Sumranwanich T., Choktaweekarn P., Tangtermsirikul S. (2013). Effect of Free Lime Content on Properties of Cementâ&#x20AC;&#x201C;Fly ash Mixtures. J Construction and Building Materials Res. 38, 829-36.

Annual Book of ASTM Standards. (2003). ASTM C33 Standard Specification for Concrete Aggregates.

Annual Book of ASTM Standards. (2000). ASTM C151 Standard Test Method for Autoclave Expansion of Portland Cement.

Annual Book of ASTM Standards. (2003). ASTM C1260 Standard Test Method for Potential Alkali Reactivity of Aggregates (Mortar Bar Method).

194 | P a g e Development of Method to Determine Water Retainability of Porous Fine Aggregate Noulananh Lathsoulin* a, Pakawat Sancharoen b, Somnuk. Tangtermsirikul a,b a

School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b Construction and Maintenance Technology Research Center, Sirindhorn International Institute of Technology, Thammasat University Keywords: Porous fine aggregate; Test method; Water retainability; Absorbtion

ABSTRACT Bottom ash from coal power plant is a porous material and its particle size is comparable to that of fine aggregate used in concrete. So it is considered to be used as porous fine aggregate.

However, current standard method for determining water absorption of fine aggregate cannot be used effectively with porous fine aggregate due to it is difficult to justify the saturated surface dry condition of it. So water reainability was determined instead. However, current method to determine water retainability is time consuming. Therefore, new method was developed based on available laboratory equipment and short testing time. In this study, various bottom ash samples were tested by different persons and different laboratory to evaluate the consistency of the developed method. Testing parameters such as testing time, collected sample depth and limit the length of container were studied. Sample of bottom ash was fully submerged a few days to reach the over saturated condition before being vibrated-drained for 24 minutes. The results show that developed method showed water retainability value similar to traditional method. However, testing time was 3 times shorter. The result can be used to determine water retainability of bottom ash and practically adjust amount of concrete mixing water. *khaek_noulananh@hotmail.com INTRODUCTION Nowadays, porous materials or water retaining materials such as light-weight aggregate have been used worldwide to be as low cost replacement materials due to a shortage of natural resources, economical benefits, environmental problem and may enhance the properties of concrete [1,2]. The main problem for the use these porous materials is the moisture content of porous aggregate is always higher than normal aggregate. Previous studies have reported that concrete mixed with bottom ash and recycled concrete-crush was very stiff and nonworkable due to a significant water demand from their porous properties [3,4]. There was a certain difficulty in determining the exact moisture properties of bottom ash [5].

Water retainability has been presented by some researchers that water required to completely fill in pores and dependently restricted on the surface of aggregate particles under gravitational condition is defined as the water retainability. Water retainability of bottom ash included absorption and water adsorption which increased moisture properties of concrete causing affects the free water amount in fresh concrete [1]. For concrete incorporating porous aggregate, it is appropriate to apply water retainability instead of water absorption to adjust amount of mixing

195 | P a g e water according to concrete mix proportion.

The purpose of this study is to develop of practical method to determine water retainability of porous fine aggregate based on available laboratory equipment and short testing time due to the previous test method was time consuming.

METHODOLOGY The study was separated into two parts; in the first part, the proposed test method was conducted with different vibration time, collected sample depth and types of container. Water retainability obtained by traditional method and proposed test method were compared and bottom ash type A was used. In the second part, to prove the proposed test method, the bottom ashes type A and B were tested by different persons and different laboratories.

A. Material Bottom ash was classified as types A and B that were collected from Mae Moh electricity generating plant, Thailand. Two bottom ashes with different burning process had been tested in this study. The bottom ash type A was obtained from power plant with 150MW capacity and type B was obtained from power plant 300MW capacity. All particles of bottom ash had to pass sieve openings 4.75mm (No.4) prior testing. Sieve analysis and physical properties of bottom ash type A and B are showed in Figure 1. and Table 1, respectively.

Figure. 1. Sieve analysis of bottom ash

B. Traditional test method B.1 Sample preparation Obtain a sample with a size of approximately of the quantity required to fill the cylinder (3â&#x20AC;&#x201C;5 kg) and dry it to a constant mass. Mix dry bottom ashes with a specified amount of water throughout the mixture in a container, and wrap the container with a plastic sheet to prevent water evaporation.

B.2 Testing Compact the prepared sample into the cylinder pipe by separated into three layers and hammering for 30 times in each layer. Vibrate the container by ordinary fine aggregate sieving machine for 24 minutes

196 | P a g e Remove two separate portions of the test samples from both ends of the cylinder each, with minimum weight of 500±10g. Find moisture content of both sample as showed in equation (1):

Where Mi is the moisture content of aggregate (%) at the top or bottom of cylinder, A and B are weight (gram) of oven-dried of sample and weight (gram) of the sample after test, respectively. Ovendried of sample at 110±5ºC for 24 hour and allow it to cool to comfortable handling temperature.

Obtain the difference of the moisture content of the tested aggregate between the top and bottom portions as showed in equation (2): Where Δm is the different moisture content of the tested aggregate (%) and mt, mb are the moisture contents (%) of the top and bottom portions, respectively as obtained from equation (1).

The water retainability of aggregate is the cross value at x-axis where Δm is equal to zero. A criterion has been set to use the data points in the range of Δm values between 0.5 and 5, since in this range the line is sufficiently linear. A linear line was drawn from the data points in that range and the xintercept value was defined as the water retainability.

Figure. 2. The criteria set to determine water retainability by traditional method [6].

C. Proposed test method C.1 Sample preparation Prepared the sample with the quantity required to fill the cylinder (5-6 kg). Mix the sample with the water in a container, wrap the container and immerse the sample for 3 days. Before testing, sample should be drained around 30 minutes before filling into the apparatus.

C.2 Testing The cylinder pipe had an inner diameter of 75 mm and 850 mm in length. Compacting sample into the cylinder pipe was separated into three layers, then hammering 30 times for each layer. Level the surface of the aggregate with a straightedge. Seal the top end and connection joint of the cylinder with sealant materials for preventing moisture loss.

Vibrate the cylinder pipe by ordinary fine aggregate sieving machine at various duration as 10, 15, 20, 24, 30 and 35 minutes respectively.

Collected the sample from the top of container and obtained the moisture content of the sample for every 3 centimeter thickness by using equation (1) as showed above.

The water retainability is the value of the moisture content from the top of the containers.

197 | P a g e Two types of bottom ash had been tested as bottom ash type A and bottm ash type B. The sample had been tested three times per one type of bottom ash at each laboratory. To prove the proposed method testing, same samples were examined by different persons and laboratory. Rajamangala University of Technology Thanyaburi, Thailand was selected to be another testing laboratory.

RESULTS AND DISCUSSION A. Effect of vibration time To find the appropriate time for water retainability, vibration time affected water retainability of porous fine aggregate as showed in Fig. 3(a). When the sample was vibrated with the shortest time, the sample showed the highest water retainability. The short testing time as 10, 15 and 20minutes, the samples showed water retainability 38.54%, 37.83% and 36.19% respectively which were decreasing.

In contrast, testing time as 24 to 35 minute, the water retainability started to be constant and had the values of water retainability around 36%. Finally the appropriate time for vibration was set as 24 minute.

Figure. 3. (a) Effect of vibration time on water retainability (b) Effect of collected sample depth on water retainability by proposed test method B. Effect of collected sample depth The thickness of collected sample was a significant point. If sample is collected too thick, it would be the value of over water retainability. So, the collected sample depth was very important in this part. It could be concluded from the Figure. 3(b) that the water retainability of bottom ash increased with thicker collected sample depth which was removed from the top of cylinder pipe. The collected sample depth with 0-1, 1-2 and 2-3 centimeter had the water retainability nearly the same around 35%, which were constant, but the collected sample depth from 3-4 and 4-5 centimeter had water retainability 36.18% and 37.76% respectively which were increasing. It could be observed from this point that the water retainability increased with increasing of the collected sample depth after 3 centimeter. Finally the water retainability was obtained by collecting the sample with 3 centimeter thickness from the top.

