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Volume 1908

8th International Conference Conference collection on Global Resource Conservation (ICGRC 2017) Conference collection Green Campus Movement for Global Conservation

Malang, Indonesia 19–20 July 2017

Editors Widodo, Chandrakhant Salunkhe, Akira Kikuchi, Bhupal Govinda, Yoga Dwi Jatmiko and Dian Siswanto

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8th International Conference on Global Resource Conservation (ICGRC 2017)

Vol. 1908

17-11-2017 09:44:23



8th International Conference on Global Resource Conservation (ICGRC 2017) Green Campus Movement for Global Conservation Malang, Indonesia


19–20 July 2017

Editors Widodo

University of Brawijaya, Malang, Indonesia

Chandrakhant Salunkhe

Krishna Mahavidyalaya, Maharashtra, India

Akira Kikuchi

University of Technology, Johor, Malaysia

Bhupal Govinda

Kathmandu University, Khulikhel, Nepal, India

Yoga Dwi Jatmiko Dian Siswanto

University of Brawijaya, Malang, Indonesia

All papers have been peer reviewed.

Melville, New York, 2017 AIP Conference Proceedings

Volume 1908

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Editors Widodo

University of Brawijaya Biology Department Faculty of Mathematics and Natural Science Veteran Street Malang, East Java 65145 Indonesia Email: widodo@ub.ac.id

Chandrakhant Salunkhe

Yoga Dwi Jatmiko Dian Siswanto

University of Brawijaya Biology Department Faculty of Mathematics and Natural Science Veteran Street Malang, East Java 65145 Indonesia Email: jatmiko_yd@ub.ac.id diansiswanto@ub.ac.id

Krishna Mahavidyalaya, Rethare Bk. Post Graduate Center of Botany Shivnagar, Maharashtra 415108 India Email: Chandrakantsalunkhe62@gmail.com

Akira Kikuchi

University of Technology, Malaysia Department of Environmental Engineering Skudai, Johor, 81310 Malaysia Email: akira@utm.my

Bhupal Govinda

Kathmandu University Department of Biotechnology Block 07, School of Science GPO Box 6250 Dhulikhel, Nepal India Email: bgs@ku.edu.np

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AIP Conference Proceedings, Volume 1908 8th International Conference on Global Resource Conservation (ICGRC 2017) Green Campus Movement for Global Conservation Table of Contents Preface: The 8th International Conference on Global Resource Conservation (ICGRC 2017) 010001 Conference Photo: The 8th International Conference on Global Resource Conservation (ICGRC 2017) 010002 ZOOLOGY A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources Gatot Ciptadi, M. Nur Ihsan, Sri Rahayu, D. H. K. Widjaja, and Mudawamah Mudawamah

020001

From little known area to the extinction race: A survey of herpetofauna in Prevab, Kutai National Park (KNP), Indonesia Nia Kurniawan, Noviati Roziah, Muhammad Alif Fauzi, and Agung Sih Kurnianto

020002

ECOLOGY The effect of organic farming systems on species diversity Amin Setyo Leksono

030001

Farmers’ perception of the role of some wild plants for the predatory coccinellidae (Adalia bipunctata L and Coccinella septempunctata L) in developing refugia in the agricultural field Bagyo Yanuwiadi

030002

The improvement of the quality of polluted irrigation water through a phytoremediation process in a hydroponic batch culture system Catur Retnaningdyah

030003

Green technology innovation in a developing country Chairat Treesubsuntorn, Rujira Dolphen, Prapai Dhurakit, Dian Siswanto, and Paitip Thiravetyan

030004

Important value index and biomass (estimation) of seagrass on Talango Island, Sumenep, Madura Citra Satrya Utama Dewi and Sukandar

030005

A study on the utilization of forest policy to review from the aspect of climate change I. Putu Gede Ardhana

030006

The implementation of biological monitoring working party average score per taxon (BMWP-ASPT) in a water quality analysis at Kalibokor drainage in Surabaya region Kristiandita Ariella and Atiek Moesriati

030007

Managing biodiversity for a competitive ecotourism industry in tropical developing countries: New opportunities in biological fields Luchman Hakim

030008

Skeleton microstructure of Porites lutea in Kondang Merak, Malang, East Java Oktiyas Muzaky Luthfi, RM. Agung M. Rizqon Sontodipoero, Andik Isdianto, Daduk Setyohadi, Alfan Jauhari, and I. Nyoman Januarsa

030009


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Copper (Cu) content in Porites lutea at South Java Sea: Case study at Pantai Kondang Merak, Malang, Indonesia Oktiyas Muzaky Luthfi, Sigit Rijatmoko, Andik Isdianto, Daduk Setyohadi, Alfan Jauhari, and Ali Arman Lubis

030010

The effectiveness of leachate remediation in the implementation of unvegetated constructed wetland Sophia Laily, Catur Retnaningdyah, and Bagyo Yanuwiadi

030011

Geo Techno Park potential at Arjuno-Welirang Volcano hosted geothermal area, Batu, East Java, Indonesia (Multi geophysical approach) Sukir Maryanto

030012

Study of Peak Ground Acceleration (PGA) by means of microzonation data: Case Study on Batubesi Dam of Nuha, East Luwu, South Sulawesi, Indonesia Sunaryo 030013 Integrating between Malay culture and conservation in Green campus program: Best practices from Universitas Riau, Indonesia Suwondo, Darmadi, and Mohd. Yunus

030014

BOTANY Design and construction of a vertical hydroponic system with semi-continuous and continuous nutrient cycling Dian Siswanto and Wahyu Widoretno

040001

Exotic plant species attack revegetation plants in post-coal mining areas Muhammad Yusuf and Endang Arisoesilaningsih

040002

Preliminary study: Genetic population of Calcinus elegans in the South coast of Java Island based on sequence COI Gene Muliawati Handayani and Imai Hideyuki

040003

The cutting effect of male flower on the size of the fruit cob, the size of the fruit and seeds in Porang (Amorphophallus muelleri Blume) Nunung Harijati, Hikma Isnailul Navisya, and Ying Diao

040004

Traditional pattern of cashew cultivation : A lesson from Sumenep-Madura, Indonesia Nurul Jadid, Sutikno, Dyah Santhi Dewi, Tutik Nurhidayati, Nurlita Abdulgani, Farid Kamal Muzaki, Byan Arasyi Arraniry, Rizal Kharisma Mardika, and R. Yuvita Rakhman

040005

The effect of tomato juices and bean sprout extracts on vitro shoot regeneration of Physalis angulata L. Retno Mastuti, Aminatun Munawarti, and Mufidatur Rosyidah

040006

The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. – A medicinal plant Retno Mastuti, Aminatun Munawarti, and Elok Rifqi Firdiana

040007


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Secretory structure and histochemistry test of some Zingiberaceae plants Serafinah Indriyani

040008

Response morphology and anatomy of tobacco (Nicotiana tabacum L.) plant on waterlogging Tutik Nurhidayati, Selfrina Puri Wardhani, Hery Purnobasuki, Sucipto Hariyanto, Nurul Jadid, and Desy Dwi Nurcahyani

040009

The phenetic relationships of Amorphophallus sp. Based on their morphological characteristics in Laren subdistrict, Lamongan Regency Gustini Ekowati, Praptomo Dwi W., and Rodliyati A.

040010

BIOTECHNOLOGY Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease Anggia N. Ramadhani, Sapti Puspitarini, Anissa N. Sari, and Widodo

050001

Genetic variability of Indonesian local chili pepper: The facts Estri Laras Arumingtyas, Joni Kusnadi, Dewi Ratih Tirto Sari, and Nursita Ratih

050002

The effect of temperature and Pasteurization time on Staphylococcus aureus isolates from dairy products Maria Nia Yaniarti, Charis Amarantini, and Tri Yahya Budiarso

050003

PHYLOGEOrec: A QGIS plugin for spatial phylogeographic reconstruction from phylogenetic tree and geographical information data Maulana Malik Nashrulloh, Nia Kurniawan, and Brian Rahardi

050004

Potency of Bacillus thuringiensis isolates from bareng Tenes-Malang City as a biological control agent for suppressing third instar of Aedes aegypti larvae Nihayatul Lutfiana and Zulfaidah Penata Gama

050005

Utilization of ultrasonic waves (Acheta domesticus) as a biocontrol of mosquito in Malang Agricultural Institute Sama’ Iradat Tito

050006

Molecular detection of Staphylococcus aureus resistant to temperature in milk and its products Stephani Valentina Harda Sutejo, Charis Amarantini, and Tri Yahya Budiarso

050007

Assessment of probiotic properties of lactic acid bacteria isolated from Indonesian naturally fermented milk Yoga Dwi Jatmiko, Gordon S. Howarth, and Mary D. Barton

050008

Species composition of mosquito and public perception about Dengue vector of hemorrhagic fever in Bareng Tenes Malang Zulfaidah Penata Gama and Jenvia Rista Pratiwi

050009

BIOMEDICINE Influence of CSN1S2 protein from Caprine milk Etawah Breed (EB) on histology of microglial cells in rat (Rattus norvegicus) Type-2 diabetes mellitus (T2DM) Margareth Rika and Fatchiyah

060001


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Comparative method of protein expression and isolation of EBV epitope in E.coli DH5α Nadya V. M. Anyndita, Nurul Dluha, Karimatul Himmah, Muhaimin Rifa’i, and Widodo

060002

Tapak liman (Elephantopus scaber L) extract–induced CD4+ and CD8+ differentiation from hematopoietic stem cells and progenitor cell proliferation in mice (Mus musculus L) Muhammad Sasmito Djati, Hindun Habibu, Nabilah A. Jatiatmaja, and Muhaimin Rifa’i

060003

BIOLOGY EDUCATION Field study learning model to introduce environmental health problems to medical students at the faculty of medicine, University of Brawijaya, Malang, Indonesia Lilik Zuhriyah, Nanik Setijowati, and Sri Andarini

070001


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PREFACE: The 8th International Conference on Global Resource Conservation (ICGRC 2017) Environmental issue is a popular topic in many countries. Academic communities give a huge concern to this issue by providing green technology to recover natural resources. In Indonesia, the governmental and private universities have collaborated to enhance the green products in order to have a better life. Some universities have a focus on the green campus program such as save energy, paperless, and zero-waste program. Sustainability of the program is a major concern to maintain the environment. This collaboration may encourage any researchers from multidiscipline area to have a collaboration. Merging basic and applied sciences to solve complex environmental issue is one of the important agenda for accelerating the contribution to society. Scientific meeting for facilitating the expert from biology, biotechnology, ecology, agriculture, and biophysics is one of strategy to overcome the recent issue in environments. The 8th International Conference on Global Resource Conservation (ICGRC 2017) with theme Green Campus Movement for Global Conservation is a consolidation effort to provide scientific forum for biologist from Indonesia and abroad to share their research interest related to environmental issues. Besides, it aims to support the green campus program in Indonesia. The editors proudly present the selected papers of ICGRC 2017. By publishing these papers, we hope to give a contribution to solve any environmental issues. Furthermore, we would like to encourage people to start a green lifestyle by using green products as well as promote green campus program. This movement will give a positive impact for global conservation. Malang, 23 October 2017

Nia Kurniawan, Ph.D. Chairman of ICGRC 2017

8th International Conference on Global Resource Conservation (ICGRC 2017) AIP Conf. Proc. 1908, 010001-1–010001-1; https://doi.org/10.1063/1.5012697 Published by AIP Publishing. 978-0-7354-1600-0/$30.00

010001-1


010002-1

A Feasibility Study of Prepubertal and Over Mature Aged Local Goat in Relation to Results of In Vitro Growth Culture to Obtain Additional M-II Oocyte Resources Gatot Ciptadi1,a), M. Nur Ihsan1), Sri Rahayu2), D. H. K. Widjaja3) and Mudawamah Mudawamah4) 1

2

Faculty of Animal Husbandry, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 3 Faculty of Animal Husbandry, Kanjuruhan University, Malang 4 Faculty of Animal Husbandry, Islamic University of Malang, Malang a)

Corresponding author: ciptadi@ub.ac.id

Abstract. The aims of this research are to study the potential source of mature (M-II) oocytes of domestic animals using follicles isolated from prepubertal and over mature aged Indonesian local goats, resulting from an in vitro growth (IVG) method. This method of IVG could provide a new source of M-II oocytes for embryo production. In Indonesia, a very limited number of a good quality oocytes are available for research purposes, as there is a limited number of reproductive females slaughtered, which is dominated by prepubertal and old mature aged animals. IVG culture systems could be improved as an alternative method to provide a new source of a good quality oocytes for in vitro maturation of M-II oocytes. From a number of prepubertal and mature aged goats slaughtered in a local abattoir, the small oocytes in the preantral follicles were cultured in vitro to normal oocyte growth. The methods used in this research are experimental. Follicles were isolated, cultured in vitro for 14 days individually using a sticky medium containing 4% (w/v) polyvinylpyrrolidone in TCM 199 10% Fetal Bovine Serum supplemented with Follicle Stimulating Hormone, which was then evaluated for their follicle development and oocyte quality. The research results showed that a minimum follicle size and oocyte diameter is needed (>100 um) for early evaluation of maturation to be achieved, meanwhile oocytes recovered from IVG after being cultured in vitro for maturation resulted in a very low rate of maturation. However, in the future, IVG of the preantral follicles of Indonesian local goat could be considered as an alternative source of oocytes for both research purposes and embryo production in vitro.

INTRODUCTION The animal ovary contains a huge number of small follicles of various sizes and shapes,with each follicle enclosing a small immature oocyte. In vitro growth (IVG) culturing of preantral follicles and small oocytes will provide a new source of mature oocytes (M-II). In the field of livestock production, IVG of small ovarian oocytes will provide a large number of mature oocytes. Using an IVG culture system, non-growing mammalian oocytes in primordial follicles could possibly grow to their final sizes and acquire full developmental competence. However, the IVG system for domestic species, in which follicles developed to a much larger size requires a longer period of time, has to be established especially for local goats. A small number of oocytes grow from a minimum diameter size of 30 μm to the final size of 120–125 μm. Among large animals, it has been reported that offspring were produced from ovarian oocytes by IVG culture 1. Porcine and bovine oocytes do not complete their growth in the early antral follicle until they develop into the late antral stage with a diameter of about 5 mm 2. The follicle size and stage in which oocyte growth is completed differ among species. The local goats slaughtered at this area of Malang, Indonesia were dominated by a younger female or prepubertal animals and old mature aged goats.


020001-1

The aims of this research are to study the potential source of an oocyte of Indonesian local domestic animals, namely the Etawah crossing grade goat, of prepubertal and old mature aged goats using an IVG method. If the small oocyte in the preantral follicles could be well cultured in vitro, similar to normal oocyte growth in vivo, it will provide a new source of M-II oocytes for embryo production. Recently, a very limited number of a good quality oocytes became available in our laboratory in Indonesia because of a small number of females slaughtered locally, which were dominated by prepubertal and old mature aged goats. So far for research purposes, the oocytes were isolated by aspiration technique of the antral follicles. Meanwhile, IVG systems for culturing preantral follicles and growing small sized oocytes inside could be improved as an alternative source of oocytes both for research purposes, as well as in vitro culture system research and development.

EXPERIMENTAL DETAILS The goat ovaries were collected from a local slaughterhouse in Malang city. Preantral follicles containing small growing oocytes were used as the material for IVG culture. Follicles with the diameter of 2.0–3.0 mm were isolated and selected using a micro-dissecting method 3. Preantral follicles of different groups of aged (prepubertal and old mature aged) goat were cultured individually for 14 days in 20 ul drops of sticky medium containing 4% polyvinylpyrrolidone 4 in TCM 199, supplemented with 10% heat activated Fetal Bovine Serum (FBS), 10% follicular fluid, 0.1 IU/ml Follicle Stimulating Hormone (FSH), pen-strep, under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37 oC. The follicle sizes cultured were classified and selected for a small category (2.0–3.0 mm of diameter). Evaluation of follicle development was done under TV monitor (scaled and calibrated) connected to an inverted microscope (Olympus). The variables observed included the number follicles obtained per ovary, morphology and size of an ovary, oocyte diameter size and quality of oocytes before and after IVG (μm). Data were analyzed descriptively for morphological and t-test analysis for a comparison before and after IVG culture.

RESULT AND DISCUSSION Preantral Follicle

Recovered

The potential of preantral follicles isolated from the local goat is relatively very low (TABLE 1). The follicle sizes recovered from the ovaries of goats is considered as having a large variation in follicle number obtained per ovary, which might be the resultof random sampling and the limited number of goats slaughtered and isolated from their ovaries. Among these local goats, there are different sizes of ovaries with different numbers of follicles being recovered. TABLE 1.Preliminary result describes the potential of IVG result of the Indonesian local goat of PE breed in alocal slaughtered house in Malang.

No 1 2

Group of ages and number of the ovary (n). Prepubertal Goat (80) Over Matured Goat (78)

Follicle phase

Follicle obtained/ovaries.