C. Effect of height of container The height of apparatus significantly affected the result as showed in Figure. 4(a). The height of container as 85, 70 and 50 centimeters, the sample showed water retainability 35.63%, 35.78% and 35.76 respectively which were quite constant. In contrast, the height of container as 25 and 35 centimeters, the sample showed water retainability 37.86% and 36.41% respectively which increased with decreasing of height of container. However, the sample with the 35cetimeter in length of container, it was tested more than 24 minute as 30, 40 and 50 minute. It was observed from the Figure. 4(b) that the sample with vibration time 40 and 50 minute started to be the same value of water retainability as the long length. So the length of container for water retainability testing should

198 | P a g e be not less than 50 centimeter. If it is less than 50centimeter, the sample had to vibrate more than 24 minute.

Figure. 4. (a) Effect of the length of container on water retainability; (b) Effect of vibration time on the pvc pipe 35cm in length on water retainability D. Comparison between water retainability obtained by traditional test method and proposed test method The comparison of test result between traditional and proposed test method was shown in Figure. 5. It was observed that the proposed test method had water retainability around 35.63% as showed in Figure. 5(b) ,which showed slightly higher than the traditional test method that had water retainability around 32.01 % as showed in Figure. 5(a). The traditional test method was time consuming which the period of time was around 2-3 weeks and the water retainability value also depended on the extrapolation. In contrast, the proposed test method was faster than the traditional one which the period was less than an hour and the obtained water retainability was also consistent. Moreover, the sample can obtain the consistent values even testing for various times as showed in Figure. 5(b).

Figure. 5. (a) Water retainability by the traditional test method; (b) Water retainability by proposed test method.

E. Comparison the water retinability by different persons and different laboratories The sample had been tested three times per one type of bottom ash and also one lab as results showed in the table 2. the bottom ash type A showed that the results of water retainability were nearly the same around 36% and the bottom ash type B also showed that the water retainability was around 46%.

Both laboratories showed that the obtained water retainability is less than 2% in different. So, it could be concluded that the proposed method is acceptable for the water retainability determination.

199 | P a g e Table 2. Comparison the water retinability of the various bottom ash samples by different persons and different laboratory

CONCLUSIONS Water absorption of aggregate was necessary for design and adjust of concrete mixtures containing the conventional aggregate. However, when using porous fine aggregates, the water retainability of the aggregate was more important.

Based on the experiment results presented in this study, it was concluded that the proposed method had water retainability slightly higher than the traditional test method, but it was more effective because the proposed method could be used to determine and adjust amount of mixing water of porous fine aggregate more practically. The proposed method showed the appropriate vibration time for 24 minutes, collected sample depth about 3 centimeter and the height of container should not less than 50 centimeter. In addition, test results conducted by different persons and laboratories showed that the proposed test method for water retainability gave better consistency of the results.

ACKNOWLEDGEMENTS The authors would like to thank the Center of Excellence in Material Science, Construction and Maintenance Technology, Thammasat University, the Higher Education Research Promotion and National Research University Project, the Office of the Higher Education Commission of Thailand and AUN-SEED NET for their financial and scholarship support.

REFERENCES [1]. R.Kasemchaisiri and S.Tangtermsirikul, (2007)"A method to determine water retainability of porous fine aggregate for design and quality control of fresh concrete." Construction and Building Materials 21.6: 1322-1334.

[2]. Andrade et al (2009). "Influence of coal bottom ash as fine aggregate on fresh properties of concrete." Construction and Building Materials 23.2: 609-614.

[3]. Bucholc J and Ghafoori N. Investigation of lignite-based bottom ash for structural concrete. J Mater Civ Eng, American Society of Civil Engineer 1996;8(3):128.

[4]. Ghafoori N and Bucholc J (1997). Properties of high-calcium dry bottom ash. ACI Mater J;94(2):90.

[5]. Landgren R. Unit weight, specific gravity, absorption, and surface moisture. In: Klieger Paul, Larmond Joseph F, editors. Significance of tests and properties of concrete-making materials, ASTM STP 169C. Philadelphia (1994): American Society for Testing and Materials;p. 421.

[6]. Kinaanath Hussain (2011) â&#x20AC;&#x153;DEVELOPMENT OF A MINIMUM CURING CONCRETE BY INTERNAL CURING PROCESS â&#x20AC;&#x153;Thesis CE-MS. Sirindhorn International Institute of Technology.

Thammasat University

200 | P a g e Advantages of Symbolic Hessian Matrices in Sizing Optimization of Trusses Chantrea Lean a, Pisith Sam a, Pruettha Nanakorn*,a a)

School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University

ABSTRACT When a conventional nonlinear programming (NLP) method is used to solve optimization problems of trusses, the gradients and Hessians of the objective and constraint functions are generally required.

When the gradients and Hessians are not analytically available, the solver will have to estimate them by approximation methods. In this paper, the advantages of the analytical Hessians of the objective and constraint functions in solving sizing optimization problems of trusses by a conventional NLP method are investigated. The investigation is done in MATLAB by using two benchmark problems.

The analytical gradients and Hessians of the constraint functions are obtained from symbolic finite element solutions. The performance of a conventional NLP method in MATLAB with the analytical gradients and Hessians of the objective and constraint functions is found to be better than the performance of the same method without the analytical gradients and Hessians. The results obtained from the employed conventional NLP method compare favourably with the results from a genetic algorithm provided in MATLAB and the literature.

Keywords: Gradient, Hessian, Genetic algorithm, Truss optimization, Symbolic finite element. *nanakorn@siit.tu.ac.th INTRODUCTION In general, truss optimization problems can be classified into three types of problem, namely sizing, shape, and topology optimization problems (Christensen and Klarbring 2008). Most frequently, the objective of a truss optimization problem is to minimize the weight of a truss structure that is subject to stress and displacement constraints. Truss optimization problems are mostly nonlinear programming (NLP) problems. Two groups of methods that are usually used to deal with these problems are conventional NLP and metaheuristic optimization methods. Conventional NLP methods are gradient-based methods. In structural optimization, the analytical forms of constraint functions are usually difficult to obtain. As a result, when a conventional NLP method is employed, the gradients and Hessians of constraint functions have to be numerically approximated. In contrast, metaheuristic optimization methods do not generally require the gradients or Hessians of the objective and constraint functions. Most of these methods are derived from natural phenomena. Examples of metaheuristic optimization methods include genetic algorithms (GAs), particle swarm optimization (PSO), firefly algorithms (FAs), and ant colony optimization (ACO). Since metaheuristic algorithms do not require the gradients and Hessians of the objective and constraint functions, they can be easily used with the finite element method (FEM) to solve structural optimization problems.

Nowadays, there are computational platforms that allow symbolic computations to be performed by computers. Examples are MATLAB and Mathematica. These computational platforms can be used to create symbolic finite element (FE) programs. Symbolic FE programs can analytically provide displacements and stresses of trusses in terms of member areas. Analytical displacements and stresses subsequently allow analytical constraint functions to be constructed. These analytical constraint

201 | P a g e functions allow conventional NLP methods to be efficiently used to solve truss optimization problems.

In this paper, the advantages of analytical Hessian matrices in solving sizing optimization problems of trusses by a conventional NLP method are investigated. The investigation is done in MATLAB. The analytical gradients and Hessians of the constraint functions are obtained from symbolic FE analysis.

In the investigation, two benchmark optimization problems of 2D truss structures (Deb and Gulati 2001, Luh and Lin 2008, Farshi and Alinia-ziazi 2010, Li et al. 2007) are used. The performances of a conventional NLP method in MATLAB with and without the analytical gradients and Hessians of the objective and constraint functions are compared. In addition, the obtained results are also compared with those from a genetic algorithm (GA) provided in MATLAB and those from the literature.

TRUSS OPTIMIZATION PROBLEMS

A typical truss optimization problem can be written as Minimize

â&#x2C6;&#x2018;

Subject to

| (

)|

| (

)| ,

where Weight of the truss, Weight density of the material of member Length of member , Area of member Stress of member , Displacement of degree of freedom Allowable stress, Allowable displacement Minimum area of member , Maximum area of member The number of members, The number of degrees of freedom.

SOLVING OPTIMIZATION PROBLEMS IN MATLAB MATLAB provides commands for solving constrained nonlinear optimization problems. The command that is mostly used is fmincon. This command allows several conventional NLP methods to be selected such as interior-point, active-set, sequential-quadratic-programming, and trust-regionreflective algorithms. In this study, the interior-point method is used. The command fmincon allows the explicit gradients and Hessians of the objective and constraint functions to be used if they are available. The syntax of fmincon can be shown as follows: [x,fval]=fmincon(fun,x0,A,b,Aeq,beq,lb,ub,nonlcon,options)

202 | P a g e Here, fun is the objective function. The constraint functions and their gradients, if they are considered, are specified via nonlcon. In addition, options is used to specify optimization options. The set of options when the gradients and Hessians are not used can be specified as options = optimoptions('fmincon','Algorithm',... 'interior-point','GradObj','off','GradConstr','off')

The set of options when the gradients and Hessians are used can be specified as options = optimoptions('fmincon','Algorithm', ... 'interior-point','GradObj','on','GradConstr','on', ...

'Hessian','user-supplied','HessFcn',myhess)

The Hessians are specified through myhess. In this study, a GA in MATLAB is also used. The command ga is used for this purpose, i.e. [x,fval]=ga(fitnessfcn,nvars,A,b,Aeq,beq,LB,UB,nonlcon,options) The details of fmincon and ga can be found in MATLAB’s documentation.

RESULTS A. Truss with six members and five nodes

Figure 1 shows a truss with six members and five nodes (Deb and Gulati 2001, Luh and Lin 2008) to be optimized. The problem parameters are as follows: Young’s modulus , the weight ⁄ , the allowable stress density , the allowable displacement , and the limit cross sectional areas .

Figure 1: Problem A--truss with six members and five nodes The results are shown in Table 1. The results from fmincon without and with the gradients and Hessians are shown as fmincon1 and fmincon2, respectively. The results from fmincon without and

203 | P a g e with the gradients and Hessians are found to be the same and comparable to the results from ga and the literature. Note that the results by Deb and Gulati (2001) and Luh and Lin (2008) are from topology optimization. The comparison with the literature is only to validate the present results.

Figure 2 shows the convergence rates from fmincon without and with the gradients and Hessians and from ga. It can be seen that the convergence rate from fmincon when the gradients and Hessians are used is the fastest and that from ga is the slowest.

Table 1: Results of Problem A Area (in2) Present study

Member

Deb and Gulati (2001)

Luh and Lin (2008)

fmincon1

fmincon2

GA

1

05.219

05.428

05.4000

05.4000

05.4375

2

20.310

20.549

20.3647

20.3640

20.3168

3

14.593

14.308

14.4000

14.4000

14.4452

4

07.772

07.617

07.6368

07.6368

07.6105

5

28.187

28.876

28.8000

28.8000

28.6804

6

20.650

20.265

20.3647

20.3647

20.4341

Weight of truss (lb)

4731.650

4730.824

4730.4000

4730.4000

4730.4598

11000

fmincon1

Best weight (lb)

10000

fmincon2 GA

9000 8000 7000 6000 5000 4000 0

5

10

15

20

25

Iteration number Figure 2: Convergence rates of Problem A

B. Truss with ten members and six nodes

204 | P a g e Figure 3 shows a truss with ten members and six nodes (Farshi and Alinia-ziazi 2010, Li et al. 2007).

The problem parameters are the same as the previous problem except for , which is equal to in this problem. The applied loads are given as and

Figure 3: Problem B--truss with ten members and six nodes

Table 2: Results of Problem B Area (in2) Li et al.

(2007)

Farshi and Alinia-ziazi (2010)

Present study fmincon1

fmincon2

1

23.353

23.5270

23.5307

23.5307

20.9862

2

00.100

00.1000

00.1000

00.1000

00.1000

3

25.502

25.2941

25.2851

25.2851

24.7515

4

14.250

14.3760

14.3745

14.3745

16.9858

5

00.100

00.1000

00.1000

00.1000

00.1196

6

01.972

01.9698

01.9697

01.9697

01.9682

7

12.363

12.4041

12.3906

12.3906

12.2274

8

12.894

12.8245

12.8277

12.8277

13.2960

9

20.356

20.3304

20.3286

20.3286

20.9993

10

00.101

00.1000

00.1000

00.1000

00.1000

Weight of truss (lb)

4677.29

4677.80

4676.92

4676.92

4710.40

Member

GA

The results are shown in Table 2. The results from fmincon without and with the gradients and Hessians are found to be the same and comparable with those from the literature. The result from ga is

205 | P a g e not as good as those from fmincon. However, it may be possible to adjust the GA parameters in ga to obtain better results. Note that the results by Li et al. (2007) and Farshi and Alinia-ziazi (2010) are from sizing optimization. From Figure 4, it can be seen that the convergence rate from fmincon when the gradients and Hessians are used is the fastest while the convergence rate of ga is the slowest.

16000

fmincon1

Best weight (lb)

14000

fmincon2

12000

GA

10000 8000 6000 4000 0

10

20

Iteration number

30

40

Figure 4: Convergence rates of Problem B CONCLUSIONS This study demonstrates the advantages of symbolic Hessians in sizing optimization of trusses. Two benchmark sizing optimization problems of 2D trusses are solved with a conventional NLP method provided in MATLAB. The analytical gradients and Hessians of the constraint functions are obtained from symbolic FE solutions. It is found that the results obtained from the employed conventional NLP method without and with the gradients and Hessians of the objective and constraint functions are the same. However, the convergence rates of the solutions when the gradients and Hessians are provided are found to be faster. The results obtained from the conventional NLP method in this study compare favourably with the results from a genetic algorithm provided in MATLAB and the literature.

ACKNOWLEDGEMENTS This study is supported by the scholarships from the ASEAN University Network/the Southeast Asia Engineering Education Development Network (AUN/SEED-Net) and Sirindhorn International Institute of Technology (SIIT).

REFERENCES Christensen, P. W. and Klarbring, A. (2008). An introduction to structural optimization, Springer Science & Business Media.

Deb, K. and Gulati, S. (2001). Finite Elem. Anal. Des. 37(5), 447-465.

Luh, G.-C. and Lin, C.-Y. (2008). Struct. Multidiscip. Optimiz. 36(4), 365-379.

Farshi, B. and Alinia-ziazi, A. (2010). Internat. J. Solids Structures. 47(18â&#x20AC;&#x201C;19), 2508-2524.

Li, L. J., Huang, Z. B., Liu, F., et al. (2007). Comput. & Structures. 85(7â&#x20AC;&#x201C;8), 340-349.

206 | P a g e Modeling Dynamics of Traffic States on Urban Road Network Using Artificial Intelligence Mongkut Piantanakulchai a* ,Soknath Mil a ,Sornthep Vannarat b, Kimheang Ly a, Chhivhout Sor a a

School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b Large Scale Simulation Research Laboratory, National Electronics and Computer Technology Center (NECTEC) Keywords : Traffic States, Time Series, Artificial Intelligence, Artificial Neural Network (ANN)

ABSTRACT Dynamics of traffic states in urban road network was studied by using an artificial intelligence technique. Traffic states of urban road network in Bangkok were obtained from available web-based traffic information service. Traffic states were collected every 10 minutes interval for several months.Traffic states are modeled using Artificial Neural Network (ANN). Performance of the traffic state forecasting model was evaluated with real observations. *mongkut@siit.tu.ac.th INTRODUCTION Recent pervasiveness of smart phones and wireless communication services available in the city allows us to obtain traffic states (traffic condition) on roads in real time from probe vehicles whose drivers use smart phones. Using this kind of data, sometimes combining with data from other sources, several service providers offer real time traffic information on their online mapping service, to name a few, Google (maps.google.com), Microsoft (www.bing.com/maps/), Nokia (www.here.com), TomTom NV (maps.tomtom.com). This allow us to continually monitor traffic information of the road network, whole city, almost at no costs. This kind of information is very beneficial to developing countries such as Thailand where investment in conventional traffic sensors is still limited. Previous attempts to utilize this kind of data included Tostes et al. (2013) who used the traffic data from Bing Maps to investigate the city-wide traffic flows and also to predict the future traffic state on the road.