Pre antral Pre antral

5.6 + 2.3 11.4 + 3.2

These results, based on the follicles obtained (TABLE 2) may clearly demonstrate the potential of IVG culture systems as an alternative source of matured oocytes because of limited data. However, this method is necessary to develop for both potential animal livestock production and research in relation to providing more recipient oocytes, for example for nuclear transfer purposes. The ovary of the cow contains a huge number of non-growing and growing oocytes. Approximately 10–000 primordial follicles are contained in the cow ovary, with 300 of them developing to follicles 1,5. A huge number of small oocytes are contained in the ovary of a cow. A small number of them grow from the minimal size of 30 um in diameter to the final size of 120–125 μm, then mature and are ovulated 3. A large number of the remaining oocytes do not enter the growth phase or degenerate in the ovary. The potential number of these local goat oocytes could be managed. In general, oocyte grows ranges from 30 μm to 120–125 μm to reach maturity, and a baby calf has been successfully produced from oocytes grown from 90–99 μm in diameter. If such small oocytes in the ovary could be

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better managed for growth, they will provide a new potential source of mature oocytes for recipient cells for nuclear transfer or animal in vitro fertilization programs 1. Bovine follicles with a diameter of 0.2–0.3 mm containing 70–90 μm diameter oocytes, have been cultured in the sticky medium of TCM 199 for two weeks and then evaluated for oocytes. Almost all the oocytes recovered were enclosed by compact granulosa cells. The oocytes recovered were further cultured for maturation in the different treatment of FBS in TCM 199 stock. A comparison of two different age level (prepubertal and old mature aged) goat showed that there are important characteristic differences between prepubertal and older goats (TABLE 2). TABLE 2.Different character morphometric of theovary of different level of age of local goat.

No 1. 2. 3.

Parameters Length (mm) Shape (mm) Ovary volume size (mm2)

Prepubertal age 13.14 + 2.40 9.14 + 1.90 97.56 + 32.40

Over matured age of goat 20.66 + 2.40 13.80 + 2.70 226.49 + 56.80

Oocyte Growth and Quality after IVG A comparison between oocytes before and after IVG are presented in TABLE 3. IVG of the preantral follicle of the local goat could be considered as an alternative oocyte source for research in the near future based on the developing oocyte capacity. The final size of the oocytes after IVG reaches about the same size approximately as the matured oocyte, but further information about the proper maturity of these cells is required. Preantral follicles from goats of diameter 0.2–0.3 mm were cultured for 2 weeks, then evaluated for the diameter of oocytes and their morphological quality of cumulus-granulosa complex. TABLE 3.The morphological character of ovary and oocyte development and quality resulted from IVG.

No 1. 2. 3.

Parameters Qualiy of oocyte base on the expanded level of Cumulus oocyte complex The average final size of oocyte after IVG (μm) Oocyte growth rate (μm) Prepubertal:before and after 14 days culture IVG Old Matured: before and after 14 days of IVG culture

Prepuberty 0.82 + 0.80 a

Aged of age 1.47 + 0.6 a

120.26 + 33.00a

128.07 + 33.58b

54.24 + 8.10a

134.08 + 8.7b

60.9 + 9.0a

149.94+ 12.50b

*Different subscript in the same row mean very significant different (P< 0.01). The expanded cumulus-oocyte complex (coc) : 2 and 1 = developed, expanded, 0 = not developed:

The oocytes after culture in vitro for maturation resulted in a lower rate of maturation, based on the expandedcumulus-oocyte complex. Further research needs to confirm these matured oocytes using an IVF test of their competence. Early antral follicles of cows have been investigated regarding their competence to mature in vitro6, meanwhile reports from Guiterez et al. 7 mentioned that oocytes resulting from IVG culture systems had not yet determined their maturation potential perfectly. The result regarding IVG from this research is relatively low and faces a significant problem regarding contamination. The culture system requires considerable improvement to increase its efficiency, especially when using large species of domestic animals whose oocytes take a much longer time to reach their final size. Miyano 1 mentioned that maintenance of the viability of the oocyte and the surrounding granulosa cells is a major problem. Porcine and bovine oocytes grow to a volume 3.5–5.0 times larger than those of mouse oocytes. In bovine IVG culture systems, supplementation with a meiotic arresting substance such as hypoxanthine is essential. In this culture system, we used supplementation of follicular fluid in a fixed presentation (5%) rather than hypoxanthine. Down et al. 9 reported that hypoxanthine in follicular fluid has been identified as one such meiosis arresting substance. For the near future, the potential of IVG culture systems for oocytes is expected to provide a new source of a large population of M-II oocytes. The results reported regarding several species is relatively promising both for research purposes as well for embryo production in vitro (TABLE 3). For future research, we need to confirm the potential of M-II oocytes through calcium dynamics during maturation of goat oocytes after 24 hours of culture

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using confocal laser scanning (CLSM-fluo-3). We analyzed the calcium intensity base on the histogram profile of oocyte and spermatozoa considered. There is quite a variation in the oocyte calcium intensity.9 Results reported from IVG culture systems from different species of the mammalian oocyte 1,10, from 1989 to 2015 show a different potential of different species including mouse, pig, and cow with matrix collagen, resulting in a positive result for IVM or IFV methods and obtaining offspring. In domestic species, the first successful IVG culture system that supports the growth of oocytes from mid-growth phase in preantral (late secondary) follicles to the final size was reported by Hirao et al. 11 (Table 3). It has been reported that sheep preantral follicles grow to the antral stage in serum-free conditions after one month of culture and a small number of in vitro grown oocytes mature to metaphase (M-II) 12.

SUMMARY IVG of Indonesian, local goat using preantral follicles and small size oocytes will provide a potential source of M-II oocytes in vitro for both research development and livestock production purposes. The follicle and oocyte size is an important parameter to consider in the early evaluation of their quality. The results showed that follicle sizes are significantly different between two groups (prepubertal and mature aged) of agoat. The number of follicles obtained from the two age groupswas not different. The minimum follicle size and oocyte diameter needed (>100 μm) for early evaluation of maturation were achieved. The final size of oocyte after IVG reaches about the same size approximately as a matured oocyte. Meanwhile, oocytes recovered from IVG after culture in vitro for maturation resulted in a very low rate of maturation to M-II. However, in the near future, IVG of the preantral follicle of Indonesian local goat could be considered as an alternative source of oocytes for both research purposes and embryo production. The oocytes recovered from IVG after culture in vitro for maturation resulted in a very low rate of maturation (IVM). Further research is suggested on IFV testing of oocytes resulting from IVG culture systems.

ACKNOWLEDGMENTS This research is a part of research funded by Minister of Research and the Higher Education Republic of Indonesia, Research Grant BERBASIS KOMPETENSI Contract No. 252.102/UN10.21/PG/ 2016, 18 February 2016. I wish to thank my previous student (master degree: Helly Nurul Karima, Supriandono, Kholifah Holil, Dinda H.K. Wijaya and Ardyah Ramadhina I. P. for involved in most of the work and it's related. I am very grateful to my technician Setyawati for her hard work, and also the staff of Sukun slaughter house of Malang.

REFERENCES 1. 2. 3. 4.

T. Miyano, J. of Reproduction and Development 51, 169 – 176 (2005). Y. Hirao, Y. Tsuji, T. Miyano, A. Okano, M. Miyake, S. Sato and R. M. Noor, Zygote 3, 325 – 332 (1995). T. Miyano and Y. Hirao, J. Mammal Ova Research 20, 78 – 85 (2003). Y. Hirao, T. Itoh, M. Shimizu, K. Iga, K. Aoyagi, M. Kobayashi, M. Kacchi, H. Hoshi and N. Takenouchi, J. BiolReprod 70, 83-91 (2004). 5. R. G. Gosden and E. E. Telfer, J. Zool, London 211, 169 – 175 (1987). 6. S. Senbon and T. Miyano, Zygote 10, 301 – 309 (2002). 7. C. G. Gutierrez, J. H. Ralhp, E. E. Teffer, I. Wilmut and R. Webb, Biol Reprod. 62, 1322 – 1328 (2000). 8. S. M. Downs, D. I. Colemen, P. F. Ward-Bailey and J. J. Eppig, Proc. Nat Acad Scie. USA 82, 454 – 458 (2004). 9. A. A. G. Adam, Y. Takahashi, S. Katagiri and M Nagano, J. of Reproduction and Development 50, 579 – 586 (2004). 10. Y. Hirao, Y. Tsuji, T. Miyano, A. Okano, M. Miyake, S. Sato S and R. M. Noor, Zygote 3, 325 – 332 (1995). 11. S. Cecconi, B. Barboni B, M. Coccia and M. Mattioli, J. Biol Reprod 60, 594 – 601 (1999). 12. A. Hasegawa, A. N. Mochida and H. Kasumi, J. Mammam. Ova Research. 24: 8 – 12 (2004)

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From Little Known Area to the Extinction Race: A Survey of Herpetofauna in Prevab, Kutai National Park (KNP), Indonesia Nia Kurniawan1, a), Noviati Roziah1), Muhammad Alif Fauzi1) and Agung Sih Kurnianto2) 1

Biology Department, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Jl. Veteran, Malang 65145, East Java, Indonesia 2 Program of Environmental Resources Management and Development, Postgraduate School, University of Brawijaya, Jl. MT. Haryono, Malang 65145, East Java, Indonesia a)

Corresponding author: wawanunibraw@gmail.com

Abstract. We develop research focused on the environmental quality through a survey of herpetofauna in a threatened area with minimum data. The collected data supported by literature was used to predict the highest threat in the Prevab forest and to recommend a conservation area according to priority. The survey was conducted on three main sites: Senadam lake, camp area, and Buntu river. Each location was surveyed using a Visual Encounter Survey in a 1 km transect, from 7 to 11 p.m. for 3 days (21–23 October 2016). This study has identified 25 species herpetofauna from 76 individuals, consisting of 3 orders: Squamata, Anura, and Crocodilia. The highest number of individuals found were Chalcorana raniceps (n:18), Hemidactylus frenatus (n:7), Hemidactylus platyurus (n:10), Eutropis sp. (n:5), Cyrtodactylus yoshii (n:4), Crocodylus porosus (n:4), Hylarana erythraea (n:3), Varanus salvator (n:3). Two species were categorized as ‘Threatened’ species in the IUCN Red List, and one (Ptychozoon horsfieldii) was categorized as ‘Data Deficient.’ One species, Limnonectes paramacrodon, was categorized as ‘Near Threatened’. There were 2 species categorized as Appendix II CITES: Varanus salvator and Crocodylus porosus. Only one species, Crocodylus porosus, was protected by Indonesia’s regulation. Chalcorana raniceps and Limnonectes paramacrodon were found in high numbers in Buntu river, while the camp area was mostly visited by Hemidactylus platyurus, Hemidactylus frenatus, and Ptychozoon horsfieldii. Senadam lake was found to be the habitat for Crocodylus porosus.

INTRODUCTION The tropical rainforest provides a huge ecological and economical service for humans. It is estimated that at around USD 3,813 is donated for the surrounding civilization economy development each year.1 However, the tropical rainforest has also become the most exploited region, and currently, faces many risks of extinction everywhere.2 Land conversion, palm cultivation, and agricultural activities are the main causes of tropical rainforest deforestation and degradation in Southeast Asia.3-4 In Indonesia, Kutai National Park (KNP) is one of the most threatened tropical rainforest 5,6, in which there was more than 4000 ha area used for settlement and 26,524.6 ha converted to agriculture or settlement area of 60,000 ha total area.7-8 Threats against KNP’s rainforest are expected to increase in the future, due to the increasing rate in needs for food, space for living, fuel, minerals, and raw ingredients.2, 9–11 We proposed an environment quality assessment by conducting a herpetofauna survey in the most threatened and the least surveyed area in KNP: Prevab.7,13 By using herpetofauna as the focal organism, we illustrated Prevab as an


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area that is rich in biodiversity but is beginning to experience major disturbance.13–16 Deforestation is reported as the main cause in the declining Darwin’s frogs Rhinoderma darwinii and R. rufum population.17 In Australia, habitat modification closely correlates with the declining population of 18 out of 40 species of threatened frog species, and also the main cause of population decreasing of 11 out of 12 lowland threatened species.18 Phelsuma antanosy is one example of a reptile that can’t survive in a degraded habitat.19 The decreasing population of the endangered Spider Tortoise (Pyxis arachnoudes) is closely correlated with the loss of vegetation in Madagascar Island. 20 Herpetofauna is significantly benefit both ecologically and economically. For example the secretion of three Australian frogs (Litoria caerulea, L. Chloris, and L. genimaculata) is able to inhibit HIV.21 In addition, tadpoles are able to maintain water quality by eating algae, preventing them from blooming22, while the adults consume various invertebrates, including disease vectors such as mosquitoes23. After collecting the data of benefits and threats of tropical rainforest herpetofaunas, the survey should be conducted based on conservation action. The collected data and literature review were used to illustrate the threats faced by the Prevab forest area and recommend areas that need to be conserved according to priority.

EXPERIMENTAL DETAILS A survey was conducted by the authors for 2 weeks. Survey activities were focused on 3 main locations: Senadam lake, Prevab camp, and Buntu river. All of these locations were located inside Kutai National Park (KNP), East Kalimantan, Indonesia (Figure 1). These locations were selected because they are able to represent different vegetation in the KNP area. Senadam lake is a basin that disconnected Sangatta river due to mud sedimentation. Prevab camp is a residential area for the researchers and directly adjacent to the forest. Buntu river is an area with the most covered vegetation, which separated forests and functioned as observation track for orangutan (Pongo pygmaeus morio) behavior.

FIGURE 1. Locality sites observed from satellite imagery. A. Location in Kalimantan, Indonesia; B. Prevab surrounded by traces of human activities. Key: square (township); triangle (palm oil plantation, inside and outside KNP); round (primary forest); pentagon (industrial areas and coal mines); C. Detail locality site. Key: DS (Senadam lake), C (Camp), SB (Buntu river).

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A Visual Encounter Survey was conducted in each location, with a transect lane within one kilometer for each site. The activity was conducted from 7 to 11 p.m. for 3 days (21–23 October 2016). We did not use Time Constrained Studies (TCS) in which a survey was done on a time variation basis, depending on species discovery. Periodically, a survey was conducted by searching on or under rocks, woods, humuses, shrubs, and fallen branches. A survey was conducted without disturbing the microhabitat. 24 Herpetofauna was collected using bare hands, a hook, or grab stick, then documented using a Canon DSLR 1100D camera. All specimens were then released in the origin habitat. Photo results were compared with references.25–27

RESULTS AND DISCUSSIONS The present survey resulted in 25 herpetofauna species, consisting of 3 orders: Squamata, Anura, and Crocodilia. The Squamata found consisted of 6 families: Scincidae, Agamidae, Gekkonidae, Natricidae, Colubridae, and Varanidae (Table 1). Anura consisted of 4 families: Rhacoporidae, Microhylidae, Ranidae, and Dicroglossidae. Crocodilia only consisted of a family, which is Crocodylidae. As many as 76 species were found. The most frequent species was Chalcorana raniceps (Anura: Ranidae, n:18), Hemidactylus frenatus (Squamata: Gekkonidae, n:4), Hemidactylus platyurus (Squamata: Gekkonidae, n:10), Eutropis sp. (Squamata: Scincidae, n:5), Cyrtodactylus yoshii (Squamata: Gekkonidae, n:4), Crocodylus porosus (Crocodilia: Crocodylidae, n:4), Hylarana erythraea (Anura: Ranidae, n:3), Varanus salvator (Squamata: Varanidae, n:3). Two species are included in the IUCN Red List of Threatened Species. One species, Ptychozoon horsfieldii, is categorized as ‘Data Deficient.’ Another species, Limnonectes paramacrodon, is categorized as ‘Near Threatened.’ There are two species that are categorized in Appendix II CITES: Varanus salvator and Crocodylus porosus. Only one species is protected by Indonesian Law, Crocodylus porosus. Chalcorana raniceps found during the survey has 2 variants, green-brown back with black spot and green coloration on its lateral side. The second variant has a dark brown on its back and green-brown coloration on its lateral sides. This species was abundant on Pandanus sp. leaves that were located around Buntu river and its flows. Few variations were found in the Perupuk tree (Coccoreras borneense) on the banks of Senadam lake. A survey showed that the research location is a suitable habitat for Chalcorana raniceps. This species has a high tolerance toward the changing vegetation. It can be found on the pandan plant and in the open vegetation on the bank of the river.

TABLE 1. Herpetofauna recorded in Prevab. Key: Species protected under a law written in bold, *= listed on Appendix II CITES, DD= Data Deficient, NT= Near Threatened, SB= Buntu river, C=Camp, DS=Senadam lake.

Species Eutropis multifasciata Eutropis sp. Eutropis sp. 2 Eutropis rudis Gonocephalus borneensis Draco melanopogon Gecko sp. Cyrtodactylus yoshii Ptychozoon horsfieldii DD Hemidactylus frenatus Hemidactylus platyurus Hemidactylus sp. Psammodynastes pictus

Locality Site SB C DS x x x x x x x x x x x x x x x x -

Species Chalcorana raniceps Amnirana nicobariensis Hylarana erythraea Meristogenys sp. Limnonectes paramacrodon NT Limnonectes finchi Oligodon purpurascens Varanus salvator* Crocodylus porosus* Rhacophorus pardalis Unidentified Rhacophoridae Microhyla sp.

Locality Site SB C DS x x x x x x x x x x x x x x -

Although Hemidactylus platyurus and Hemidactylus frenatus were abundant, their distribution was limited in the camp. During the survey, both species were found in the camp’s wall near the forest. Generally, they could be found under big rocks or on rotting wood, tree trunk, even on building walls.

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Every location in Prevab gave a distinct illustration of herpetofaunal character. Buntu river was categorized as an area with little disturbance due to its function as observation track for orangutan behavior. This location was also covered with a dense vegetation and lack of sunlight. Few herpetofauna, especially amphibians such as Chalcorana raniceps and Limnonectes paramacrodon, were found to be abundant in this area. They were found in small rivers. The researcher’s camp provides a suitable habitat for few herpetofaunas from the family Gekkonidae. The presence of lamplight attracts various insects, thereby also attracting Hemidactylus platyurus, Hemidactylus frenatus, and one unique reptile namely Ptychozoon horsfieldii. Although it has been affected by human activities, the survey sites were adjacent to the natural forest; therefore it gives a sharp gradation between forest and open space. In addition, Senadam lake is known as the most open area. The vegetation primarily constituted of Poaceae. Slow streams and enough sunlight exposure make this area a suitable habitat for certain herpetofauna, such as saltwater crocodile, Crocodylus porosus. Based on observation and local people’s knowledge, crocodiles often visit Senadam lake due to its calm water and a direct passage from Sangatta River and Prevab forest’s edge. Prevab is located along Kutai National Park and conterminal with wood industry and plantation area. It makes Prevab vulnerable to human influences. Mining sites near KNP pose a direct or indirect threat to Prevab. Sudiyono 8 mentioned the area that must be maintained as a unit of Kutai National Park was about 60,000 ha, but this area continuously decreased in terms of original forest cover, and now only 33,475.40 ha remain. The opening of the Bontang–Sangatta road, which was agreed in 1990, became the main reason for the emergence of a settlement and plantation area, and the increase of human traffic in the National Kutai Park.28 In point of fact, there were 1700 people who already had land inside Kutai National Park, with a total area about 4,577 ha.7 Prevab became more threatened by the opening of access roads from the border of the national park. This access can be passed easily because of several factors including the absence of a buffer zone to prevent human pressures, lack of awareness in obeying the regulation for entry restrictions, and less support from the government to maintain the integrity of KNP as the conservation area.8, 29–30 Many pressures on Prevab occur in Senadam lake due to the water transportation activity in Sangatta River and the oil palm plantation which is located at the opposite sides. Offenders could easily carry out illegal activities and transport timber or animals through the transportation route. Meanwhile, the other two areas, Camp and Buntu River, are more secure because they are close to the camp area of KNP (office). However, recently, various human activities have been revealed to be mostly carried out in the inner part of Prevab forest. Based on the information from the KNP officers, geckos, snakes, and tortoises are herpetofauna that is mostly hunted by hunters and sold in both national and international markets. 31–32 As one of the animals that are vulnerable to the changes of environment quality, herpetofauna in Prevab is also more threatened due to the impact of land clearing, which pollutes soil and destroys natural vegetation. 7, 13 Herpetofauna could be considered as one of the most strong natural bioindicators to determine the quality of water and environment33 as it shows the physiological conditions, individual development, regeneration abnormality, or population that are affected by the changes in the micro temperature.34–35 This condition is getting worse due to the limited mobility of herpetofauna in order to avoid the pressures on the population and its habitat. Therefore, some population of herpetofauna is isolated, and it affects their global conservation status.36

Species of Particular Interest Two species discovered in this research were considered interesting (Figure 2). Horshfield’s Flying Gecko, Ptychozoon horsfieldii, is one of the Gekkonidae species with data deficient status (DD). It has a snout-vent length of up to 8 mm [37]. It has dorsals with grey or brown coloration (black dot pattern) and doesn’t have tubercles. The sides of its head have a black line which is elongated from snout to tympanium. There are 3 wavy patterns, 2 oval spots in the front of its body, and black marking resembling ‘butterfly shapes’ in its axillar. The chin has a yellow coloration with a few black spots. All of its toes have a wide web, which can optimize for gliding.38

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(a)

(b)

FIGURE 2. (a) Ptychozoon horsfieldii; (b) Limnonectes paramacrodon.

Ptychozoon horsfieldii distribution includes Southeast Asia, specifically Myanmar, Thailand, Peninsular Malaysia, Singapore, Sumatra, and Borneo. This species inhabits lowland of tropical rainforests, and it is even found near settlements37, specifically in the Prevab. In this survey, one of this species was found at the camp’s wall, when waiting for insects that were attracted to the lamp (n:1, 19.04 WIB). Its voice behavior was not detected. Deficient Data (DD) status version 3.1 in Ptychozoon horsfieldii shows that this species has an unidentified distribution. Several populations are known to be distributed in Thailand, Malaysia, Java, and Borneo, in a disturbed habitat, such as agricultural and palm plantation area. Regarding the explained problem, further study is needed to be conducted for this species, so the conservation status can be revised as Concern or Threatened.39 Other species, Limnonectes paramacrodon, was one of the species with Near Threatened status (version 3.1). This species belongs to the Dicroglossidae family with round elongated snout. Generally, the SVL of males reaches 60–75 mm, while females reach 55–66 mm.40 It has robust dorsal and small tubercles. There is a black diamondshaped area in its postocular, with a clearly visible tympanium and black line elongated from the tip of the snout until its nares. The ventral side and its femur have pale coloration, while its head, upper body, and lateral side have a grey or reddish-brown color. The tip of its hind limb toes are rounded (with a web between the toes, except the 4th toe), but its forelimbs were not. Although this species has a relatively wide distribution (Brunei Darussalam, Indonesia, Malaysia, Singapore, and Thailand), this species often depends on the river near the swamp forest. So, the habitat of this species becomes narrower (< 2000 m2). In addition, the degradation of environmental quality has led to this species losing its habitat. The result of this observation showed that Limnonectes paramacrodon inhabited an area near the river. This species was generally found in the forest area and swamp in lowland areas, with a narrow river. Some adult phases of this species were found along the clay shelf and graze on the edge of the cascade, and mark it as a breeding site. Limnonectes paramacrodon is quite tolerant of deforestation, but this species can’t adapt to a heavily modified habitat. This trend will make this species attain vulnerable status if it continues.41

SUMMARY Approximately 25 species herpetofauna, which consisted of 3 orders of Squamata, Anura, and Crocodilia, were identified in the survey sites. Among 25 species, one is categorized as ‘Near Threatened’ according to IUCN (Limnonectes paramacrodon) and one is protected by Indonesian law (Crocodylus porosus). Further study needs to be conducted in Kutai National Park regarding its limited data about herpetofauna diversity. Furthermore, complete and structured data would be a great help for the government or local institution to develop a regulation that is suitable for Kutai National Park.

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ACKNOWLEDGMENTS We are grateful to Mr. Haryadi of Prevab ranger, and also to field staffs for their support, and to Balai Taman Nasional Kutai for their approval of research permit and other courtesies. Thanks also to Anggun Sausan Firdaus (Animal Diversity Laboratory) for first-precious revision of this manuscript

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R. Constanza, R. d’Arge, R. de Groot, S. Farber, M. Grasso, B. Hannon, K. Limburg, S. Naeem, R. V. O’Neill, J. Paruelo, R. G. Raskin, P. Sutton, M. van den Belt, Nature 387, 253-260 (1997). S. Chakravarty, S. K. Ghosh, C. P. Suresh, A. N. Dey, G. Shukla, Global Perspectives on Sustainable Forest Management, 1- 27 (2011). N. Myers, “Tropical deforestation: rates and patterns,” in The Causes of Tropical of Tropical Deforestation. The economic and statistical analysis of factors giving rise to the loss of the tropical forest, edited by. K. Brown and D. Pearce (UCL Press, 1994), pp 27-40. S. S. Scrieciu, Economic causes of tropical deforestation- a global empirical application. Development Economics and Public Policy Working Paper 4. (Institute of Development Policy and Management, University of Manchester, 2003) W. D. Sunderlin and I. A. P. Resosudarmo, CIFOR occasional paper 9, 1-19 (1996). R. Burgess, M. Hansen, B. Olken, P. Potapov, and S. Sieber. Quarterly Journal of Economics 127(4), 17071754 (2011). Subarudi, Info Sosial Ekonomi 2(1), 29-35 (2001). Sudiyono, Jurnal Masyarakat dan Budaya 7, 119-142 (2005). C. P. Brooks, C. Holmes, K. Kramer, B. Barnett, and T. H. Keitt, PLoS ONE 4(6), e5783 (2009). J. J. Swenson, C. E. Carter, J. C. Domec, and C. I. Delgado, PLoS ONE 6(4): e18875 (2011). V. Vijay, S. L. Pimm, C. N. Jenkins, S. J. Smith, PLoS ONE 11(7), e0159668 (2016). Tnkutai. Prevab dan Mentoko. http://Tnkutai.com. Accessed on 24 September 2016 (2015). J. N. Fulton and W. H. Smith. “Herpetofauna as indicator species in the health of riparian buffer zones,” in Methamorphosis, COPLAC Proceeding (2013). K. R. Lips, Conservation Biology 13,117-125 (1999). Jr. M. M. Cohen, American Journal of Medical Genetics 104, 101-109 (2001). T. Hayes, A. Collins, M. Lee, M. Mendoza, N. Noriega, A. A. Stuart, and A. Vonk. Proc of the Nat Aca of Scie 99, 5476-5480 (2002). C. Soto-Azat, A. Valenzuela-Sánchez, B. Collen, J. M. Rowcliffe, A. Veloso, A. A. Cunningham. PLoS ONE 8(6), e66957 (2013). J. M. Hero and C. Morrison. The Herpetologica Journal 14, 175-186 (2004). C. J. Raxworthy and R. A. Nussbaum, Conservation Biology 10, 750–756 (1996). R. C. J. Walker, Herpetologica 66, 411–417 (2010). S. E. VanCompernolle, R. J. Taylor, K. Oswald-Richter, J. Jiang, B.E. Youree, J.H. Bowie, M.J. Tyler, J.M. Conlon, D. Wade, C. Aiken, T.S. Dermody, V.N. KewalRamani, L.A. Rollins-Smith, and D. Unutmaz, Journal of Virology 79, 11598-11606 (2005). A. W. Ranvestel, K. R. Lips, C. M. Pringle, M. R. Whiles, and R. J. Bixby, Freshwater Biology 49, 274-285 (2004). M. J. Greenlees, G. P. Brown, J. K. Webb, B. L. Phillips, and R. Shine, Animal Conservation 9, 431-438 (2006). S. Pradhan, D. Mishra, and K. R. Sahu, International Journal of Pure and applied zoology 2 (3), 241-245 (2014). R. Whitaker and A. Captain. Snakes of India, the field guide. 1st Ed, (Draco Books, Chengalpet, 2008). S. K. Dutta, M. V. Nair, P. P. Mohaptra, and A. K. Mohaptra. Amphibians and Reptiles of Similipal Biosphere Reserve (Regional Plant Resource Center, Bhubaneswar, 2009). I. Das. A Field Guide to the Reptiles of South-East Asia (Bloomsbury Publishing Plc, London, 2015). R. Siburian, Jurnal Masyarakat & Budaya 10(2), 89-115 (2008). M. Diego. Electronic Green Journal 1(15), 1-18 (2001). G. S. M. Andrade and J. R. Rhodes, Ecology and Society 17(4): 14 (2012).

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31. D. T. Iskandar and W. R. Erdelen, Amphibian and reptile Conservation 4(1), 60-87 (2006). 32. C. R. Sheperd and V. Nijman. An overview of the regulation of the freshwater turtle and tortoise pet trade in Jakarta, Indonesia: A TRAFFIC Southeast Asia Report (TRAFFIC Southeast Asia, Petaling Jaya, 2007). 33. E. Simon, P. Miklos, B. Mihaly, and T. Bela. Frogs and toads as biological indicators in an environmental assessment (Nova Science Publishers, Inc. USA, 2011). 34. A. R. Blaustein and P. T. J. Johnson. Frontiers in Ecology and the Environment 1, 87-94 (2003). 35. D. W. Sparling, G. Linder and C. A. Bishop, Ecotoxicology of amphibians and reptiles. (Pensacola, FL: Society of Environmental Toxicology and Chemistry (SETAC), 2000), pp. 904. 36. R. A. Blausein, K. B. Lisa, H. O. Deanna, M. G. David, L. R. Terry, M. K. Joseph, Conservation Biology 5(6), 1804-1809 (2001). 37. I. Das. A field guide to the reptiles of South-East Asia (New Holland Publisher, United Kingdom, 2010). 38. R. Malkmus, U. Manthey, G. Vogel, P. Hoffmann, and J. Kosuch. 2002. Amphibians and reptiles of Mount Kinabalu (North Borneo). A. R. G. Gantner Verlag K.G., Ruggell. Liechtenstein (2002). 39. R. F. Inger, R. M. Brown, and L.L. Grismer. Ptychozoon horsfieldii. The IUCN Red List of Threatened Species 2010: e.T178723A7603187. http://dx.doi.org/10.2305/IUCN.UK.2010-4.RLTS.T178723A7603187.en. Accessed on 04 July 2017 (2010). 40. R. F. Inger and R.B. Stuebbing. A field guide to the frogs of Borneo (Natural History Publication, Kinabalu, 2005) 41. P. P. Van Dijk, D. Iskandar, R. Inger, N. Yaakob, L. T. Ming, and Y. Chuaynkern. Limnonectes paramacrodon. The IUCN Red List of Threatened Species 2004: e.T58363A11771741. http://dx.doi.org/10.2305/IUCN.UK.2004.RLTS.T58363A11771741.en. Downloaded on 9 February 2017 (2004).

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The Effect of Organic Farming Systems on Species Diversity Amin Setyo Leksono1a) 1

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145. a)

Corresponding author: amins28@ub.ac.id

Abstract. Organic farming systems have been well known to support the diversity of a wide range of taxa, including microorganisms, arable flora, invertebrates, birds, and mammals, which benefit from organic management leading to increases in abundance and/or species richness. The objective of this paper is to review the effect of organic farming on species diversity reported in several articles and compare this with the current study in Gondanglegi, Malang. A review of several studies showed that organic farming systems have been reported to increase species diversity, including that of mammals, birds, arthropods, vascular plants and arbuscular mycorrhizal fungi. The researchers about arthropod groups consisted of carabid beetles, butterflies, wasps, predators, and bees. Agricultural landscape, habitat type, farming system, landscape composition and connectivity all contribute to explaining species biodiversity and richness. Moreover, based on current and relevant studies, the results showed that the application of refugia blocks has increased arthropod diversity and composition.

INTRODUCTION Ecological damage caused by intensive agriculture systems can be seen at the local, regional and global levels, because of the application of chemical pesticides and fertilizers. The aim of these systems is to increase food production twofold by 2050 1. However, intensive conventional farming systems have led to a decline in agricultural landscape diversity 2. Locally, these farming systems degrade soil quality, increase erosion, decrease biodiversity, destroy habitats and trigger deforestation, while at the regional level they create groundwater contamination and eutrophication. Meanwhile, at a global level, this intensive agriculture causes air pollution and contributes to climate change issues 3. With so many problems resulting from intensive agriculture, agroecological methods should be applied to reverse trends that threaten the loss of biodiversity and to create a healthier agroecosystem environment. Organic systems are considered environmentally friendly agriculture systems for producing foods and have received agri-environmental payments for conserving biodiversity 4. However, due to the apparent trade-offs on declining yields, this situation has led to an alternative strategy known as “land sparing and land sharing” 5. Green et al. 5 describe land sparing as an approach to sustain diversity whereby agricultural land is farmed as intensively as possible, with wildlife being conserved within separate “nature reserves.” In contrast, “land sharing” is described as an attempt to conserve biodiversity within the agricultural system. The application of blocks of refugia consisting of locally grown herb species is an alternative of land-sharing effort. The objective of this paper is to review the effect of organic farming on species diversity reported in several articles and compare this with the current study in Gondanglegi, Malang.

Organic Farming System Organic farming is a farming system with a righteous and holistic perspective method that involves growing and nurturing crops 6. This system strives for sustainability and enhances soil fertility and biological diversity whilst, with rare exceptions, prohibiting synthetic fertilizers, pesticides, antibiotics, genetically modified organisms and growth hormones. The concept of organic agriculture is closely related to the concept of agroecology 7 and dynamic


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agriculture. This system supports the protection of plants and soil by using the practice of nutritional input of organic matter, crop rotation, and pest and weed control of mechanically and biologically. It is estimated that 40% of the earth’s surface, or about half of the area that can be inhabited, comprises agricultural land 8,9. Conservation biologists consider an increase in the agricultural land area through conventional cultivation systems to be the main threat indicator of species diversity 9. According to the International Federation of Organic Agriculture Movements, in 2014 there were 172 countries that possessed data on organic agriculture. Across the world, 43.7 million hectares of agricultural land were organic (including conversion areas). The area had increased by 0.5 million hectares compared to the previous year. Almost 1 percent of agricultural land was organic, with 11 countries having more than 10 percent of the organic agricultural land. Over 2.3 million producers were reported, with more than three-quarters of these being in developing countries10. An organic farming system is built on two important concepts. The first minimizing the impact of the system on the ecosystem and producing high-quality food. This concept means that organic farming has to produce healthy products without residues that can be harmful to humans and other animals. The second is the implementation of water management practices because water is a staple crop requirement and management practice 11. Water quality greatly affects the quality of agricultural crop production. Contaminated water can reduce the quality of rice produced because plants can absorb harmful pollutants. In addition, while organic farming maintains good physically, chemical and biological soil properties, the quality of the product increase. Therefore, in organic farming, land management is preferred to minimize erosion, increase soil organic matter content and encourage diversity of soil biology. Several recent studies have shown that organic agriculture produces higher carbon sequestration and less leaching of nutrients12 while decreasing the erosion rate and pesticide levels in water 13,14. Based on the characteristics of the organic farming system referred to above, this system depends entirely on natural processes such as the decomposition of soil organic matter, using various techniques such as organic fertilizers, livestock feces, and compost. This approach aims to replace nutrients lost from the soil by previous agricultural systems. The biological process is controlled by various decomposer microorganisms including bacteria, fungi, and mycorrhizae. The presence of mycorrhizae allows the production of natural nutrients in the soil. Organic farming employs various methods to improve soil fertility, including the use of cover crops belonging to Leguminosae and mulch. Legume plants such as peanuts can fix nitrogen to increase the nitrogen content in the soil. Cover plants may act as refugia for arthropods and reduce the rate of soil erosion. Mulch plays a role in maintaining soil moisture so that living organisms in the soil surface find habitats that have appropriate moisture levels. Organic systems are also applied by reducing soil treatment that can disrupt the activity of soil microorganisms, such as soil cultivation, the use of chemical compaction pesticides and other forms of soil tillage. By reducing the forms of soil processing, the process of soil reversal that causes the exposure of soil to the air decreases. This causes volatile nutrients such as less nitrogen and carbon to disappear. In addition, the use of vegetable pesticides and biological and natural controls is also highly recommended in supporting the success of organic farming systems.