Morgul et al. (2014) reviewed methodology for web-based real-time data collection for transportation analysis.

By our observation, it is possible to obtain the updated information quite frequently such as every 5 or 10 minutes. The data become enormous when traffic information is continually collected from the whole city for some period of time. It is challenging how we could utilize this kind of data to understand the complex traffic phenomena in the city. Artificial Neural Network (ANN) is one of analytical techniques which can take advantage from enormous data while the relationships among them are complex, not well established or understood. This study attempts to investigate the performance of ANN to deal with the traffic state prediction problem. This study is in the preliminary state of research which scope of the study includes the prediction of the average traffic condition at city level during 4 periods. The periods of traffic state prediction are whole day average (7:00-20:00), morning average (7:00-10:00), noon average (10:00-15:00), and evening average (15:00-20:00).

207 | P a g e PROBLEM STATEMENT Traffic states may be influenced from many factors, such as recurrent pattern of travel activities of travelers in the city or external factors such as traffic accidents or weather. In this study only the historical records of traffic states were used while other information of external factors are neglected.

Therefore, the traffic state prediction problem is viewed as the time series prediction problem. Time series is a sequence of measurements of one or more visible variables resulting from an underlying dynamic system, whose state are change with time (Frank et al., 2001). The state of change theoretically can be continuously with time, such as the temperature. However in practice, it is convenience to sampling a series of discrete data points, equally spaced in time. Thus time series data in naturally temporal ordering. The problem of time series prediction is the used of model to predict future values based on past observed values. As a result, this can be state as finding a function f: RN→R to obtain a predicted value at time (t+d) using N time steps backward from current time t: x(t + d) = f [(x(t), x(t -1),..., x(t - N)] In this study, ANN was used to model the time series prediction problem with one step ahead prediction (the value d was set to 1).

DATA COLLECTION AND PREPARATION This study collected traffic states of urban road network in Bangkok from web-based traffic information services. Road network in Bangkok was marked at about every 200 meters, resulted in 15,559 artificial traffic sensors over the city. Traffic states were collected every 10 minutes during 7:00-20:00. The current study covers the period of 8 months (September 1, 2014 - April 30, 2015).

The congestion index of the whole city is calculated by averaging the indices obtained from all artificial sensors in the city. The data collected every 10 minutes are averaged over the city and over the period of prediction. The periods of traffic state prediction in this study includes whole day average (7:00-20:00), morning average (7:00-10:00), noon average (10:00-15:00), and evening average (15:00-20:00). Linear mapping was performed to present the traffic intensity in 0-10 scale (0 = lightest traffic and 10 = heaviest traffic). Figure 1 show the daily traffic intensity pattern of Bangkok during 8 months of the study period. There are two noticeable periods of uncommonly low intensity, the New Year (December 31th and January 1st) and the Songkran (Thai’s New Year) festival (April 13rd-15th).

Figure 1. Bangkok’s Traffic Intensity Patterns During the Period of Study ANN: NETWORK ARCHITECTURE AND TRAINING SCHEME Common three-layer feedforward type of network architecture was chosen for this study. A general process of feedforward neural network is to receive the input data from input layer, pass them into the

208 | P a g e hidden layer after multiplying by a weight. At a hidden neuron the inputs are summed up, associate it with a bias and then passes the results through a non-linear transfer function. Before its application, the network must be trained. At this stage the output from the network are compared to the desired target and the differences are minimized by adjusting the weights through specified algorithms.

Backpropagation training algorithm, namely, scaled conjugate gradient method , was used in this study. A set of N values of previous traffic states is denoted as the input layer and a single value output as a predicted value (Figure 2). The method is called sliding window technique as the input vectors (N time steps) slides over a full training set.

Figure 2. Network Architecture of the ANN in This Study ANN: RESULT OF TRAININGS AND MODEL VALIDATION After few trials, 7 input nodes in the input layer and 5 nodes in one hidden layer were applied in this study. To investigate the performance of the ANN model, data were randomly drawn to construct the models 10 times. For each run, training of the network was done with 70% of the whole data, while 15% were used for internal validation (to avoid over fitting). The rest 15% were used for external validation. Figure 3-6 show examples of result obtained from a single run of ANN (morning case).

Figure 3 shows comparison of predicted and observed results. Figure 4 shows the distribution of errors. Figure 5 shows the optimal number of trainings by internal validation process. Figure 6 shows result of the fitness of the model by using R-squared. This study utilized MATLAB as a computing tool in ANN modeling.

Figure 3. Comparison of Predicted and Observed Values of Traffic Intensity (Morning Model)

209 | P a g e

In order to obtain the statistics of the performances of the prediction models, each model was run 10 times as described earlier. Average results of the Mean Absolute Percentage Error (MAPE) and Rsquared of the external validations are shown in Table 1. On average, each model provided reasonable accuracy of predictions. MAPE is generally below 20% and R-squared are between 0.63-0.76.

210 | P a g e CONCLUSIONS This study presented the application of ANN to predict average the traffic state of the whole city.

Results from a case study using Bangkok’s traffic state data revealed that application of ANN can provide reasonable accuracy of prediction. This study only applied historical data of traffic states in the model. Further study is recommended to include additional information such as reports of traffic accidents and weather condition to the prediction model to further improve the model’s accuracy.

REFERENCES Tostes, A. I. J., de LP Duarte-Figueiredo, F., Assunção, R., Salles, J., Loureiro, A. A. (2013).

From data to knowledge: city-wide traffic flows analysis and prediction using bing maps. Proceedings of the 2nd ACM SIGKDD International Workshop on Urban Computing. !

Morgul, E., Yang, H., Kurkcu, A., Ozbay, K., Bartin, B., Kamga, C., & Salloum, R. (2014).

Virtual Sensors. Transportation Research Record: Journal of the Transportation Research Board, 2442, 106-116. doi:10.3141/2442-12

211 | P a g e Sustainable Construction Practice in Thailand via Service Life Design Somnuk Tangtermsirikul*,a, Chalermchai Wanichlamlerd b, Kritiya Kaewmanee b, Taweechai Samranwanich c a

School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University b Construction and Maintenance Technology Research Center, School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University c Department of Civil Engineering, Burapha University Keywords: Durability, Service life design, Carbonation, Steel corrosion, Sulfate attack

ABSTRACT Sustainable development concept for concrete structure construction via service life design in Thailand is described. Observation on previous and current practices showed lacks of attentions, leading to some low quality and short life structures. Problems are identified and solutions are proposed for all four practicing steps, i.e. 1) analysis and design, 2) materials 3) construction, and 4) maintenance, for partly achieving sustainable practices. This paper describes some efforts in the aspect of design. The new Thailand Standard launched by the Department of Public Works and Town & Country Planning is one of them. The concept covers design considering durability and service life. It was published for the first time in Thailand in 2009 and had its first revision in 2012.

The types of severe environment considered are chloride, carbonation and sulfate environments.

Design equations together with tables and charts were prepared to facilitate design process for practical engineers. *somnuk@siit.tu.ac.th INTRODUCTION To prolong the service life of structures and to properly use cement replacing materials are two of the components of sustainable construction practices. It has been reported that many low quality structures caused by inadequate attentions have been found in many countries including Thailand [1],[2]. There are still many opportunities to reduce burdens on environment by proper construction practices. Many countries have been spending big amount of money for maintaining their structures.

Those low quality structures were normally caused during one or more of the following 4 steps of practice; 1) analysis and design, 2) materials selection and specification, 3) construction and 4) maintenance. Since it is not possible to elaborate all issues in this paper, only some examples of efforts to establish good practices in Thailand for issues 1) and 2) will be briefly introduced.

SUSTAIABILITY PROBLEMS AND SOLUTIONS Inspection on deterioration of concrete structures in different areas in Thailand showed that various types of damages were observed. Structural damages caused by loading were found in some special cases where unexpected loads took place. Although some severe load-inducing structural damages call for immediate repairs, they were found in much smaller number of cases when compared with the damages due to durability problems. This fact highly emphasizes that the current practices are not

212 | P a g e sufficient to guarantee service life of structures and so leads to needs of huge amount of maintenance works. Therefore, it is obvious that practices which enable us to obtain durable structures should be employed as the standards of practice and the new standards should be applicable to various construction and environmental conditions.

A. Proposed Solutions The authors have been spending efforts to promote the sustainable construction practice in Thailand during the last two decades. The following practices are emphasized.

- practicing a design considering durability and service life - selecting proper materials and mix proportion of concrete - practicing good construction control and inspection - practicing good maintenance It is clear that to have good quality structures with satisfactory service life, all 4 mentioned steps of practice should not be ignored. Here, only some examples of efforts in Thailand in the step of design as well as material selection and specification will be elaborated. Without good practices in the first 3 steps, good quality structures cannot be obtained right after the construction. In addition, lack of proper maintenance also results in deterioration with the rate faster than expected. In many western countries, the ratios of budget used for maintaining structures to total construction budget are higher than 50%. In Thailand, the authors estimated the figure at approximately 30%. It has been observed that the maintenance budget in Thailand rarely moves along with the economic condition while the total construction budget usually does. Higher of this figure means there is less budget for new construction. Such situation is considered unacceptable for developing economies.

DESIGN SOLUTION: DURABILITY AND SERVICE LIFE DESIGN In this section, a concept of service life design, introduced by the Department of Public Works and Town and Country Planning of Thailand is briefly introduced. Most of the design equations, charts and tables were established based on the works done by the authorâ&#x20AC;&#x2122;s research group [3],[4],[5] together with those modified from some existing standards. Only three major deteriorations i.e. chloride-induced corrosion, carbonation and sulfate attack are briefly introduced here. The items concerned in durability design are based on selection of binders, and mix proportion in addition to the cover thickness, depending on the involved durability problems.

A. Steel corrosion due to Chloride In the design, the chloride content at the surface of reinforcing bars must be controlled to prevent initiation of steel corrosion as shown in Equation 1.

Cd â&#x2030;¤ Clim

(1)

where,

Clim : Critical chloride content that causes steel corrosion (see Table 1) C d : Chloride content at the surface of steel bars in concrete (%wt. of binder) coming from external environment, and can be estimated from

213 | P a g e

   c   C Cd  (C s  C0 ) 1  erf  0     2 Da t r  

(2)

where, c : Thickness of concrete cover (cm) Da :

Apparent chloride diffusion coefficient (cm2/year)

tr : Repair-free service life of concrete structure (year)

C s : Chloride content on the surface of concrete structure (%wt. of binder) (see Table 2) C0 : Initial chloride content at the surface of steel in concrete (%wt. of binder)

Table 1. Critical Chloride Content for Different Type of Binder Critical Chloride Content Type of Binder (%wt. of total binder) Cement (OPC) only

0.45

Cement (OPC) + Limestone powder (LP) - LP to total binder ratio is not more than 0.15

0.45

Cement (OPC) + Fly ash (FA) - FA to total binder ratio is less than 0.15

0.45

- FA to total binder ratio is not less than 0.15 but less than 0.35

0.35

- FA to total binder ratio is not less than 0.35 but not more than 0.50

0.30

For OPC+LP+FA, if LP to total binder ratio is less than 0.15, the values of OPC+FA shall be used.

Table 2. Chloride Content at the Surface of Concrete Structures ( C s ) Service Life(years) Distance from Seashore (m) Splash 0m 100 m 250 m 500 m 1000 m

10 0.971 0.299 0.135 0.061 0.041 0.040

20 30 40 Chloride content, C s (%wt. of concrete) 1.233 0.389 0.176 0.079 0.054 0.052

1.391 0.453 0.205 0.092 0.063 0.060

1.504 0.506 0.229 0.103 0.070 0.067

50 1.592 0.551 0.249 0.112 0.076 0.073

Figure 1 shows an example of the relationship between the apparent chloride diffusion coefficient of a non-cracked cement-only concrete and water to binder ratio.

214 | P a g e B. Carbonation Spalling of concrete cover due to carbonation is observed in many transportation infrastructures in Bangkok. Quality and thickness of concrete cover of these structures should be sufficiently provided to ensure that the corrosion of reinforcement due to carbonation does not take place during its designed repair-free service life. The thickness of concrete cover should not be smaller than the depth of carbonation during the designed repair-free service life (see Equation 3).

20 30 40 50

CO2 concentration (ppm)

10

of non-cracked

Chloride diffusion coefficient

Service life [yr]

Water to binder ratio (w/b)

High severity

Medium severity

Relative Humidity (%)

Figure 1. Relationship between Apparent Chloride Diffusion Coefficient of Uncracked Cement-Only Concrete and Water to Binder Ratio

Figure 2. Degree of Severity of Carbonation Environment

Xc ≤ c

(3)

where, c : Thickness of concrete cover (mm)

X c : Depth of carbonation (mm) which can be estimated from

X c  1 2 kt 0.5

(4)

where,

1 : Effect of rain subjection (see Table 3)

 2 : Effect of severity of the environment (see Table 4).

The severity of the carbonation environment is specified from average relative humidity and average CO2 concentration of the environment as in Figure 2. t is the repair-free service life (years). k is the carbonation coefficient which can be derived from

k  1.5k r (w / b) 3 where,

w / b : Water to binder ratio

(5)

215 | P a g e

k r : Effect of fly ash on carbonation coefficient (see Figure 3).

C. Sulfate Attack Different types of sulfate have different attacking mechanisms and the new code includes this fact.

Sodium sulfate causes expansion while magnesium sulfate mainly causes conversion and dissolution of calcium silicate hydrate with less expansion [5]. Different recommendations are given for each type of sulfate. It is noted that only a few codes have provided separate sulfate resisting specifications for different types of sulfate. Table 5 shows classification of level of severity of environment and recommended types of cementitious material and conditions of mix proportion for sodium sulfate resisting concrete. In case that fly ash is used to improve sodium sulfate resistance, the minimum amount of fly ash, which gives an equivalent expansion to that of the OPC 5 concrete, can be estimated from Figure 4. Table 6 shows classification of level of severity of magnesium sulfate environment and recommended types of cementitious material and conditions of mix proportion for magnesium sulfate resisting concrete.

Table 3. Effect of Rain Subjection on

Table 4. Effect of Severity of

Carbonation Depth Condition of Rain Subjection Can be wetted by rain or water

ď Ą1

Level of Severity of Carbonation Environment

ď Ą2

0.95

Typical

0.65

1.00

Medium Severity

0.85

High Severity

1.00

Fly ash

Minimum fly ash replacement

No possibility of rain/water subjection

Carbonation Environment

w/b 0.40 w/b 0.45 w/b 0.50 w/b 0.55

Fly ash replacement percentage CaO content of fly ash

Figure 3. Coefficient k r for Each Type of Fly Ash

Figure 4. Minimum Fly Ash Replacement Percentage to Obtain Equivalent NaSO4 Resistance to Type 5 Cement

216 | P a g e Table 5. Classification of Level of Severity of Sodium Sulfate Environment and Recommendation for Mixture Condition Concentration of Sulfate (SO42-) Level of

Soluble Sulfate

Recommended

Content in Soil

Binder Type

Max. w/b

In Water Severity (ppm) (% by weight of soil) Low

Less than 150

Less than 0.1

Medium

150-1,500

0.1-0.2

-

-

OPC 2 or OPC5 or 0.5 OPC 1 with pozzolan OPC5 or Severe

1,500 –10,000

0.2-2.0

0.45 OPC 1 with pozzolan

Very severe

Higher than 10,000

OPC 1 with pozzolan or Higher than 2.0

0.40 OPC 5 with pozzolan

Table 6. Classification of Level of Severity of Magnesium Sulfate Environment and Recommendation for Mixture Condition Level of Severity

Concentration of Sulfate

Low

Recommended Binder Type

Max w/b

Less than 300

-

-

Medium

300 – 1,000

OPC 1 or OPC 2 or OPC 5

0.5

Severe

1,000 – 3,000

OPC5

0.45

Very severe

Higher than 3,000

OPC 5

0.40

in Water (ppm)

CONCLUSION A concept of sustainable practice for concrete construction is described. As an example, the durability design concept discussed in this paper is an important component to obtain more sustainable infrastructure development. Only design for chloride-induced corrosion, carbonation-induced corrosion and sulfate resistance specification were introduced.