Effect of Organic Farming Systems on Species Diversity of Mammals, Birds, Arthropods, Vascular Plants and Arbuscular Mycorrhizal Fungi Organic farming systems have been reported to increase species diversity. The advantages of these systems are linked to the increased diversity of taxa 15,16 and functional diversity 17 as well as the more complex linkage network of pollinators 18. Birds, mammals, arthropods, and plants benefit from the production of plants that apply this system with biological control applications, maintaining the presence of natural enemies and pollinators 19,20. Plenty of bird species and territories, butterflies and herbaceous plants, and bumblebees are more abundant on small than that on large farms. The largest differences can be found between small organic and large conventional farms. Differences are also noted between small and large organic farms: 56% more bird species have been found on small organic farms. The overall effect of organic agriculture on biodiversity includes factors affected by farm size 21. Organic farming practices in this system, such as crop rotation, intercropping and soil cultivation, are things that can increase the biodiversity of animal species by providing healthy habitats for many species. Several studies have also reported benefits from organic farming for the diversity of insects. In general, on average more than 50% of organisms are more abundant in organic farming systems, but the results vary between studies and groups of organisms. Another study with a meta-analysis approach was conducted by Hole et al. 19 and Bengtsson et al. 22. The study draws on a variety of different research methods and scales, finding that organic plants are related to the abundance and wealth of various high taxonomic groups. Natural enemy

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groups also increase with organic farming systems 20. In addition, Sandhu et al. 23 concluded that organic crops retain ecosystem services such as pollination. Non-predatory and pest insects did not respond positively. Both organic farming and landscape heterogeneity significantly increased butterfly species richness and abundance. There was also a significant interaction between farming practice and landscape heterogeneity because organic farming only significantly increased butterfly species richness and abundance in homogeneous rather than heterogeneous landscapes. The species richness and abundance of butterflies can be enhanced by actions aimed at both promoting organic farming and increasing landscape heterogeneity 24. Several studies showed that organic farming practices can improve the characteristics of microorganisms as well as increase the content of soil organic matter including arbuscular mycorrhizal fungi (AMF). The spore abundance and species diversity of arbuscular mycorrhizal fungi were significantly higher in the organic than in the conventional systems. Furthermore, the AMF community composition differed between the conventional and organic systems. Glomus species were similarly abundant in all systems, while the spores of Acaulospora and Scutellospora species were more abundant in the organic systems. Some of the AMF species present in the natural environment are maintained under an organic farming system. The other species diminished under conventional farming, indicating a potentially severe loss of ecosystem function under conventional farming. These results suggest that agricultural practices significantly influence the AMF community structure and mycorrhizal inoculum potential 25. A previous study reported that roots in arable fields were colonized by mycorrhizal fungi species belonging to Glomus 26. This led to the conclusion that agricultural practices significantly affect mycorrhizal fungi abundance and community structure 27.

Effect of Block Refugia on the Diversity and Abundance of Arthropoda Based on a land-sharing concept proposed by Green 5, biodiversity in organic farming is managed within the agricultural system. The application of blocks of refugia consisting of is an alternative of land-sharing effort. This could be done by providing an alternative habitat for arthropod diversity. A block refugium is an area on farmland that is engineered to grow local herb species that provide shelter, food sources, and other resources for natural enemies such as predators and parasitoids 28. A refugium is a microhabitat capable of contributing to the conservation of natural enemies. Some of the plants planted in these blocks are commonly known as wild plants or even weeds, such as Mimosa pudica, Chromolaena odorata, Brachiaria mutica and Panicum repens 29,30. Wild plants provide an alternative habitat for the survival of a particular organism. Weeds are the basis of agricultural food webs, providing food to many living organisms. The abundant presence of weeds can improve regulatory and pollination services by ensuring the survival of honeybees due to the absence of oil seed crops 31. Wild plants that grow around plantations are not all harmful. There are several species of wild plants that are able to provide benefits; however, the selection of these plants needs to be studied seriously. This is because wild plants can also be an alternative habitat for pests. Wild plants will provide a favorable habitat for predators and parasitoids. Studies on a laboratory scale show that the parasitoid of cabbage headlines (Cotesia plutellae) is highly dependent on the nectar produced by plants. With the availability of sufficient sugar derived from nectar, the female parasitoid has a long life span and gets enough nutrients for egg ripening and oviposition success 32. A verified habitat can also provide nectar and pollen for insects, especially insect pollinators, and can serve as a temporary shelter. This leads to the presence of insects in these habitats 33. Pollen and honey from weeds or wild flowering plants can serve as an alternative food source for insects, especially insect pollinators. The performance and effectiveness of natural enemies can be enhanced by manipulating habitats. Past research has shown that some wild plants such as Bidens pilosa, Mimosa pudica and Vernonia cinerea very interesting couple of insect natural enemies of the Syrphidae family that act as pollinators and predators in rice, among others: Pocota personata and Eristalis tenak 34. Other studies mentioned Cyperus rotundus, Capsicum frutescens, and Bidens pilosa as also being known to attract insect pollinators, especially in the cultivation of apples 35. Species of insects of interest include Phygadeuon sp., Spathius sp., the Syphidae fly, the dome beetle and Apis mellifera. The species are attracted to the apple or bush flower surrounding it. Therefore it is necessary to conduct further research in order to see the pattern of interest and combination of bushes that can be used against natural enemies on organic farmland.

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EXPERIMENTAL DETAIL The current study in Gondanglegi, Malang was conducted between March and June 2017. The aim of this study is to analyze the effect of refugia blocks on arthropod diversity. Observations were carried out by using visual encounter surveys conducted 11 times on each of six plots in a paddy field with and without refugia as control starting from 37 days after planting (DAP) and up to 103 DAP. The types of refugia plants used were chili (Capsicum frutescens), ridge gourd (Luffa acutangula), tomato (Solanum lycopersicum) and long beans (Vigna unguiculata). These species were chosen because they have flowers that attract the presence of natural enemy insects. These species were planted alongside a small dike.

RESULT AND DISCUSSION The results showed that the abundance, taxa richness and diversity of arthropods in the paddy field with refugia were higher than those in the control, but statistical analysis showed this result was not significant. Peak arthropod abundance occurred at 71 DAP with 408 individuals in the treatment area and 307 individuals in the control, which may be related to the beginning of the generative period 36. Previous studies conducted by Maisyaroh et al. 37 and Purwantiningsih et al. 38 mentioned that the wild plants Mimosa pudica, Vernonia cinerea, Marsilea crenata, Pistia stratiotes, Bidens pilosa, Capsicum frutescens, Commelina diffusa and Ageratum conyzoides have been shown to provide support for the existence of arthropods around farmland and also on other cultivated land. The diversity of pollinating insects in a habitat is influenced by the availability of food sources and environmental factors 39. In a study conducted by Abidin et al. 40, the structure of a community of pollinators in an apple orchard was attracted to Ageratum conyzoides, Bidens pilosa and Capsicum annuum. This was composed of the Apidae, Sphecidae, Formicidae and Syrphidae families. Other studies conducted by Mustakim et al. 41 and Muhibah et al. 42 using refugia blocks in the form of a combination of Ageratum conyzoides, Capsicum frutescens and Tagetes erecta showed that arthropods on control blocks were dominated by Syrphidae, Tabanidae, Apidae, Papilionidae, Braconidae and Tachinidae.

SUMMARY Review from several studies showed that the organic farming system has been reported to increase species diversity, including mammals, birds, arthropods, vascular plants, and arbuscular mycorrhizal fungi. The research about arthropod groups were consisted of carabid beetles, butterflies, wasps, predators and bees. Agricultural landscape, farming system, habitat type, landscape composition and connectivity, contribute to the explanation of biodiversity and species richness. Moreover, based on current and relevant studies, the result showed that application of refugia block has increase arthropod diversity and composition.

ACKNOWLEDGMENTS I would like to express my gratitude to the Director Directorate Research and Community Empowerment, Directorate General Research and Development Strengthening, Head of Research and Community Empowerment Institute University of Brawijaya, Director of the Postgraduate University of Brawijaya, Dean of Faculty of Science University of Brawijaya. This research is funded by University Excellent Research Grant with Decentralization scheme.

REFERENCES 1. 2. 3. 4. 5. 6.

S. Butler, J. Vickery, and K. Norris. Science 315: 381-384 (2007). T. G. Benton, J. A., Vickery, and J. D. Wilson, Trends Ecol. Evol. 18, 182-188 (2003). P. Matson, W. Parton, A. Power and M. Swift, Science 227: 504-509 (1997). G. Rahmann, Agric. For. Res., 3, 189-208 (2011). R. E. Green, S. J. Cornell, J. P. W. Scharlemann, and A. Balmford, Science 307, 550–555 (2005). A.N. Mannion, Agriculture and environmental change: temporal and spatial dimensions (Wiley, Chichester, 1995).

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M. A. Altieri, and C. I. Nicholls,“Applying agroecological concepts to the development of ecologically based best management systems” in Proceedings of a Workshop “Professional societies and ecological based best management systems”. (Nat. Res. Council, Washington, DC. 2000). pp. 14-19. R. DeFries, F. Jonathan and G. Asner. Frontiers in Ecol.Environ. 2, 249-257 (2004). P. Donald, and A. Evans, J. Appl. Ecol. 43, 209-218 (2006). H. Willer, and J. Lernoud, (Eds.) The World of Organic Agriculture. Statistics and Emerging Trends. FiBL and IFOAM, (2016). K., Mondelaers, J. Aertsens, and G. Van Huylenbroeck, British Food J. 111, 1099-1120 (2009). L.E. Drinkwater, D.K. Letourneau, F. Workneh, A.H.C. van Bruggen, and C. Shennan, Ecol. Appl. 5, 1098– 1112 (1995) J. Kreuger, M. Peterson, and E. Lundgren, Bull. Environ. Con. Tox. 62, 55 – 62 (1999). P. Mäder, A. Fliessbach, D., Dubois, L., Gunst, P. Fried, and U. Niggli, Science 296, 1694 –1697 (2002) R. Feber, P. Johnson, L. Firbank, A. Hopkins, and D. Macdonald, J. Zool. 273, 30-39 (2007). L. Salazar, and A. Salvo, Neo. Entomol. 36, 765-773 (2007). D. Letourneau, and B. Goldstein, J. Appl. Ecol. 38, 557-570 (2001). E. F. Power and J. C. Stout, J. Appl. Ecol. 48, 561–569 (2011). D. Hole, A. Perkins, J. Wilson, I. Alexander, P. Griceand and A. Evans, Biol. Conserv.122, 113-130 (2005). M. P. D. Garratt, D. J. Wright, and S. R. Leather, Agric. Eco. and Environ. 141, 261-270, (2011). K. Belfrage, J. Björklund, and L.Salomonsson, AMBIO: A J. Hum. Environ. 34, 582-588 (2005). J. Bengstsson, J. Ahnstom, and A. Weibull, J. Appl. Ecol. 42, 261-269 (2005). H., Sandhu, S., Wratten, and R. Cullen, Environ. Sci. Pol. 13, 1-7 (2010). M. Rundolf and H. G Smith, J. Appl. Ecol. 43,1121–1127 (2006). S. I. Lee, E. H. Lee, and A. H. Eom, Mycobiology. 36, 19–23 (2008). T. Helgason, T. J. Daniell, R. Husband, A. H. Fitter, and J. P. W. Young. Nature 394, 431 (1998). S. I. Lee, and A. H. Eom, Mycobiology. 37, 272–276 (2009). W. Nentwig, and H. M. Poehling. Insektengesllscften auf Selbstbegrünten und eingesäten Ackerbrachen (Paul Haupt Berne, Switzerland, 1994). S. Karindah, B. Yanuwiadi, L. Sulistyowati, and P.T. Green, Agrivita 33, 133-141 (2011). A. N. Alifah, B. Yanuwiadi, A. S.Leksono, and Z. Penatagama,“Effect of Refugia Blog to Spatial and Temporal Distribution of Natural Enemies in Rice Field,” in Proceeding of 1st ICBS. (2011). V. Bretagnolle and S.Gaba, Agron. Sustain. Dev. 35, 891–909 (2015). T. Mitsunaga, T. Shimoda, and E. Yano, Appl. Entomol Zool. 39, 691-697 (2004). D. A. Landis, S. D. Wratten and G. M. Gurr, Annu. Rev. Entomol. 45, 175–201 (2000). B. Yanuwiadi, S. Nandini and Z. P. Gama, “Tanaman Liar untuk Manipulasi Habitat dalam Konservasi Syrphidae Predator dan Pollinator,” Proceeding Simposium PEI. (Cisarua, 2000). A.S. Leksono B. Yanuwiadi, M. A. Hasyim, B, Purwantiningsih, and F.L. Apituley, Trends in Entomology 8, 75 – 83 (2012). A. L. Leksono and J. Batoro, “The effect of refugia block on the Arthropod diversity in paddy fields in Malang, East Java. Abstract. The 2nd International Conference On Life Science and Biotechnology (ICOLIB). (2017). In Press. W. Maisyaroh, B. Yanuwiadi, A. S. Leksono, and Z. P. Gama, Agrivita 34, 67-74 (2012). B. Purwantiningsih, A. S. Leksono, and B. Yanuwiadi. Berk. Penel. Hayati 17, 165–172 (2012). D. Banjo, O. A. Lawal, and S. A. Aina. J. App. Sci. Res. 2, 858 -863 (2006). Z. Abidin, A. S. Leksono, and Z. Kusuma, 2013. J. Bio. Env. Sci.3, 20-24 (2013). Mustakim, A. S. Leksono, and Z. Kusuma, Natural B. 2, 248-253 (2013). T. M. Muhibah, and A. S. Leksono, Biotropika 3, 123-127 (2015).

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Farmers’ Perception of the Role of Some Wild Plants for the Predatory Coccinellidae (Adalia Bipunctata L and Coccinella Septempunctata L) in Developing Refugia in the Agricultural Field Bagyo Yanuwiadi1a) 1

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 a)Corresponding author: yanuwiadi@ub.ac.id Abstract. The decreases in plant and animal diversity in intensive agricultural practice have been caused by the application of new and very broad-spectrum pesticides. This situation motivated some researchers to observe the attractiveness of wild plants for some predatory Coccinellids. This was done with a view to improving the agricultural ecosystem. Previous research results showed that Blumea sp. (L), Tagetes erecta L. and Bidens pilosa L. could attract predatory Coccinellidae: Adalia bipunctata L. and Coccinella septempunctata L. But, unfortunately, there were no research results showing how farmers accepted those related new improved strategies for controlling pests. The research was conducted to analyze what they felt about introducing the function of wild plants for attracting beneficial arthropods. To this end, 60 farmers were selected purposively as respondents in the south of Malang and interviewed in depth with the main question focusing on their knowledge of the general function of wild plants in agriculture. Then, more specifically, they were asked about the role of wild plants in the agricultural field for attracting these beneficial arthropods. Their answers were grouped into the following categories: they don’t know, they know a little, they know enough, they know a lot about the general function of wild plants in general and more specifically as attractants for some beneficial arthropods. The results showed that the majority of farmers know only a little about the function of wild plants in general. None of the farmers realized that wild plants can be used as ground-covering plants. Most of them knew only about the use of wild plants for cattle feed. The majority of them did not know that some of the wild plants that can be found in their agricultural fields can be used as attractants in looking for beneficial arthropods. Farmers, as the frontier in the agricultural field, must become knowledgeable about the specific use of the wild plants in their fields to help them control pests naturally.

INTRODUCTION The decreases in plant and animal diversity in intensive agricultural practice have been caused by the application of new, very broad-spectrum pesticides .1 This situation motivated some researchers to observe the attractiveness of wild plants for some natural enemies of pests.2 Some of these natural enemies are predatory arthropods .3 A study of the function and killing capability of predatory arachnids was conducted by Yanuwiadi et al. 4 with a view to improving the agricultural ecosystem. The complexity of the food web in the ecosystem depends on the diversity of the producers. In the terrestrial ecosystem, the producers are green plants and crops. Previous research results showed that Blumea sp (L), Tagetes erecta L. and Bidens pilosa L. could attract predatory Coccinellidae: Adalia bipunctata L. and Coccinella septempunctata L. 5-7 The existence of these Coccinellid predators can act as controlling agents for some pests. If people want to reduce the use of pesticides so that their negative impacts can also be minimized, these research results should be understood and implemented by farmers. But, unfortunately, there are no research results showing what farmers think about those related new improved strategies for controlling pests. The research was conducted to


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analyze farmers’ perceptions of the introduction of the function of wild plants for attracting beneficial arthropods for their agricultural practices.

EXPERIMENTAL DETAILS To understand farmers’ perceptions of the introduction of the function of wild plants for attracting beneficial arthropods for their agricultural practices, the following social research was carried out. In this activity, 60 farmers were selected purposively as respondents in the south of Malang. They were interviewed in depth with the main question concerning the introduction of the function of wild plants for attracting natural enemies in relation to their knowledge of the general function of wild plants agriculture. Then, more specifically, they were asked about the role of wild plants in the agricultural field for attracting these beneficial arthropods. Their answers were grouped into the following categories: they don’t know, they know a little, they know enough, they know a lot about the general function of wild plants in general and more specifically as attractants for some beneficial arthropods. The following activities were conducted if the answer of the respondents to the question above was positive, and they were asked whether they were ready to use the useful plants for attracting the natural enemies in their agricultural field or not. The answers to these questions were grouped into the following categories: they are not willing to use the plants at all, they are reasonably willing to use the plants, they use the plants already, they will keep using the plants for attracting some beneficial arthropods.

RESULT AND DISCUSSION The wild plant species that were shown to the farmers as respondents can be seen in Table 1. All of them are wild plants grown in surrounding agricultural fields. Almost all of the farmers knew their local name well. Some of them are well known as weeds that have a negative impact on the main cultivated crop. All of the wild plant species show good potency in attracting the natural enemies of the pests of the cultivated plants 8,9. TABLE 1. The wild plants that were showed to the farmers

Number

Wild plant species

1

Bidens pilosa L

2

Marsilea crenata P

3

Ageratum conyzoides (L)

4

Eupatorium odoratum L

5

Ocimum sp. L

6

Ipomoea aquatica F

7

Centrosoma pubescen (Bth)

Table 2 shows that more than half of all the respondents that were interviewed were males aged 31–64 years. This was the most productive phase in their lives and this influenced their responses to the information. The majority of the respondents had only a basic school educational background (40 out of 60 participants). Of course, this played an important role in their acceptance of new knowledge. They tended to have a simple way of thinking 10.

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TABLE 2. The respondent (farmers) characters

No Characters

Criteria

Number of Respondent

Percentage (%)

1

Age (Years)

≤ 30 31–64 ≥65

10 35 15

16.7 58.3 25

2

Education Level

Basic School Elementary School High School Graduate

40 10 10 -

66.6 16.7 16.7 -

3

Intensity of getting Guidance (time per year)

2 3 4 5 6

6 35 17 2 -

10 58.3 28.3 3.3 -

Fortunately, 54 of the respondents received guidance from the government more than three times a year. This can cause a rapid increase in their knowledge from time to time.11 Governmental support sometimes has a positive impact on the people in a certain district. 12 Table 3 shows that the majority of farmers know only a little about the function of wild plants in general. In fact, wild plants have a lot of ecological functions.13,14 None of the farmers realized that wild plants can be used as ground-covering plants. In reality, they can play an important role in the ecosystem.15,16 Most of them knew only about the use of wild plants for cattle feed. The majority of them did not know that some of the wild plants that can be found in their agricultural fields can be used as attractants in looking for beneficial arthropods.2,3 Farmers, as the frontiers in the agricultural field, must become knowledgeable about the specific use of wild plants in their field to help them control pests naturally, as was mentioned by Yanuwiadi et al. 9 If their knowledge level is low, it will be difficult for them to be willing to use wild plants in their agricultural practices. 17 This can also be seen clearly in Table 3. TABLE 3. The respondent perception and willingness to use the wild plant

Respondent Perception of Wild Plant

Criteria

Number of Respondents

Percentage (%)

General Knowledge Ground cover plant about Plant Function as:

not know

60

100

know little

-

-

they know enough

-

-

know well

-

-

not know

-

-

know little

-

-

they know enough

-

-

know well

60

100

not know

-

-

know little

-

-

they know enough

-

-

know well

-

-

not know

-

-

know little

5

8.4

Fresh Air

Shielding Effect

Green Fertilizer

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Respondent Perception of Wild Plant

Cattle Feed

Criteria

Number of Respondents

Percentage (%)

they know enough

10

16.7

know well

45

75

not know

-

know little

-

they know enough

-

know well

60

100

55

91.7

know little

5

8.4

they know enough

-

-

know well

-

-

Special Function as Attractant for Natural Enemies not know

Willingness respondent to use the wild plant for attracting the natural enemies

Having no 55 willingness to use the plants at all

91.7

having a little 5 willingness to use the plant

8.4

using the plant directly

-

-

Having the willingness to keep using the plant

-

-

SUMMARY After considering the discussion above, the conclusions of this research are as follows: 1. The majority of farmers know only a little about the function of wild plants in general. 2. None of the farmers realize that wild plants can be used of as a ground-covering plant. Most of them only know about the use of wild plants for cattle feed. 3. The majority of them did not know that some of the wild plants that can be found in their agricultural fields can be used as attractants in looking for beneficial arthropods. Only a small number of farmers use the wild plants in their field to help them control pests naturally.

ACKNOWLEDGMENTS I would also like to show my gratitude to the Head Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya to support and allow me to conduct this field studies.

REFERENCES 1. 2. 3.

E. S. Harsanti, S. Y. Jatmiko and A. N. Ardiwinata, “Insecticide Residue on East Java Irrigated Rice Ecosystem”, Proceedings of Glass House Gases Emission Research and Increasing Rice Productivity at Lowland Rice (1999). W. Maisyaroh, B. Yanuwiadi, A. S. Leksono and Z. P. Gama, Agrivita. 34, 67-74 (2012). S. Karindah, B. Yanuwiadi, L. Sulistyowati and P. T. Green, Agrivita. 33, 133-141 (2011).

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

B. Yanuwiadi, M. Schade, S. Permana and A. Rasetyoaji, “Entwicklung und Frassleistung von Plexippus paykulli (Audouin) (Arachnoidea, Salticidae) gegeÜber Drosophila melanogaster (macquart)”. Internationale Entomologen-Tagung DgaaE, SEG, öEG, XVI.SIEEC (Basel, 1999). E. A. L. Sari, F. Rohman and B. Yanuwiadi. “Study of the Visiting Frequency of Arthropods to the Wild Plants Bidens pillosa L. and Ageratum conyzoides L. in Tea Plantation in Wonosari Malang”. Proceeding of 2th International Conference on Global Resource Conservation (ICGRC) (2011). D. D. R. Turista, F. Rohman and B. Yanuwiadi. “Study of Visiting Frequency of Arthropods on the Wild Plants Centella asiatica and Synedrella nodiflora (L) Gaertn in the Wonosari Tea Plantation in Malang”. Proceeding of 2th International Conference on Global Resource Conservation (ICGRC) (Malang, 2011). A. Wulansari, F. Rohman and B. Yanuwiadi, “The Frequency of Arthropods Visiting Borreria repens DC. and Setaria sp. in Wonosari Tea Plantation of Malang”. Proceeding of 2th International Conference on Global Resource Conservation (ICGRC) (Malang, 2011). D. Suheriyanto, Soemarno and B. Yanuwiadi, Applied Mechanics and Materials 747, 341-344 (2015). B. Yanuwiadi, H. Riniwati, Z. Zulia and A. M. Rachim, “The Importance of Research Result Distribution for Supporting of Implementation of Adiwiyata Award in High School in Blitar”. Proceeding of International Conference on Global Resource Conservation (ICGRC) (Malang, 2015) Sumarni, D. M. Dewi and Herawati, Women farmers group perception to the introduction of innovations processed food from cassava in Central Sulawesi. Prosiding Seminar Hasil Penelitian Tanaman Aneka Kacang dan Umbi BALITBANG (Kementerian Pertanian, 2014). A. S. K Asri and B. Yanuwiadi, J-PAL 6, 42-47 (2015). Z. Zulia and B. Yanuwiadi, JITODE 3, 45-52 (2015). S. Wantouw, Antariksa, B. Yanuwiadi and Z. Tamod, IJAS 4, 108-113 (2014). Sopingi, A. Suman, Soemarno and B. Yanuwiadi, World Environment. 5, 39-45 (2015). M. Y. Rahmaddin, T. Hidayat, B. Yanuwiadi and Suyadi, Resources and Environment. 5, 97-105 (2015). M. Y. Rahmaddin, T. Hidayat, B. Yanuwiadi and Suyadi, J. Appl. Psychol 5, 96-102 (2015). R. Hidayat, B. Yanuwiadi, & B. Prasetyo, J-PAL 8, 53-61 (2017).

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The Improvement of the Quality of Polluted Irrigation Water through a Phytoremediation Process in a Hydroponic Batch Culture System Catur Retnaningdyah1a) 1

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 a)

Corresponding author: caturretnaningdyah@gmail.com

Abstract. The objective of this research was to determine the effectiveness of a phytoremediation process using some local hydro macrophytes to reduce fertilizer residue in irrigation water in order to support healthy agriculture and to prevent eutrophication and algae bloom in water. A phytoremediation process was carried out in a hydroponic floating system by using transparent plastic bags of 1 m in diameter and 1 m in height that were placed in collecting ponds before they were used for agricultural activities. Paddy soils were used as substrates in this system. The irrigation water was treated with nutrient enrichment (Urea and SP-36 fertilizers). Then, the system was planted with remediation actors (Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata, polyculture of those hydro macrophytes and control). The improvement of the water quality as a result of the phytoremediation process was characterized by a decline in the concentration of some physicochemical parameters, which were measured at 7 days after incubation, as well as an increase in the plankton diversity index value. The results showed that all of the hydro macrophytes used in this research, which was grown in the hydroponic batch culture system for a period of 7 days, were able to significantly improve the irrigation water quality, which was enriched by the synthetic fertilizers Urea and SP36. This was reflected by a significant decrease in the concentration of water TSS, nitrate, BOD, COD and total phosphate and an increase in the value of water DO at 7 days after incubation. Improvement of the water quality is also reflected in the increasing plankton diversity index value as a bioindicator of water pollution indicating a change in the pollution status from moderately polluted to slightly polluted at 7 days after incubation.

INTRODUCTION The increasing human population has led to an increasing food demand. To meet this demand, the Indonesian government’s policy is to intensify agriculture, which is achieved, among other things, by water regulation through irrigation and the intensive use of synthetic fertilizers and pesticides. Irrigation is the providing, arranging and disposing of irrigation water to support agricultural activity. In Indonesia today, there is approximately 5 million ha of irrigated rice fields.1 The irrigation water is distributed to farm plots, with the quantity and quality of water being adapted to the needs of the cultivated crops, and excess water being diverted to other places so as not to damage the plants. Irrigation water in Indonesia still relies mostly on surface water sources such as rivers, springs, waterfalls, reservoirs, and lakes.2 The results of previous studies show that surface water sources in Indonesia, in general, have been polluted due to industrial activities as well as settlement.1,3,4,5 In line with the Healthy Indonesia 2015 program and obstacles, problems, and complaints related to the intensification of agriculture, switching farming back to traditional organic farming was the target of the government in 2010. Unfortunately, this has not been achieved. One of the constraints in organic farming is that water used for general irrigation is generally contaminated with pesticide residues and synthetic fertilizers. Farmers generally use synthetic fertilizers mainly containing major nutrients such as nitrogen (N) and phosphorus (P), which have been known to have a negative impact on the environment, especially in triggering eutrophication in rivers, which in turn can trigger algae bloom in the waters.6, 7, 8, 9 Two fertilizers often used by


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farmers in Indonesia are Urea and SP36. Synthetic fertilizer residues can increase the concentration of N and P in irrigation water. Thus this is a major obstacle in terms of fulfilling the requirements of organic agriculture. Synthetic fertilizer residues can be controlled in various ways, one of which is the use of plant hydromacrophytes.10 The phytoremediation process using hydro macrophyte plants is the right choice. This is due to it being relatively cheap and because the diversity of hydro macrophyte plants in Indonesia is high. In addition, phytoremediation technology with local hydro macrophytes can be applied by organic farmers themselves in order to improve water quality for agriculture. The phytoremediation process can be carried out by organic farmers in ponds through a hydroponic batch culture system using local hydro macrophytes. The objective of this research was to determine the effectiveness of the phytoremediation process performed in a hydroponic batch culture system using some local hydro macrophytes (Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata and polyculture of those hydro macrophytes) to reduce fertilizer residue (Urea and SP36) in irrigation water in order to support healthy agriculture and prevent eutrophication and algae bloom in the water. As a result of the phytoremediation process, the water can be further used as a source of organic agriculture irrigation water which exists in the surrounding areas. The success of the phytoremediation process is known, from some physicochemical parameters of water and phytoplankton diversity, as a bioindicator of water pollution. Phytoplankton or algae are an ecologically important group in most aquatic ecosystems and have been an important component of biological monitoring programs. Phytoplankton are ideal for assessing water quality because they belong to primary producers and are the basis of the food web in the aquatic ecosystem, have a short life cycle and rapid reproduction, and respond quickly to water quality changes, so they can be used as a bioindicator of water pollution.11, 12

EXPERIMENTAL DETAILS Research Design This is a quasi-experimental research with the completely randomized design. The independent variables in this experiment were some local hydro macrophyte species as remediation actors, included Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata, polyculture of those hydro macrophytes and control (no plant). The dependent variables in this experiment were water quality and plankton diversity as a bioindicator of water quality. This research was conducted in a phytoremediation pond belonging to an organic farmer located in the Kepanjen district of Malang, East Java, Indonesia. Physicochemical analyses of water quality and identificat ion and counting of plankton were performed in Laboratorium of Ecology and Animal Diversity at Brawijaya University. Twelve hydroponic floating systems were assembled by using transparent plastic bags. Each system had a diameter of 1 m and a height of 1 m and they were all placed in collecting ponds before being used for agricultural activities (FIG 1). This is a batch culture system. Five kilograms of paddy soil was used as a substrate in this system. The irrigation water (200 L) was treated by nutrient enrichment (Urea 100 ppm and SP36 fertilizer 50 ppm). Then, the systems were planted with remediation actors (Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata, polyculture of those hydro macrophytes and control).

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a

b

c

d

e

f

g

h

FIGURE 1. Preparation and planting hydro macrophytes (a,b); plant after two weeks incubation (c. No plant (control), d. Polyculture, e. Ipomoea aquatica, f. Limnocharis flava, g. Azolla sp., h. Marsilea crenata).

Monitoring the Effectiveness of the Phytoremediation Model Monitoring of the water quality was carried out immediately after planting the remediation actors and 7 days after planting. Improvement of the water quality as a result of the phytoremediation process was characterized by a decline in the concentration of some physicochemical parameters, which were measured at 7 days after incubation, as well as the increasing plankton diversity index value. The physicochemical parameters measured in this research included DO, pH, TSS, nitrate, total P, BOD, and COD, which were determined based on standard methods for the examination of water and wastewater.13 Plankton sampling was done by filtering 1 liter of sample water using a plankton net with a mesh size of 406 pores per inch and a diameter of 12 cm. The filtered plankton sample was 15 mL, which was inserted into a flacon bottle, then preserved with 4% formalin. To maintain the chlorophyll in the phytoplankton, five drops of saturated CuSO4 solution were added to each sample. The plankton could then be identified and the amount of each species per liter calculated using a Sedgewick Rafter Counting Chamber and a microscope. Plankton identification was based on the identification guidebook.14, 15 16, 17 .

Data Analysis Plankton data (composition and density) was then used to determine the Important Value Index (IVI), taxa richness and Shannon-Wiener diversity index as bioindicators of water quality in the hydroponic floating system after the phytoremediation process.12 The difference in water quality between treatments was determined by ANOVA and this was followed by Tukey’s HSD test at the 0.05 level, which was done with the package SPSS for Windows, Release 16. The grouping of general water quality among the systems was determined using cluster analysis and a biplot from principal component analysis (PCA).

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RESULT AND DISCUSSION The Effectiveness of a Phytoremediation Model in a Hydroponic Floating Batch Culture System Using Some Local Hydromacrophytes to Improve the Physicochemical Quality of Polluted Irrigation Water Some local hydro macrophytes such as Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata and polyculture from those hydro macrophytes that were grown in the hydroponic batch culture system for 7 days were able to significantly reduce the levels of TSS, nitrate, total phosphate, BOD and COD and increase the concentration of water dissolved oxygen (DO) due to residues from the synthetic fertilizers Urea and SP36 (FIG 2). All of the hydro macrophytes used in this experimental treatment and control without plants were capable of reducing total suspended solids (TSS) from 60.00–159.07 ppm to 47.00–56.75 ppm. The Indonesian government regulation No. 82/2001 requires that the maximum limit for TSS for agriculture (class II) is 50 ppm. This meant that further effort was needed to fulfill this requirement. Hydro macrophytes play a role in precipitating and filtering the sediment in water.18 A control treatment without plants in the hydroponic system was also capable of decreasing the levels of TSS. Paddy soil is a substrate capable of precipitating suspended solids in the water. This is because of the batch culture used in this system. Therefore the absence of flow can result in the occurrence of solid precipitation in the paddy soil as a substrate in this treatment. The residue of synthetic fertilizers present in irrigation water can be reflected from phosphate and nitrogen content in water either in the form of soluble phosphate, total phosphate (TP), nitrate, ammonium or total nitrogen (TKN). Nitrates and total phosphate are water quality characteristics of irrigation enriched by Urea and SP36 fertilizers. Enrichment of the water irrigation with 100 ppm Urea synthetic fertilizer did not increase the nitrate content enough to exceed the Indonesian government’s standard quality of water No. 82/2001, which requires a maximum of 10 ppm. The concentration of nitrate in this experiment decreased from 5.49–7.97 ppm immediately after planting to 2.54–4.31 ppm after incubation for 7 days. The phytoremediation process is able to reduce the nitrate level to less than 5 ppm, which is a good quality category and may be used for any crop 19, while this phytoremediation is capable of decreasing the total phosphate from 2.10–2.21 ppm to 1.57–1.83 ppm. Enrichment of the irrigation water with 50 ppm SP36 synthetic fertilizer resulted in an increase of TP levels in the water, thereby exceeding the predetermined standards. The maximum limit of total phosphate Class II for agriculture is 0.2 ppm, whereas it is 1 ppm for Class III. However, TP levels greater than 0.1 ppm belong to the category of hypertrophic waters.20 This suggests that agricultural activity may be the main cause of eutrophication in downstream river waters, including waters from reservoirs. Planting of some local hydro macrophytes in a hydroponic batch culture system for 7 days was able to reduce the level of nitrates in the water but was still not able to reduce the TP to meet the levels set by the Indonesian government. Hydro macrophytes from the species Azolla sp. has also been proven to reduce dissolved N and P in the greenhouse through a batch culture system as well as inhibiting the blooming of Microcystis sp.21 All treatments in this study were able to increase the levels of DO and pH in the water. The concentrations of dissolved oxygen levels and pH at the beginning of the study ranged between 2.93–3.17 ppm and 7.53–8.02, whereas after 7 days’ incubation the value of pH ranged between 9.11 and 9.61 and DO increase to 5.34–5.71 ppm, which met the government standard quality Class II that requires a minimum DO of 4 ppm.