217 | P a g e ACKNOWLEDGEMENTS

The first author would like to acknowledge all his research colleagues as well as his former and present students for their contributions to the research outputs. Appreciations are also given to all sponsoring organizations for financial supports to the authorâ&#x20AC;&#x2122;s research group.

REFERENCES [1] Tangtermsirikul, S., Kaewkhluab, T., and Kaewmanee, K. (2003). Information acquisition and structural health monitoring of bridges in Bangkok. Proceedings of the Second International Symposium on New Technologies for Urban Safety of Mega Cities in Asia. 30-31 October 2003, Tokyo, Japan, pp.133-140.

[2] Tangtermsirikul, S. (2004). Durability design for concrete structures for urban safety in Thailand. Proceedings of the 3rd International Symposium of New Technologies for Urban Safety of Mega Cities in Asia, 18-19 October 2004, Agra, India, pp.177-184.

[3] Khunthongkeaw, J. and Tangtermsirikul, S. (2005). Model for simulating carbonation of fly ash concrete. ASCE Journal of Materials for Civil Engineering. 17(5), 570-578.

[4] Samranwanich, T. and Tangtermsirikul, S. (2004). A model for predicting time dependent chloride binding capacity of cement-fly ash binder system. Materials and Structures, RILEM. 37(270), 387-396.

[5] Krammart, P. and Tangtermsirikul, S. (2004). Expansion, strength reduction and weight loss of fly ash concrete in sulphate solution. ASEAN Journal on Science and Technology for Development. 21(4), 373-390.

218 | P a g e Deterioration of RC Structure due to Chlorine Attack: A Case Study Pakawat Sancharoen a,*, Tamyot Somyapakdeeb, Somnuk Tangtermsirikul a,c a

Construction and Maintenance Technology Research Center, Sirindhorn International Institute of Technology, Thammasat University b Repcon Service Company Limited c School of Civil Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University

ABSTRACT Reinforced concrete structure can be deteriorated by many mechanisms. Chlorine attack is one of them and rarely found. In Thailand, chlorine gas is used to disinfect potable water supply because of its low cost compared to other disinfectants such as ozone or chlorine dioxide. Due to its high oxidizing capacity, chlorine gas can be deteriorated RC structure. In this paper, a case study of chlorine gas attack is presented. It was observed that RC structure can be severely deteriorated due to chlorine gas by two major mechanisms. One is surface concrete erosion due to low pH of chlorine gas attacks concrete directly as acid attack. Second is corrosion of reinforcing steel due to chlorine gas transforming to chloride ion and diffusing into concrete. Repair and prevention methods are also proposed. *pakawat@siit.tu.ac.th Keywords: chlorine attack, corrosion, maintenance

INTRODUCTION Reinforced concrete (RC) structure can be deteriorated by many mechanisms. This paper presents one of rarely found deterioration mechanisms due to chlorine attack. Chlorine gas is still used in water chlorination in order to disinfect the water supply as well as sodium hypochlorite. However, chlorine gas is considered as strong oxidizing agent. When reacted with water, it will produce hypochlorous acid and hydrochloric acid as shown in eq.(1). The ratio between hypochlorous acid and hydrochloric acid will depend on pH of solution. And further hydrochloric acid will break to chloride ion and hydrogen ion as shown in eq.(2). Even the dosage of chlorine gas is very small in water disinfection process, due to no ventilation of structure, high concentration of acid and chloride ion can be condensate on concrete surface. As a result, damages of RC structure can be observed. In this study, inspection results and maintenance option of example deteriorated structure due to chlorine gas is presented.

Cl2  H 2O  HCl  HOCl

(1)

HCl  H   Cl 

(2)

219 | P a g e METHODOLOGY A. Target structure Target structures are RC clear water storage tank of water purification process which has around 40 years in service. Designed compressive strength of concrete is 250 ksc (cylinder). Chlorine gas is dosed to disinfect treated water before discharging. An overview of target structure is shown in Figure 1. As shown, water tank is fully enclosed and ventilation is very limited inside. Maximum water storage level is 0.50 m. below bottom surface of roof RC slab.

Figure 1: Target structure B. Inspection methods To identify cause of damage, firstly, visual inspection was conducted to record damage pattern including crack and spalling. Compressive strength was estimated by rebound hammer. Reinforcing steel covering depth was measured by covermeter. Chloride content profile of concrete was measured by collecting concrete powder sample by drilling at different depth from concrete surface. Then chloride content was measured by titration technique. Structure was inspected by divided to each member as roof slab, column, beam and wall. Five locations of high or low level of damage for each member were compared.

RESULTS AND DISCUSSION A. Visual inspection Result of visual inspection is shown in Figure 2. Locations of concrete cracking and spalling observed on bottom of roof slab were recorded. Please be noted that only half of structure was inspected due to a limitation of access. As shown, a number of crack and spalling was observed. Although, this structure is not exposed to marine environment of high carbonation concentration, corrosion cracking and spalling were observed.

220 | P a g e

Figure 2: Damage mapping B. Rebound number Concrete compressive strength was estimated by rebound number test at different structural members and level of damage. Result is shown in Table 1. As shown, rebound number value is not significantly different between high damage level and low damage level. For different structural members, wall and column showed a little lower rebound number. This may due to effect of moisture. Because wall and column have a section submerged in water. Therefore, moisture content of concrete is higher which causes lower rebound number. However, please be noted that damage location is concentrated only on surface of beam and roof slab. There is no damage observed on wall and column members.

Based on compressive strength conversion equation, strength of concrete is higher than designed strength at 250 ksc. Therefore, low compressive strength of concrete is not a cause of observed damage.

Table 1: Result of rebound number

C. Covering depth Reinforcing steel covering depth was measured and shown in Table 2. As shown, covering depth of reinforcing steel is higher than 40mm which is acceptable value in the past 40 years. However, comparison of covering depth between high and low damage level is shown that high damage level area has lower covering depth. Therefore, ability of steel corrosion protection is lower.

221 | P a g e Table 2: Result of covering depth

D. Chloride content Based on visual inspection result, corrosion of reinforcing steel can be one of causes of damage.

Therefore, chloride content was measured and shown in Table 3. It is shown that chloride content in concrete is high at the concrete surface and low at the inner part of structure. This shows that chloride penetrated in to concrete from the surface. Moreover, at depth of reinforcing steel around 4 cm, chloride content is higher than critical chloride content which is equal to 0.05% by weight of concrete, especially at the location with high level of damage. Please be noted that allowable chloride content in water stored in this structure is only 250ppm (0.025%) which is significantly lower than chloride content measured in concrete. Therefore, there is a chance that chloride ion from dosed chloride gas will concentrate on concrete surface, especially the structure that is not exposed to water such as concrete above the maximum storage water level.

Table 3: Result of chloride content in concrete

222 | P a g e CONCLUSION Corrosion of reinforcing steel due to chloride was evidence in water storage tank that limit chloride concentration in water only 250ppm. Inspection results show that chloride ion as a by-product from chlorine gas used in disinfection process condensed and concentrated on concrete surface, especially at location above maximum water level. This causes chloride ion penetration and initiated corrosion of reinforcing steel. In order to prevent this deterioration, ventilation to prevent condensed chloride ion should be conducted.