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FIGURE 2. The average value of DO, TSS, nitrates, BOD, COD and total phosphates water in hydroponics batch culture system at a moment after planting some local hydro macrophytes (day 0) and seven days after incubation (day 7). (Note: Treatment: 1. Control (no plant), 2. Limnocharis flava, 3. Azolla sp., 4. Marsilea crenata, 5. Polyculture, 6. Ipomoea aquatica. The same notation on each parameter showed no significant difference by ANOVA test followed by Tukey HSD α 0.05)

The existence of organic matter in the waters can be known from the parameter values of BOD and COD. BOD and COD levels are the levels of oxygen needed to degrade organic materials biologically (BOD) and chemically

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(COD). Enrichment of the irrigation water with 100 ppm and 50 ppm Urea and SP36 synthetic fertilizer, respectively, resulted in an increase of COD and BOD concentration in the water of between 42.61–50.41ppm and 14.42–12.67, respectively. This concentration exceeded the Indonesian government standards. The maximum limits of BOD and COD Class II for agriculture are 3 ppm and 25 ppm, respectively. Planting of some local hydro macrophytes in the hydroponic batch culture system for 7 days in this research was able to significantly reduce the levels of BOD and COD to 7.53–9.23 ppm and 18.90–24.74 ppm, respectively. Therefore, this phytoremediation model has succeeded in reducing the COD level to meet the standards, although the BOD content still exceeds the established standard. Thus the phytoremediation model still requires improvement.

The Effectiveness of a Phytoremediation Model in a Hydroponic Floating Batch Culture System Using Some Local Hydromacrophytes to Improve the Quality of Polluted Irrigation Water Based on the Plankton Diversity Index as a Bioindicator The improvement of the water quality in this research treatment is also reflected in the increasing plankton diversity index value. The results of identification and counting of each plankton species abundance in the system immediately after planting and 7 days after incubation were then used for calculating the important value index, taxa richness and Shannon-Wiener diversity index (FIG 3). A total of 13 taxa of phytoplankton found in the culture system was used in this research. There was no significant difference in the composition and abundance of the plankton after 7 days of incubation. However, the results of IVI calculations (FIG 3) showed that on the 7th day of incubation, in general, there were decreases in the value of Chroococcus as an indicator of moderate organic pollution and Microcystis as an indicator of high organic pollution22.

Important Value Index (%)

200 180 160 140 120 100 80 60 40 20 0 Day 0

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Pediastrum

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Scenedesmus

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(c)

FIGURE 3. Important Value Index/ IVI (a), taxa richness (b) and Shannon Wiener diversity index (c) of plankton in hydroponics batch culture system at a moment after planting some local hydro macrophytes (day 0) and seven days after incubation (day 7) (Note: Treatment: 1. Control (no plant), 2. Limnocharis flava, 3. Azolla sp., 4. Marsilea crenata, 5. Polyculture, 6. Ipomoea aquatica.

Taxa richness at the beginning of all treatments with the phytoremediation model ranged from 7 to 12 species. This value tended to be almost the same 7 days after planting hydro macrophytes with the number of plankton taxa being 9–11. This was in contrast to the results of Shannon-Wiener’s diversity index calculation, which showed an increase in value from 0.97–1.71 to 1.88–2.22. This value indicated an increase in water quality from moderately polluted to slightly polluted12 except for the treatment with Limnocharis flava. Therefore, it can be concluded that all treatments with the phytoremediation model (except for Limnocharis flava) in this study were effectively able to improve water quality from moderately polluted to slightly polluted after incubation for 7 days. The effectiveness of the phytoremediation process in a hydroponic batch culture system using some local hydro macrophytes can be seen from the improvement of water quality on the 7th day of incubation compared with day 0, which was shortly after planting. If the phytoremediation process can effectively improve water quality, there will be significant physicochemical differences reflected by the decreasing value of pollutant parameters and plankton diversity index as bioindicators of water quality after 7 days of incubation. Water quality differences can generally be seen from the results of water quality data analysis based on cluster and biplot analysis as seen in FIG 4. Based on cluster analysis, it can be seen that there were significant differences in the water quality between day 0 and day 7. Therefore it can be concluded that the phytoremediation process in this research treatment was able to improve water quality based on physical, chemical and biological parameters of plankton diversity as a bioindicator of water quality. Based on biplot analysis, it can be seen that water quality shortly after treatment with enrichment of Urea and SP36 fertilizer is characterized by high values of nitrate, BOD, COD and TP, and low values of DO, pH, and plankton Shannon-Wiener diversity index. The 7-day phytoremediation process using several local hydromacrophytic species and also treatment without hydro macrophytes are considered highly effective for improving water quality as reflected by decreased levels of nitrate, BOD, COD and TP and increased DO, pH and Shannon-Wiener’s plankton diversity index. Hydromacrophytes contribute to increased infiltration of water into the soil and are also expected to be utilized in the phytoremediation process to degrade toxic contaminants such as fertilizer and toxic pesticide residues where applied to agricultural land.23 Thus the results of the phytoremediation process using a model such as in this study in the future can be used to irrigate surrounding organic rice fields. However, it is still necessary to improve the phytoremediation process by reducing the BOD and TP levels to meet the standards that have been set by the government.

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FIGURE 4. Group of water quality in hydroponics batch culture system at a moment after planting some local hydro macrophytes (day 0) and seven days after incubation (day 7) based on some physics chemical parameters and plankton diversity using cluster analyses and biplot from Principal Component Analysis (PCA)

SUMMARY The phytoremediation process of enriching irrigation water with 100 ppm and 50 ppm Urea and SP36 synthetic fertilizers, respectively, in a hydroponic batch culture system planted with various kinds of local hydro macrophytes, such as Azolla sp., Ipomoea aquatica, Limnocharis flava, Marsilea crenata and polyculture from those hydro macrophytes, for a period of 7 days can significantly improve water quality. This was reflected by decreasing the concentration of water TSS, nitrate, BOD, COD and total phosphate significantly and increasing the value of water DO after 7 days of incubation. The improvement of water quality is also reflected in the increasing plankton diversity index as a bioindicator of water pollution indicating the change in the pollution status from moderately polluted to slightly polluted after 7 days of incubation.

ACKNOWLEDGMENTS I thank family of Mr. Pudji Raharjo for permitted to use the pond. This research is a part of Institutional Research Grant that is funded by Directorate General of Higher Education of Indonesia through the University of Brawijaya, so I would like to say thank you very much to National Education Ministry of Indonesia and Rector of Brawijaya University.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

G. Kurnia, “Efisiensi Air Irigasi untuk Memperluas Areal Tanam. Prosiding Seminar Nasional Multifungsi Lahan Sawah”, (Balai Penelitian Tanah, Bogor, 2001). Direktorat Pengelolaan Air, Dirjen Pengelolaan Lahan dan Air, Pedoman Teknis Pengembangan Irigasi Air Permukaan (Departemen Pertanian, Jakarta, 2010). C. Retnaningdyah and E. Arisoesilaningsih, “An indication of Water Springs Pollution in Malang Area of Upper Brantas Watershed Using Biotic Index from Benthic Macroinvertebrates”, Proceeding of National Seminar on Biodiversity 2013, (UNS, Solo, 2013). R. Rahmawati and C. Retnaningdyah, J. Biotropika 3, 50-54 (2015). R. Wimbaningrum, S. Indriyani, C. Retnaningdyah and E. Arisoesilaningsih, J. Ind. Tour. Dev. Std. 4, 81-90 (2016). I. Panagopoulos, M. Mimikou and M. Kapetanaki, J. Soils Sediments 7, 223–231 (2007). F. Yin, B. J. Fu and R. Z. Mao, China. J. Soils. Sed. 7, 136–142 (2007).

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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23.

C. Retnaningdyah, “Correlation between Water Quality and Population Dinamics of Microcystis spp. In Sutami Reservoir, Malang, East Java. Indonesia”, Proceedings of National Seminar of Limnologi IV, (Limnology Research Center -LIPI, Bogor, 2008). C. Retnaningdyah, Suharjono, A. Soegianto and B. Irawan, J.Trop.Life.Science 1, 42-46 (2010). K. Ivansyah and C. Retnaningdyah, J. Biotropika 1, 80-84 (2013). W. M. W. Omar, Trop.Life Sci. Res. 21, 51–67 (2010). N. Wu, B. Schmalz dan N. Fohrer, Austin J. Hydrol 1, 9 (2014). L. S. Clesceri, A. E. Greenberg and A. D. Eaton, Standard Methods for the Examination of Water and Waste Water. 20th Ed., (Washington, 1998). W. T. Edmondson, Freshwater Biology. Second Ed. (John Wiley and Sons Inc., New York, 1959). W. Whipple, Fresh Water Biology. 2nd Ed. (John Wiley and Sons Inc., New York, 1959). G. W. Prescott, How to Know the Fresh Water Algae. 3rd Ed. (Wm.C. Brown Company Publisher, Iowa, 1978). C. H. Bold and M. J. Wynne, Introduction to the Algae: Structure and Reproduction. 2nd Ed. (Prentice Hall Inc Engelwood Clif, New Jersey, 1985). W. Xiang, E. Y. Xiao and Z. Rengel, Environ. Monit. Assess. 157, 277-285 (2009). R. S. Ayers and D. W. Westcot, Water Quality for Agriculture. FAO Irrigation And Drainage Paper. 29 Rev. 1. (Rome, 1994). C. C. Shoc and K. Pratt, “Phosphorus Effects on Surface Water Quality and Phosphorus TMDL Development”, Proceedings on Western Nutrient Management Conference. (Salt Lake City, UT, 2003). C. Retnaningyah, Suharjono, Budiman and Purnomo, “Control of Microcystis growth by Azolla sp. in combination with aerobic denitrifying bacteria indigenous from Indonesia reservoir”. Oral Presentation on Malaysia International Biological Symposium 2012 (ί-SIMBIOMAS 2012), Kuala Lumpur July 11th -12th, 2012. I. C. Onyema, actaSATECH 4, 93-107 (2013). Q. Chaudhry, P. Schroder, D. Werck-Reichhart, W. Grajek and R. Marecik, Environ Sci & Pollut Res 9, 4- 17 (2002).

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Green Technology Innovation in a Developing Country Chairat Treesubsuntorn1, a), Rujira Dolphen1, b), Prapai Dhurakit2, c), Dian Siswanto3, d) and Paitip Thiravetyan2, e) 1

Pilot Plant Development and Training Institute, King Mongkut's University of Technology, Bangkok 10150, Thailand. 2 School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand. 3 Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145. a) chairat.tre@kmutt.ac.th b) rujipen@hotmail.com c) prapai.dhu@kmutt.ac.th d) diansiswanto@ub.ac.id e) Corresponding author: paitip.thi@kmutt.ac.th Abstract. Developing countries rapidly grow when green technology, which is referred to as eco-friendly processes or methods, is developed in parallel. Here, some examples of green technology research and development in Thailand will be overviewed. A huge amount of agricultural waste is generated during agricultural processes. Applying these agricultural wastes in order to maximize the benefits for environmental cleanups of water, soil and air has been studied and commercialized. For example: 1) Application of agricultural waste and/or biochar developed from agricultural waste as biological adsorbents for wastewater treatment in some industries, such as textile/dye industries, and printing industries. In addition, this agricultural waste can also be applied in decolorization of sugar syrup from sugar industries; 2) The research on modified biomaterials as adsorbents and packing materials in biofilters would also be presented, and now, pilot scale biofilters have been developed and applied to solve air pollution problems in the field for future commercialization; 3) Some agricultural waste and/or biochar developed from agricultural waste in our laboratory can promote rice growth and improve rice quality via the reduction of Cd uptake and translocation in rice. Phytoremediation technology, in which plants are used to improve the environmental quality in water and air, has also been studied and would be presented. 1) Some species of native Thai plants can effectively remove heavy metals and dye from wastewater. For this research, a constructed wetland for wastewater treatment was developed and applied in a real contaminated site. 2) In air phytoremediation, some plant species harbor highly volatile organic compound (VOC) removal efficiency. In addition, plants do not only absorb organic pollutants, but also they have the innate ability to degrade organic compounds and use them as carbon sources for their growth. In addition, plant growth-promoting (PGP) bacteria inoculation into plants can enhance airborne pollutant removal. From this research, an indoor air phytoremediation system was developed in order to reduce CO2 emissions with high VOC removal efficiency.The high cost of technology transfer is a major problem, especially in developing countries, and green technology research and innovation can overcome this problem along with efficient allocation of resources and technologies.

INTRODUCTION TO GREEN TECHNOLOGY In recent years, many research and development companies have paid more attention to green technology, which focuses on the development of research, innovation and activities to prevent pollution and to enhance green capabilities.1-3 Application of green technology can improve energy efficiency, material and waste utilization and recycling.1 Based on these benefits, green technology not only enhances environmental quality but also promotes the strength of a country’s economy.4,5


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Nowadays, industrial and technological development in developing countries is growing rapidly, but environmental problems must also be considered. Therefore, green technology research and innovation needs to be carried out in order to promote environmental and economic development in parallel.6 In Thailand, green technology, focused on energy and the environment, has been developing continuously. In this review article, we aim to present effective green technology for environmental treatment in Thailand from laboratory scale to pilot scale and application.

ASPECTS OF GREEN TECHNOLOGY IN THE ENVIRONMENT Since the bioresources in tropical developing countries have high biodiversity, this can be a strength for green technology research and development in Thailand. The application of agricultural waste and phyto- and bioremediation to remove water, air and soil pollution has been researched and developed. In terms of agricultural waste, materials and modified materials, for example bagasse fly ash, bamboo biochar and sawdust fly ash, can be effectively used to remove dye from contaminated wastewater in textile/printing industries.7-11 From basic research, a wastewater treatment system has been developed and commercialized. Nowadays, this treatment system has been transferred to more than 30 printing industries in Thailand. In addition, many agricultural wastes show a high capacity to adsorb VOCs (BTEX, TMA, formaldehyde, etc.) contaminated in air.12-14 The modified material from agricultural waste can be effectively used as an adsorbent and a packing material in a biofilter system.15 Modified materials supplemented with a carbon source for bacterial growth can stabilize biofilter system humidity, reduce system pressure drops and support carbon sources for bacterial growth, and the developed biofilters can be operated for long times without the addition of external nutrients. Nowadays, the system has been scaled up to test on a pilot scale and will be commercialized in the near future. The application of agricultural waste and biomaterials to improve agricultural productivity and quality has also also studied. The addition of biochar from agricultural waste and/or some endophytic bacteria species can reduce Cd uptake and translocation in rice plants and grain.16,17 This experiment can be developed into effective soil amendment for Cd reduction in rice. The application of agricultural waste and biomaterials can provide many benefits for the environment, waste minimization, agriculture, industry and energy. Phytoremediation and bioremediation, using plants and microorganisms to improve environmental quality, are very effective methods. The use of plants to adsorb dye and xenobiotic compounds from contaminated wastewater from printing industries has been studied18-21, and artificial wetland from high dye uptake plant species was developed. The system was tested in a real site of contamination. Not only water but also air pollution can be removed using phytoremediation technology. Many plant species have presented high VOC removal efficiency.13,2226 In addition, plants have the ability to transform carbon-based air pollution and use it as a carbon source for plant growth.27-31 Some native and nonnative microorganisms growing on plants can promote significant VOC removal efficiency.32 Many hormones and enzymes, produced from suitable microorganisms, have been shown to be an important factor in increasing air phytoremediation efficiency.33-38 The application of plants and microorganisms with a well-designed system can be used to remove VOC contamination in pilot experiments. In addition, an effective air phytoremediation system was designed to reduce CO 2 emissions. In the system, there are many interesting points of research and development in this area waiting for investigation.