ACKNOWLEDGEMENTS The authors would like to thank the Center of Excellence in Material Science, Construction and Maintenance Technology, Thammasat University, and the Higher Education Research Promotion and National Research University Project, the Office of the Higher Education Commission of Thailand for their financial and scholarship support.

223 | P a g e Energy Dissipation Models for Computing Statistical-Based and Spectral-Based Root-Mean-Square Wave Heights Nga Thanh Duong*,a, Winyu Rattanapitikona, Tawatchai Tingsanchalib a

Civil Engineering Program, Sirindhorn International Institute of Technology, Thammasat University, Thailand b Asian Institute of Technology, Pathum Thani, Thailand Keywords: Energy dissipation, parametric wave model, root-mean-square wave height

ABSTRACT The energy dissipation due to wave breaking is an essential requirement for computing the transformation of root-mean-square ( rms ) wave height in the surf zone. The rms wave height can be classified according to its definition based to be statistical-based rms wave height ( H rmsW ) and spectral-based rms wave height ( H rmsE ). These two definitions of rms wave height are usually assumed to be equal. However, it was shown by many researchers that the rms wave heights derived from the two definitions are significantly different. No literature has described clearly the applicability of existing energy dissipation models in simulating H rmsW and H rmsE . Therefore, the main purpose of this study is to recalibrate and examine the applicability of ten existing models in simulating the transformation of H rmsW and H rmsE , and identifying the suitable ones. A wide range of experimental data was collected for calibration and examination of the models. The examination shows that the default coefficients in all models are not the optimal values for computing both of

H rmsW and H rmsE . The optimal coefficients for computing H rmsW are different from those for computing H rmsE . The top four models with optimal coefficients for computing H rmsW and H rmsE are recommended to be used. *dthnga5890@gmail.com INTRODUCTION The transformation of root-mean-square ( rms ) wave heights has been a subject of study for decades because of its importance in studying beach deformations and the design of coastal structures.

Common methods to model rms wave height ( H rms ) transformation can be classified into four main approaches, i.e. representative wave approach, spectral approach, probabilistic approach, and parametric wave approach. The parametric wave approach is widely used for computing the transformation of H rms , especially in commercial and free software. Hence, only the parametric wave approach is considered in this study. In the parametric wave model, the energy dissipation due to wave breaking is an essential requirement for computing the H rms in the surf zone. In the past decades, various energy dissipation models have been proposed based on the parametric wave approach. Because of the complexity of the wave breaking mechanism, most of the energy dissipation models were developed based on the empirical or semi-empirical approach calibrated with the measured H rms . However, there are two approaches to describe the H rms , i.e. statistical approach (or

224 | P a g e wave-by-wave approach) and spectral approach (or energy approach). Therefore, the H rms can be classified according to its definition based to be statistical-based rms wave height ( H rmsW ) and spectral-based rms wave height ( H rmsE ). These two H rms are normally assumed to be equal.

However, it was shown by many researchers that the H rms derived from the two definitions are significantly different. It is important for engineers to understand which definition of H rms they are using in the model and which model is suitable in computing H rmsW or H rmsE . No literature has described clearly the applicability of existing energy dissipation models in simulating H rmsW and

H rmsE . Therefore, the main purpose of this study is to examine the applicability of the existing models in simulating the transformation of H rmsW and H rmsE , and identifying the suitable ones.

PARAMETRIC WAVE MODEL The parametric wave models are generally based on the work of Battjes and Janssen (1978). The approach relies on the macroscopic features of breaking waves and predicts only the transformation of

H rms . The rms wave height transformation is computed from the energy flux conservation law as: 2 g  H rms c g cos     DB 8 x

(1)

where H rms is the rms wave height,  is density of water, g is gravity acceleration, c g is the group velocity, x is the distance in cross shore direction,  is the wave angle, and DB is the energy dissipation rate due to wave breaking.

The selected ten existing DB models (based on the parametric wave approach) are shown in Table 1 together with their abbreviations. Brief reviews of the models were presented in the paper of Rattanapitikon and Shibayama (2010). It can be seen from Table 1 that the majority of existing models represent a relationship between the energy dissipation rate ( DB ) and the variables at the deepwater or local conditions, i.e. deepwater rms wave height ( H rmso ), deepwater wavelength ( Lo ), spectral peak period ( T p ), rms wave height ( H rms ), water depth ( h ), fraction of wave breaking ( Qb ), breaker height ( H b ), wave number ( k ), wavelength ( L ), and bottom slope ( mb ).

Pre-calibrations were performed to investigate the effect of coefficients on the accuracy of the models.

The pre-calibrations reveal that only one coefficient has significant effect on the transformation of different H rms . Therefore, only a coefficient ( K ) in each DB model (shown in Table 1) was introduced as the coefficient for adjusting the effect of the transformation of different H rms .

225 | P a g e Table 1 Selected energy dissipation models for parametric wave approach Sources: Abbreviation s Battjes and Janssen (1978): BJ78 Thornton and Guza (1983): TG83 Battjes and Stive (1985): BS85 Southgate and Nairn (1993): SN93 Baldock et al. (1998): BHV98

Models

DB  Qb

 gH b2 4T p

3  DB  0.51 4

DB  Qb

DB  Qb

1  Qb  H rms   lnQb  H b

 H rms   H  b

 gH b2 4T p

 gH b3 4T p h

;

   

2

 1 1  2  1  H rms H b 



1  Qb  H rms ;   lnQb  H b

;

1  Qb  H rms   lnQb  H b

Alsina and Baldock (2007): AB07

Janssen and Battjes (2007): JB07

2

  H   ; H b  Kh0.39  0.56 tanh  33 rmso  Lo    2







for H rms  H b for H rms  H b



for H rms  H b

  H 3 Hb     exp   b   4T p h  H rms  2 H rms    H rms   

 H  3 3  gH rms  b

  

   

2

 3   H   1  erf  b 4   H rms  

    ;   

   

2

 3   H   1  erf  b  4   H rms 

    ;   

    kh    

  H 3 Hb     exp   b  2 H rms    H rms   

  H H b  Kh0.39  0.56 tanh  33 rmso Lo  

; H b  KL tanh0.86kh  0.33kh

for H rms  H b

 H  3 3  gH rms  b

 4T p h  H rms 

  

;

  H  H b  KL tanh 0.57  0.45 tanh 33 rmso Lo   

DB 

    kh    

  

   H 2   g H 2  H 2 b rms exp   b      H rms   4T p  DB    2  gH b2   exp 1 4T p 

DB 



 gH 3 rms ; H b  Kh 2.5   4T p h

  H    ; H b  KL tanh 0.57  0.45 tanh  33 rmso  Lo    

   H 2   g H 2  H 2 b rms exp   b      H rms   4T p  DB    2  gH b2   exp 1 4T p    H H b  Kh0.39  0.56 tanh  33 rmso Lo  

Ruessink et al. (2003): RWS03

2

  ; H b  KL tanh 0.91kh   

226 | P a g e Rattanapitiko n and Sawanggun (2008): RS08

Apotsos et al. (2008): AREG08

DB 

gH b2 

2

H 2.096 rms  H   b 

4T

 H   1.601 rms   H   b

  H  H b  KL tanh 0.57  0.45 tanh  33 rmso Lo   

DB 

3  4

 H rms   H  b

   

2

 1  1  2   1  H rms H b 





    0.293 ;    

    kh     3   gH rms ;  2.5 4T p h  

H b  K[0.18  0.40 tanh(0.9H rmso )]h

EXAMINATION OF EXISTING MODELS Experimental data from 5 sources for H rmsW (283 cases) and 8 sources for H rmsE (1709 cases) were collected for examination of the existing models. The collected experimental data cover a wide range of wave and bottom topography conditions, including small-scale, large-scale and field experiments.

Summary of the collected experimental results of H rmsW and H rmsE are shown in Table 2.

Table 2 Summary of collected experimental data of H rmsW and H rmsE Experimental data of H rmsE

Experimental data of H rmsW Sources: Apparatus

No. of Sources: Apparatus cases

Smith and Kraus (1990): S Ting (2001): S

cases

12 Smith and Vincent (1992): S 1 Hamilton and Ebersole (2001): S

Kraus and Smith (1994): L

128 Smith and Seabergh (2001): S

Dette et al. (1998): L

138 Kraus and Smith (1994): L

Thornton and Guza (1986): F

No. of

4 Roelvink and Reniers (1995): L

4 1 11 128 95

Dette et al. (1998): L

138

Birkemeier et al. (1997): F

745

Herbers et al. (2006): F

587

Total 283 Total Remarks: S = small-scale, L = large-scale, F = field.

1,709

The basic parameter for determination of the overall accuracy of a model is the average rms relative error ( ERavg ), which is defined as:

227 | P a g e

3

ERavg   ERgj 3 ;

in which ER g  100

j 1

ng

 ( H ci  H mi ) 2 i 1

ng

H i 1

2 mi

(2)

where ER gj is the rms relative error of the data group j (the collected experiments are separated into 3 groups according to the experiment scale, i.e. small-scale, large-scale, and field experiments), i is the wave height number, H ci is the computed wave height of number i , H mi is the measured wave height of number i , and ng is the total number of measured wave heights in each data group.

It is expected that a good model should be able to predict well for the three groups of experiment scale and for H rmsW and H rmsE . Therefore, the average error from the six groups ( ERall ; three groups of experiment scale and two groups of H rms ) are used as a main criteria to examine the models.

The errors ( ERavg and ERall ) of each dissipation model for computing H rmsW and H rmsE by using default coefficients are shown in the first five columns of Table 3. However, because most models were developed without considering the difference between H rmsW and H rmsE , the coefficients in all models should be recalibrated before verifying the applicability of the models.

Table 3 The errors ( ERavg and ERall ) of each dissipation model for computing H rmsW and H rmsE by using default and calibrated coefficients default coefficients Model

calibrated coefficients

K

HrmsW

HrmsE

ERall

K

HrmsW

K

HrmsE

ERall

BJ78

0.14

14.4

13.1

13.8

0.14

14.4

0.12

11.8

13.1

TG83

0.42

18.8

10.6

14.7

0.50

18.1

0.44

10.5

14.3

BS85

0.14

11.2

8.7

10.0

0.15

10.7

0.14

8.7

9.7

SN93

1.0

16.2

11.8

14.0

1.0

16.2

0.85

9.8

13.0

BHV98

1.0

13.2

9.7

11.5

1.1

13.2

1.0

9.7

11.4

RWS03

0.14

14.3

10.2

12.2

0.16

13.7

0.15

9.8

11.8

AB07

0.14

11.2

9.0

10.1

0.15

10.9

0.13

8.9

9.9

JB07

1.0

11.4

9.3

10.3

0.99

11.4

0.93

8.7

10.0

RS08

0.14

10.4

8.9

9.7

0.15

10.4

0.14

8.9

9.7

1.0

20.0

10.9

15.5

1.5

19.0

0.96

10.8

14.9

AREG08

228 | P a g e MODEL CALIBRATION AND COMPARISON The measured data shown in Table 2 are used to calibrate the coefficients ( K ) for H rmsW and H rmsE .

The calibrations are conducted by gradually adjusting the coefficients K until the minimum error (

ER avg ) of each model is obtained. The calibrated coefficients and errors ( ERavg and ERall ) of each dissipation model for H rmsW and H rmsE are shown in the last five columns of Table 3. The results from Table 3 can be summarized as follows: (a) The optimal coefficients ( K ) of all models for H rmsW are larger than those for H rmsE . (b) After calibration, the overall accuracy of the models for computing H rmsW and H rmsE are slightly improved. Therefore, it is possible to use the same coefficient for computing both of H rmsW and H rmsE . However, as H rmsW is different from H rmsE , it is more suitable to use the different coefficients. (c) The overall errors ( ERall ) of the models for computing H rmsW and H rmsE in ascending order are the models of RS08, BS85, AB07, JB07, BHV98, RWS03, SN93, BJ78, TG83, and AREG08. The top four models (RS08, BS85, AB07, and JB07), which give nearly the same overall accuracy ( 9.7  ERall  10.0 %), are recommended to be used for computing H rmsW and H rmsE .

CONCLUSIONS This study is undertaken to find out the suitable dissipation models, which can be used to compute

H rmsW and H rmsE for a wide range of experimental conditions. The transformation of H rmsW and H rmsE were computed from the energy flux conservation law based on the parametric wave approach.

Ten existing dissipation models were recalibrated and examined their applicability in computing

H rmsW and H rmsE . The optimal coefficients ( K ) of all models for H rmsW are larger than those for H rmsE . The top four models, which give nearly the same overall accuracy (9.7  ERall  10.0%), are recommended to be used for computing H rmsW and H rmsE .

REFERENCES Alsina, J.M. and Baldock T.E. (2007) Coast. Eng. 54, 765-769.

Apotsos, A., Raubenheimer, B., Elgar, S. and Guza, R.T. (2008) Coast. Eng. 55, 224-235.

Baldock, T.E., Holmes, P., Bunker, S. and Van Weert, P. (1998) Coast. Eng. 34, 173-196.

Battjes, J.A. and Janssen, J.P.F.M. (1978) Proc. 16th Conf. on Coast. Eng., pp. 569-587.

Battjes, J.A. and Stive M.J.F. (1985) J. Geophys. Res. 90 (C5), 9159-9167.

Birkemeier, W.A., Donoghue, C., Long, C.E., Hathaway, K.K., and Baron, C.F. (1997). The DELILAH Nearshore Experiment, U.S. Army Corps of Engineers.

Dette, H.H, Peters, K. and Newe, J. (1998). MAST III – SAFE Project: Large Wave Flume Experiments ‘96/97, Leichtweiss-Institute, Technical University Braunschweig.

Hamilton, D.G. and Ebersole, B.A. (2001) Coast. Eng. 42, 199–218.

229 | P a g e Herbers, T.H.C., Elgar, S., Guza, R.T. and O’Reilly, W.C. (2006). DUCK94 Experiment Data Server: SPUV Pressure Sensor Wave Height Data, Available online at: /http://dksrv.usace.army.mi/jg/dk94dirS. Janssen, T.T. and Battjes, J.A. (2007) Coast. Eng. 54, 711-716.

Kraus, N.C. and Smith, J.M. (1994). SUPERTANK Laboratory Data Collection Project, U.S. Army Corps of Engineers.

Rattanapitikon, W. and Sawanggun, S. (2008) Songklanakarin J. Sci. Technol. 30, 333-341.

Rattanapitikon, W. and Shibayama, T. (2010) Coast. Eng. J. 52, 305-330.

Roelvink, J.A. and Reniers, A.J.H.M. (1995). LIP 11D Delta Flume Experiments: A Data Set for Profile Model Validation, Delft Hydraulics.

Ruessink, B.G., Walstra, D.J.R. and Southgate, H.N. (2003) Coast. Eng. 48, 139-149.

Southgate, H. N. and Nairn, R. B. (1993) Coast. Eng. 19, 27-56.

Smith, J.M. and Kraus, N.C. (1990). Laboratory Study on Macro-features of Wave Breaking over Bars and Artificial Reefs, U.S. Army Corps of Engineers.

Smith, M.K. and Vincent, C.L. (1992) J. Waterw. Port Coast. Ocean Eng. 118 (5), 517–533.

Smith, J.M. and Seabergh, W.C. (2001). Wave Breaking on a Current at an Idealized Inlet with an Ebb Shoal, U.S. Army Engineer Research and Development Center.

Thornton, E.B. and Guza, R.T. (1983) J. Geophys. Res. 88 (C10), 5925-5938.

Thornton, E.B. and Guza, R.T. (1986) J. Phys. Oceanogr. 16, 1165-1178.

Ting, F.C.K. (2001) Coast. Eng. 43, 183-208.