Waste Utilization and Minimization Biochars and Agricultural waste for wastewater treatment Biomaterials and modified biomaterials can be used to remove dye from contaminated wastewater in textile/printing industries.8,9,11,39 For example, chitosan, waste from industrial seafood processes, showed a high capacity to adsorb dye from printing industry wastewater. The material can strongly combine with synthetic reactive dye molecules under acidic and caustic conditions. Chemisorption has been confirmed as an important mechanism, which chitosan uses to adsorb dye molecules.39 In 2010, sawdust fly ash also presented a high ability to absorb dye in contaminated wastewater of around 90%8, which is higher than other modified materials. Not only the printing industry but also wastewater contaminated with melanoidin from the sugar syrup industry can be treated by developing activated carbon from bagasse bottom ash.9 A previous experiment showed that the application of biomaterials and modified biomaterials showed a high capability in wastewater treatment. From basic research, a

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wastewater treatment system was developed and commercialized, in which various materials would be specifically applied with different types of wastewater Modified biomaterial as a packing material in biofilters Since 2012, many plant species have been reported to have the ability to uptake benzene under light and dark conditions, where stomata under dark conditions should be closed in most of the plant.22 However, some plant species can still highly take up air pollution under dark conditions. This suggested that plants can uptake air pollution via cuticular wax on shoot and leaf surfaces. Crude wax was used to absorb benzene in a closed system to confirm the benzene absorption potential of plant wax. In 2013, the quantity and quality of wax were reported to be important factors for benzene absorption.12 The results showed that nonpolar fatty acids, > 18C fatty acids, might be the key factor for benzene adsorption when Boraphech and Thiravetyan13 reported that low carbon fatty acid (< 10 C fatty acids) plant material can adsorb highly trimethylamine (TMA). The results suggested that different pollutants have different water solubility. Benzene, a very low water soluble compound, can be adsorbed by long chain fatty acids (hydrophobic wax) while TMA, high water solubility, can be well adsorbed by short chain fatty acids (hydrophilic wax). So, different biomaterials, containing different structures, will probably have different adsorption capacities in different pollutants. The application of biomaterial for VOC absorption and adsorption was interesting to investigate. In 2016, 22 plant leaf materials were screened for benzene adsorption and used as a packing material in a biofilter system. Six leaf materials modified from leaves of Dieffenbachia picta, Acrostichum aureum, Ficus religiosa, Lagerstroemia macrocarpa, Alstonia scholaris and Dracaena sanderiana (FIG 1A) showed high benzene adsorption. Physical sorption was reported as a mechanism of benzene adsorption by these materials. Moreover, these six materials were immobilized on glass beads in order to reduce system pressure drop, and Pseudomonas putida was inoculated into the system. This developed biofilter (FIG 1B) showed high benzene removal efficiency; however, the external nutrient supply needs to be supported daily.15 Today, modified plant material and agricultural waste with glucose syrup inside the packing bead for VOC biofiltration has been developed (FIG 2D) and tested in a pilot scale experiment (FIG 2A–C), with the aim of reducing system pressure drop, increasing removal efficiency and supporting bacteria growth without the addition of external nutrient support. The biofilter was applied to remove benzene in a testing chamber (container), and the benzene removal efficiency is shown in FIG 3. In cycles 1–3, 0.6 ppm of benzene was completely removed within 10 h, while in cycles 4–6, benzene was completely removed within only 6–8 h. The results suggested that microorganisms in biofilters might require 1–3 cycles for adaptation.

A

B

FIGURE 1. Modified biomaterials from plant leaf and agricultural waste to adsorb VOCs and be a packing material in biofilter (A) and biofilter experiment (laboratory scale) to study the removal efficiency and specific maximum loading rate of each biofilter and materials (B).

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A

D

B

C

FIGURE 2 .Modified container to be a testing chamber for pilot scale experiment (A), installed turbine to equilibrate rapidly inside VOCs distribution (B), designed gas injection port and gas sampling port based on guideline from US.EPA. (C) and model of biofilter system (50 L of volume) with flow rate controller (D).

FIGURE 3. Remaining benzene concentration (ppm) in container (testing chamber) after biofilter system operation for 6 cycles

Using biochars and microorganisms to improve rice quality and productivity Rice can uptake and translocate a high concentration of Cd (toxic compound) resulting in low productivity and quality of rice grain production. In Thailand, this problem has a profound economic effect of around 7,800–14,000 tons per year.40 In a previous laboratory experiment, the application of biochars and/or microorganisms was able to significantly reduce the Cd concentration in rice roots, shoots and grains.16,17 Interestingly, all treatments that presented a low Cd concentration showed significantly high other divalent cations, for example Ca, Mg, Mn, etc. in rice roots, shoots and grains. These elements might also play an important role in controlling Cd uptake and translocation of rice resulting in a low Cd concentration in shoots.41-43 In addition, soil amendment containing high divalent cation would be developed and applied in a real field in Thailand. This technology has a high possibility of improving rice grain quality and productivity.

Phyto- and Bio-remediation Technologies Artificial wetland :Research and Development Phytoremediation for wastewater treatment is well-known as an eco-friendly technology with low operation costs. In addition, plants can transform or degrade pollutants from toxic to less toxic forms and accumulate in plant tissue or organelle, for example vacuoles, cell walls, cell membranes, etc. 28. The application of different plant species can remove different types of pollutants, and in some cases, a plant might show itself as a hyperaccumulator, especially in heavy metal-contaminated wastewater. Or, in some cases, plants can completely degrade the pollutant to CO2. In 2007, Typha angustifolia was applied to dye-contaminated wastewater. This plant can remove 60% of

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reactive color and 44% of sodium after 14 days of an experiment. Saiyood et al.19 used Dracaena sanderiana and Dracaena fragrans to remove bisphenol A (BPA) contamination in wastewater or industrial leachate. D. sanderiana can uptake more than 80% of BPA through roots and translocate it to stems but not leaves after 20 days of an experiment. In addition, plants and native microorganisms in the roots can secrete extracellular plant mucilage to reduce BPA phytotoxicity. In 2010, Echinodorus cordifolius was reported to have the ability to remove ethylene glycol (EG)-contaminated wastewater. In this study, E. cordifolius was able to rapidly reduce EG and COD in wastewater. In addition, E. cordifolius can clearly transform triethylene glycol (TEG) to diethylene glycol (DEG) and monoethylene glycol (MEG), respectively.20 In several research studies, different pollutants required different plant species to remove, and phytoremediation for wastewater treatment needed a large area and required a long time for remediation. To overcome these problems, an artificial wetland was developed by focusing on increasing removal efficiency with limited land for operation. Recently, the system was tested in a real site of contamination (FIG 4A and B).

A

B

FIGURE 4. Artificial wetland for dye contaminated wastewater treatment (A) and real model (B)

Indoor air phytoremediation

FIGURE 5 . Green liver model in plant cell including transformation, conjugation and localization, plant use to minimize phytotoxicity of pollutions .

Using plants to improve indoor air quality has been reported as an effective, eco-friendly and low operation cost method. Many plant species have shown high VOC removal efficiency.27,44-49 In 2012, eight ornamental plants – Chamaedorea seifrizii, Scindapsus aureus, Sansevieria trifasciata, Philodendron domesticum, Ixoraebarbata craib, Monster acuminate, Epipremnum aureum and D. sanderiana – were screened for gaseous benzene remediation. D. sanderiana had the highest benzene removal efficiency, and can remove around 80% of 20 ppm gaseous benzene after exposed the pollutant in a closed system for 3 days.22 In addition, stomata were proposed as a main benzene uptake pathway in this study. In 2013, Zamioculcas zamiifolia was reported as a high BTEX removal efficiency plant. This plant species can completely remove BTEX after 3-day exposure, and the plant can uptake benzene and

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toluene higher than ethylbenzene and xylene. This might be the effect of the molecular weight of the compound, with benzene and toluene having a lower molecular weight than ethylbenzene and xylene, which means they can easily diffuse to the stomata of plants.23 For other VOCs such as trimethylamine (TMA) (fishy smell) and formaldehyde, plants also showed the ability to remove these pollutants. In 2015, Sansevieria sp. was reported as a high TMA removal efficiency plant. In addition, this study confirmed that plants use stomata as a main gaseous pollutant uptake pathway. A combination of C3 and CAM plant species can be used to treat TMA efficiency because C3 can open the stomata during the daytime while CAM can open the stomata at night. Therefore, a combination of two different types of plants might promote TMA removal all the time 13,26. In 2016, Euphorbia milii was applied to remove formaldehyde in a closed system. The results showed that this plant species can effectively remove formaldehyde, especially with microorganisms.33 Not only uptake but also the transformation of air pollutants has been studied. In general, plants have a Green Liver Model (FIG. 5), where plants can use to manage toxic compounds inside the cell. This model can be explained as having three main functions: transformation, conjugation and localization. For transformation, the pollutant reacts with the enzyme or reactive compound, resulting in changing the toxic compound structure. In this step, some pollutants might be completely degraded, for example benzene, toluene, xylene and, formaldehyde. However, some toxic compounds cannot be completely degraded in the transformation step, and can combine and stabilize with phyto-compounds, for example glutathione-S-transferase (GST) or phytochelatin. These stable compounds can be localized on cell walls, cell membranes, vacuoles etc., which can be called the localization step. In a previous experiment, Ugrekhelidze27 showed that benzene can be mainly transformed to phenol, catechol and cis,cis-muconic acid in plant cells. In addition, this experiment confirmed that benzene can be completely degraded to CO2 in plants. Kvesitadze et al.28 concluded the benzene transformation mechanism by plant and important enzyme. This article indicated that P450 monooxygenase is the critical enzyme to add hydroxyl group on the benzene ring, which generates the phenol compound. Then, the phenol compound can be transformed rapidly to the catechol compound. Catechol can be continuously transformed to o-quinone and cis,cis-muconic acid. As regards other xenobiotics, Boraphech14 presented the TMA phytodegradation pathway by Sansevieria sp., and Sangthong et. al.31 showed xylene phytodegradation by Bougainvillea buttiana. With regard to formaldehyde, Khaksar et al.33-35 proposed possible phytodegradation pathways. Many researches confirmed that plants can degrade air pollution. The application of this technology to remediate indoor air pollution can be effective, low cost, easy to operate and eco-friendly.

FIGURE 6 . Model of air phytoremediation system for indoor air pollution treatment (A), comparison between toluene removal efficiency by turned off and turned on phytoremediation system in a pilot scale experiment (B)

Although plants can remove air pollution, effective application of air phytoremediation has some limitations. For example, this technology requires a large area for growing plants, and in order to remove pollutants completely, a high number of plants must be used. A high number of plants growing in indoor air conditions can produce high CO2 concentrations at night. With poor ventilation in indoor conditions, CO2 can accumulate and be harmful to human health. Therefore, using plant-plant and plant-microbe interaction is interesting. These interactions might

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promote pollutant removal efficiency and reduce the number of plants used for indoor air phytoremediation, which can directly reduce CO2 emissions from the system. In a previous experiment, plants inoculated with native epiphytic bacteria enhanced gaseous xylene phytoremediation.31 This might be the effect of a high number of microorganisms on plant surfaces, which can directly contact and uptake xylene. However, the application of epiphytic bacteria to promote plants for air pollution removal can be operated for only a short time compared with the application of endophytic bacteria.37 In 2017, the application of native and nonnative endophytic bacteria increased formaldehyde phytoremediation. The role of plant stress hormone regulation was investigated in this study.33-35,37,38 In addition, application in suitable concentrations of the exogenous hormones jasmonic acid and auxin can also increase gaseous formaldehyde remediation.38 Based on laboratory research, an indoor air phytoremediation system was developed (FIG 6A). The use of a pump to increase air ventilation together with an effective plant-plant and plant-microbe interaction system was able to rapidly remove 2 ppm benzene-contaminated gas in a pilot scale experiment within 3 h (FIG 6B). In a previous study, a botanical biofilter was developed for TMA removal. The results showed that a combination of Sansevieria sp. and native endophytic bacteria could continuously treat gaseous TMA.32 Not only high removal efficiency but also low CO2 emission is the benefit of using this system because it can be operated effectively with a low number of plants. Application of C3 and CAM plants together to minimize CO2 and increase removal efficiency may be a possible strategy. Controlling the physical environment such as light quality, humidity and temperature may also be an interesting area of research.

SUMMARY The development of innovative green technology together with economic growth in developing countries can provide many benefits, including the improvement of economic competitiveness, clean environmental promotion, sustainability of technology, etc. Many green technology researches, developments and innovations in Thailand have been successful and overcome environmental problems. For example, agricultural waste was applied in a dyecontaminated wastewater treatment system. The system has been commercialized and adopted in many industries. In addition, agricultural waste and modified agricultural waste showed benefits in reducing Cd concentrations in rice and as a high-performance packing material in a biofilter system. Not only agricultural waste but also phyto- and bio-remediation technology can clean up the environment. Artificial wetlands for dye-contaminated wastewater and indoor air phytoremediation have been developed. The systems not only adsorb pollutants but also probably degrade some xenobiotic compounds to less toxic compounds. These are some examples of innovative green technology that have been developed from academic research based in Thailand. In green technology research and development, many topics still need to be investigated, especially in developing countries that have various natural resources. This can be an important strength of research and development in developing countries.

ACKNOWLEDGMENTS The authors would like to thank the financial support provided by King Mongkut’s University of Technology Thonburi through the Kmutt 55th Anniversary Commemorative fund and International Conference on Global Resource Conservation (ICGRC) 2017, Brawijaya University for invitation and the economic international airfare and local accommodation support during ICGRC 2017.

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10. D. Inthorn, K. Tipprasertsin, P. Thiravetyan and E. Khan, J. Environmental Science and Health Part A 45, 637644 (2010). 11. M. Hata, Y. Amano, P. Thiravetyan and M. Machida, Water Environment Research 88, 87-96 (2016). 12. C. Treesubsuntorn, P. Suksabye, S. Weangjun, F. Pawana and P. Thiravetyan Water, Air, & Soil Pollution, 224, 1-9 (2013). 13. P. Boraphech and P. Thiravetyan, Journal of Hazardous Materials 284, 269-277 (2015). 14. P. Boraphech .and P. Thiravetyan, International Journal of Phytoremediation 18, 1002-1013 (2016). 15. C. Treesubsuntorn and P. Thiravetyan, Clean-Soil, Air, Water, 44, 915-921 (2016). 16. P. Suksabye, A. Pimthong, P. Dhurakit, P. Mekvichitsaeng and P. Thiravetyan, Environmental Science and Pollution Research 23, 962-973 (2016). 17. C. Treesubsuntorn, P. Dhurakit, G. Khaksar, and P. Thiravetyan, Environmental Science and Pollution Research, in press (2017). 18. S. Nilratnisakorn, P. Thiravetyan, and W. Nakbanpote, Science of the Total Environment 384, 67-76 (2007). 19. S.. Saiyood, A. S. Vangnai, P. Thiravetyan and D. Inthorn, J. of Hazardous Materials 178, 777-785 (2010). 20. P. Teamkao and P. Thiravetyan, Chemosphere 81, 1069-1074 (2010). 21. W. Sriprapat and P. Thiravetyan, International Journal of Phytoremediation 13, 592-600 (2011). 22. C. Treesubsuntorn and P. Thiravetyan, Atmospheric Environment 57, 317-321 (2012). 23. W. Sriprapat and P. ThiravetyanWater, Air,& Soil Pollution 224, 1482 (2013). 24. W. Sriprapat, P. Suksabye, S. Areephak, P. Klantup, A. Waraha, A. Sawattan and P. Thiravetyan, Ecotoxicology and Environmental Safety 102, 147-151 (2014). 25. W. Sriprapat, P. Boraphech and P. Thiravetyan, Environmental Science and Pollution Research 21, 2603-2610 (2014). 26. P. Boraphech and P. Thiravetyan, Environmental Science and Pollution Research 22, 11543-11557 (2015). 27. D. Ugrekhelidze, F. Korte and G. Kvesitadz, Ecotoxicology and Environmental Safety 37, 24-29 (1997). 28. E. Kvesitadze, T. Sadunishvili and G. Kvesitadze, Engineering and Technology 55, 458-468 (2009). 29. P. Thiravetyan, C. Treesubsuntorn and W. Sriprapat, Phytoremediation of BTEX by plants. Published in A. A. Ansari, S. S. Gill, R. Gill, G. R. Lanza, and L. Newman (Eds.). Phytoremediation: Management of Environmental Contaminants, Volume 1 (Springer, New York, 2014), pp. 71. 30. D. Siswanto, Y. Chhon and P. Thiravetyan, Environmental Science and Pollution Research 23, 17067-17076 (2016). 31. S. Sangthong,P. Suksabye and P. Thiravetyan, Ecotoxicology and Environmental Safety 126, 273-280 (2016). 32. C. Treesubsuntorn, P. Boraphech and P. Thiravetyan, Environmental Science and Pollution Research, 1013910149 (2017). 33. G. Khaksar, C. Treesubsuntorn and P. Thiravetyan, Molecular Plant-Microbe Interactions, 29, 663-673 (2016). 34. G. Khaksar, C. Treesubsuntorn and P. Thiravetyan, Plant Physiology and Biochemistry 107, 326-336 (2016). 35. G. Khaksar, C. Treesubsuntorn and P. Thiravetyan, Environmental and Experimental Botany 126, 10-20 (2016). 36. W. Sriprapat and P. Thiravetyan, International Biodeterioration & Biodegradation 113, 262-268 (2016). 37. G. Khaksar, C. Treesubsuntorn, P. Thiravetyan, Plant Physiology and Biochemistry, 1-9 (2017). 38. G. Khaksar, C. Treesubsuntorn, P. Thiravetyan, Environmental and Experimental Botany, 130-138 (2017). 39. N. Sakkayawong, P. Thiravetyan and W. Nakbanpote, J. of Colloid and Interface Science 286, 36-42 (2005). 40. C. Phaenark, P. Pokethitiyook, M. Kruatrachue and C. Ngernsansaruay, International Journal of Phytoremediation 11, 479–495 (2009). 41. N. Hayakawa, R. Tomioka and C. Takenaka, Soil Science and Plant Nutrition 57, 691-695 (2011). 42. F. Eller and H. Brix, AoB PLANTS 7, 143 (2015). 43. H. Kudo, K. Kudo, M. Uemura and S. Kawaic, Botany 93, 345–351 (2015). 44. B. C. Wolverton, A. Johnson and K. Bounds, Interior Landscape Plants for Indoor Air Pollution Abatement. Final Report. (NASA Stennis Space Centre MS, USA, 1989). 45. R. L. Orwell, R. Wood, J. Tarran, F. Torpy and M. Burchett, .Removal of Benzene by the Indoor Plants/Substrate Microcosm and Implications for Air Quality. (Plants and Environmental Quality Group, Faculty of Science, University of Technology, Sydney, Westbourne St, Gore Hill, Australia, 2004). 46. United States Environmental Protection Agency (USEPA), Phytoremediation Resource Guide (Texas, USA, 1999). 47. F. Korte, G. Kvesitadze, D. Ugrekhelidze, M. Gordeziani, G. Khatisashvili, O. Buadze, G. Zaalishvili and F. Coulston, Ecotoxicology and Environmental Safety 47, 1-26 (2000).

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Important Value Index and Biomass (Estimation) of Seagrass on Talango Island, Sumenep, Madura Citra Satrya Utama Dewi1, a), Sukandar2, b) 1

Coastal and Marine Research Centre, Institute for Research and Community Services, University of Brawijaya, Malang, East Java, Indonesia 65145 2 Department of Fisheries Resources Utilization, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 a)

Corresponding author: satryacitra@ub.ac.id b) satryacitra@gmail.com

Abstract. Seagrass is the only plant that can live in shallow waters, and in groups with the same species (monospecies), as well as with different species (heterospecies), and form a seagrass bed. Seagrass beds have an important ecological role, one of them as a carbon storage in shallow water. Seagrasses can store carbon in the form of biomass from its body. Seagrass beds can be found on Talango Island, Sumenep, Madura. Exploration of seagrass beds in this location has not been carried out, especially related to its potential in storing carbon. The aim of this study was to find out the important value index of seagrass species and to estimate their biomass. This research was conducted from October to November 2016 at two stations on Talango Island, Sumenep, Madura, using the Point Intercept Transect (PIT) method. The data taken during the research comprise the species diversity, density, covering and frequency of seagrass. Data analysis was performed to obtain the important value index and to estimate the biomass of seagrass species. The results show that Enhalus acoroides have the highest important value index. Furthermore, the highest biomass estimation value is obtained from Enhalus acoroides, which is 1,650 gram/Ha. Moreover, the total estimation of the seagrass bed on Talango Island is 1,721.250 gram/Ha.

INTRODUCTION Seagrass is the only flowering plant that lives in shallow water, and it lives together to form seagrass beds. Seagrass beds have ecological benefits, including as primary producers in shallow water, as living habitats for other organisms, as sediment traps and for stabilizing substrate. 1-3 In addition, as a high-level plant, seagrass functions actively to absorb carbon too. Furthermore, seagrass stores it in the form of biomass. Seagrass absorbs carbon dioxide to perform photosynthesis, and stores carbon in the form of biomass in its body. Seagrass biomass is divided into two, namely above the substrate and below the substrate. Seagrass biomass that is above substrates can be predicted by using the percentage value of covering species of seagrass, and the index value of biomass of each species. It shows that the percentage value of covering species aligns with the value of biomass. Moreover, it describes a linear pattern with carbon storage therein. Seagrass researches related to its biomass, productivity, and carbon storage have already been conducted and published, including in Indonesia 4-6. The status of seagrass beds can be measured by the percentage value of covering seagrass (Kepmen LH No 4, 2004). However, some parameters can describe the seagrass bed condition too, such as species density, species frequency and important value index (IVI). IVI can be used to illustrate the ecological role of one species in a community7. Seagrass beds can be found in almost all coastal and shallow waters in Indonesia. The distribution of seagrass in East Java is located in several locations, i.e. Pacitan, Malang, Lumajang, Jember, Banyuwangi, Situbondo, Lamongan, Gresik and Talango Island. The aims of this research were to find out the important value index of seagrass and to estimate the value of seagrass biomass in Talango Island.


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EXPERIMENTAL DETAILS This research was conducted from October to November 2016. Data were taken from Talango Island, Sumenep, Madura. Data analysis was conducted in the Marine Science Laboratory, Fisheries and Marine Science Faculty, University of Brawijaya. The tools used in this research were a masker, snorkel, fin, 100 m roll meter and a transect quadrant (50 cm x 50 cm). Meanwhile, waterproof paper and pencils were also needed for the research. The data were taken by walking and forming a vertical line to the shoreline, at the lowest tides. A 100 m roll meter was stretched perpendicularly to the shoreline. Furthermore, a transect quadrant was placed at every 30 m (Hutomo and Nontji, 2014). Observations and data were collected on each quadratic transect, beginning with identifying the seagrass species, then observing the species covering, species frequency and species density. Data analysis in this research was performed in FPIK UB, and involved the calculation of species covering, species frequency and species density of seagrass, using the formula of Brower et al.8: Covering of species i: ‫ ݅ܥ‬ൌ Ci: Covering of species i (%) Ai: Area covered by species i A: Total area covered by all species

௔௜ ஺

௉௜

Frequency of species i: ‫ ݅ܨ‬ൌ σ ௉ Fi: Frequency of species i Pi: Number of transect quadrant that contains species i P: Total number of transect quadrant Density of species i: ‫ ݅ܦ‬ൌ Di: Density of species I (Individuals/m2) Ni: Number of individual species i A: Area of transect quadrant (m2)

௡௜ ஺

Hereinafter, data analysis was conducted to calculate the relative covering the area, relative frequency and relative density of seagrass species. Analysis of this data was done by using the formula of Brower et al.8. So its value can be used to calculate the important value index of seagrass species. ஼௜

Relative covering of species i: ܴ‫ ݅ܥ‬ൌ σ஼௜௝ RC: Relative covering of species i Ci: Covering of species i (%) ∑Cij: Total covering of species (%) ி௜

Relative frequency of species i: ܴ‫ ݅ܨ‬ൌ σ

ி

RFI: Relative frequency of species i Fi: Frequency of species i ∑F: Total frequency of species Relative diversity of species i

௡௜

= ܴ‫ ݅ܦ‬ൌ σ

௡

RDi: Relative diversity of species i Ni: Number of individual species I (individuals) ∑n: Total number of individual species Important value index (IVI) = RCi + RFi + RDi Furthermore, the analysis tried to estimate the alleged biomass of seagrass species. This analysis used data regarding the covering of seagrass species, which was processed using the formula adopted from English et al. (1997) in Assuyuti et al.9:

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Biomass Estimation (EB) = T x 10.000 x Carbon Species Index T = Covering of species i

RESULT AND DISCUSSION Seagrass on Talango Island comprised a heterogeneous seagrass bed, composed of more than one species. The species found on Talango Island were Enhalus acoroides, Cymodocea serrulata, and Halophila ovalis. The total covering of seagrass at Stations 1 and 2 had the same value, i.e. 40%. Enhalus acoroides had the highest covering area value in both of the stations, followed by Cymodocea serrulata and finally Halophila ovalis (Figure 1). This was because of the different morphology of the leaves of each type of seagrass, with the leaves of Enhalus acoroides being larger than those of Cymodocea serrulata and Halophila ovalis. 30

24

25

21

20 15

12

10

11

6

5

5 0 Station 1 Cymodocea serrulata

Station 2 Enhalus acoroides

Halophila ovalis

FIGURE 1. Percent of Seagrass Covering Area in Talango Island, Madura (%) The seagrass frequency indicates how often a seagrass species appears in the transect quadrant. The study data showed that Enhalus acoroides were the most common species of seagrass at the two stations, followed by Halophila ovalis, and the most rarely encountered was Cymodocea serrulata (TABLE 1). TABLE 1. Frequency of Seagrass Species on Talango Island, Madura

Species

Frequency Station 1

Station 2

Cymodocea serrulata

2

1

Enhalus acoroides

5

5

Halophila ovalis

3

4

Total

10

10

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Seagrass density provides information about the number of seagrass species in each square meter. The highest seagrass density is Halophila ovalis, at 21–24 individuals/m2, then Enhalus acoroides, i.e. 11–12 individuals/m2, and Cymodocea serulata, at 5–6 individuals/m2. The total seagrass density on Talango Island reaches 39–40 individuals/m2 (FIGURE 2). 30 24

25

21

20 15

12

10

11

6

5

5 0 Station 1 Cymodocea serrulata

Station 2 Enhalus acoroides

Halophila ovalis

FIGURE 2. Density of Seagrass Species on Talango Islands, Madura (individual/m2)

The relative covering the area, relative frequency and relative density of seagrass species showed a linear value to covering are frequency, and density of seagrass species (TABLE 2). TABLE 2. Relative covering area, relative frequency, and relative density of seagrass species on Talango Islands, Madura

Species Cymodocea serrulata Enhalus acoroides Halophila ovalis

Relative Density

Relative Frequency

Relative Covering Area

1

2

1

2

1

2

0.154 0.307

0.125 0.275

0.2 0.5

0.1 0.5

0.125 0.75

0.125 0.75

0.538

0.6

0.3

0.4

0.125

0.125

Important value indexes that were generated from this study show that Enhalus acoroides have the highest value, i.e. 1.5 (TABLE 3). This means that Enhalus acoroides plays an important role in maintaining the seagrass bed on Talango Island, Madura. The existence of Enhalus acoroides would affect the ecological function of the seagrass bed. TABLE 3. Important Value Index of Seagrass Species on Talango Islands, Madura

Species

Important Value Index Station 1

Station 2

Cymodocea serrulata

0.479

0.35

Enhalus acoroides Halophila ovalis

1.558 0.963

1.525 1.125

Seagrass biomass analysis in the study showed that Enhalus acoroides biomass estimation was greater than that of Cymodocea serrulata and Halophila ovalis (TABLE 4). This value was certainly related to the morphology of the leaves and the closure of each type of seagrass. The total estimated value of the seagrass biomass found on Talango Island was 1,721,250 g/Ha.

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TABLE 4. Biomass (Estimation) of Seagrass Species on Talango Islands, Madura.

Cymodocea serrulata Enhalus acoroides Halophila ovalis

Station 1 Ci 5.00 30.00 5.00 40.00 Station 2 Ci 5.00 30.00 5.00

Total

40.00

Species Cymodocea serrulata Enhalus acoroides Halophila ovalis Total Species

T (m2) 0.05 0.30 0.05

Cs (g/m2) 62.50 550.00 80.00

EB (g/Ha) 31,250 1,650,000 40,000 1,721,250

T (m2)

Cs (g/m2)

EB (g/Ha)

0.05 0.30 0.05

62.50 550.00 80.00

31,250 1,650,000 40,000 1,721,250

Enhalus acoroides is a very recognizable type of seagrass, because it has long, wide and stiff leaves. Halophila ovalis is also an easily recognizable type of seagrass, with its oval leaf shape. Meanwhile, Cymodocea serrulata has a special feature of strap-like leaves, with a leaf width of about 1 cm, and rounded 10. The frequency of seagrass emergence in each of these transect quadrants is thought to be related to the substrate type. Enhalus acoroides are known to have the widest tolerance and can live on mud substrate, sandy mud, muddy sand and sand. Meanwhile, Cymodocea serrulata can live on sand and sand muddy substrate. Furthermore, Halophila ovalis lives on a sand substrate with a little mud, sand or sand with rubble 11. This is very appropriate for the conditions in this study’s site, which has a muddy to sand substrate. The density of seagrass on Talango Island (39–40 individuals/m2) is known to be relatively lower than that in some other locations in Indonesia. This is similar to the results presented by Dewi et al. 12 and Dewi et al. 13, where the density of seagrass on Biak Island of Papua is between 450 individuals/m2 and 898 individuals/m2 and Nusa Lembongan Island of Bali, with 397 individuals/m2. Moreover, the density of seagrass at the location of this study is also lower than that of Kondang Merak Beach, which is in Malang East Java, i.e. 6,370 individuals/m 2 14. The highest important value index of seagrass on Talango Island is that of Enhalus acoroides, indicating that the species has an important role in the sustainability of the seagrass ecosystem at the study site. Enhalus acoroides can have a profound effect on the seagrass ecosystem, especially for organism associations that live in the substrate and that adhere to the seagrass. The roots of Enhalus acoroides are relatively large and hard and will stabilize the substrate so that some types of Polychaeta can live in it 11. The wide surface of the stems and leaves provides a good attachment capability for epiphytic organisms, such as periphyton 15. The biomass of seagrass can be estimated from the seagrass covering area value in each location. The seagrass biomass estimation on Talango Island is bigger than that on Pramuka Island, DKI Jakarta, amounting to 829,986.6 g/Ha 9. Seagrass biomass can illustrate the value of carbon stored in seagrass bodies, where stored carbon values generally account for about 10% of the biomass of organisms 5,16.

SUMMARY The conclusions of this research were as follows: (1) It is known that the seagrass on Talango Island consists of three types, namely Enhalus acoroides, Cymodocea serrulata and Halophila ovalis. (2) In fact, it is known that Enhalus acoroides are a type of seagrass that has an important role in the seagrass ecosystem, characterized by the highest importance value index, i.e.1.5. (3) Furthermore, the estimated value of the seagrass biomass on Talango Island is at each station, each of which is 1,721,250 g/Ha. One suggestion to emerge from this research is that the research time should be adjusted in line with the tide at the research location to identify the best time for data retrieval.

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ACKNOWLEDGMENTS This research is funded by FPIK Research and Community Service Board (BPPM) – the University of Brawijaya, through Operational Aid Fund of State Universities (BOPTN) of FPIK Featured Research, Universitas Brawijaya 2016.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

B. Mats, F. Short, E. Mcleod and S. Beer, Managing Seagrasses for Resilience to Climate Change (IUCN, Gland, Switzerland, 2008), pp. 56. P. S. Gartside and S. F. Smith, A Review of Mangrove and Seagrass Ecosystems and Their Linkage to Fisheries and Fisheries Management (Food and Agriculture Organization of the United Nations Regional Office for Asia and the Pacific, Bangkok, 2013). P. J. Hogart, The Biology of Mangroves and Seagrasses (Oxford University Press, United Kingdom, 2015). W. Kiswara, Oseanologi dan Limnologi di Indonesia 36, 361– 376 (2010). A. Rustam, L. K. Terry, N. A. Restu, L. S. Hadiwijaya, A. Mariska, D. August, M. Peter, S. Nasir, P. R. Yusmiana, D. S. Devi and H. Andreas, J.Segara 10, 107-117 (2014). Supriadi, “Produktivitas Lamun Enhalus acoroides (LINN. F) Royle dan Thalassia hemprichii (EHRENB.) Ascherson di Pulau Barang Lompo Makassar”. Thesis, IPB, 2003. H. Kordi, Ekosistem Lamun (Rineka Cipta, Jakarta, 2011). J. E. Brower, J. H. Zar and C. N. V. Ende, Field And Laboratory Method For General Ecology Fourth Edition (McGraw-Hill Publication, Boston, 1989). Y. M. Assuyuti, F. R. Alfan, R. Firdaus and B. Z. Reza, DEPIK 5, 85-93 (2016). M. Waycott, K. McMahon, J. Mellors, A. Calladine and D. Kleine, A Guide to Tropical Seagrass of The IndoWest Pacific (James Cook University, Townsville-Queensland Australia, 2004). Den Hartog, The Seagrasses of The World (North Holland, Amsterdam, 1970). C. S. U. Dewi, B. Subhan and D. Arafar, Jurnal Ilmu Ilmu Perairan Pesisir, dan Perikanan 6, 122 – 127 (2017). C. S. U. Dewi, S. Yusuf and Widiawati, “Status Padang Lamn di Daerah Budidaya Rumput Laut Pantai Barat Nusa Lembongan Bali”. Prosiding Seminar Nasional Perikanan dan Kelautan V. (Universitas Brawijaya, Malang, 2015), pp. 142 – 145. C. S. U. Dewi, R. D. Kasitowati and A Yamindago, “Bioprospecting of Seagrass from South Malang as Marine Natural Product”. Oral Presentation. The 5th Annual Basic Science International Conference. February 11 th – 12th 2015. Malang, Indonesia (2015). A. Wibowo, Umroh and D. Rosalina, Akuatik 8, 7-16 (2014). M. Wawo, Y. Wardianto, L. Adrianto and D. G. Bengen, JMHT 20, 51-57 (2014).

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A Study on the Utilization of Forest Policy to Review from the Aspect of Climate Change I Putu Gede Ardhana1,a) 1

Department of Biology, Faculty of Science, Udayana University, Denpasar, Bali, Indonesia a)

Corresponding author: crescentbali@indo.net.id

Abstract. The purpose of this study was to review the utilization of forests from the policy aspects of climate change. This was then associated with the implementation of governmental commitment to carry out REDD+ to cope with the impact of climate change and to achieve sustainable development. Firstly, the author studied this problem from data and information about vast forest areas and conservative water areas in Indonesia. According to provincial governments, there have been several decision letters from the Minister of Forestry from different years ranging from 1999–2014. Comparing the forest areas in letters of 2005, 2008, and 2015, it can be suggested that the areas allocated as productive forest exceeded the areas of conserved or protected forest. This indicates that the utilization of forest as a development resource has occurred, and will continue to become an important element in Indonesia. Furthermore, Indonesian forests continue to suffer deforestation and forest degradation. Therefore, the author presented data and information about deforestation and forest degradation that occurred from forest damage and forest fires. Thirdly, the author presented data and information about the deforestation rate from 2000–2014. In 1989, rehabilitation activities were carried out for critical lands, and from 2012–2014, rehabilitation of forest and riverside areas occurred. This research uses descriptive methods with an approximation of legislation and an approach to librarianship. Then, this study is described in a narrative as well as an interpretive style, and compiled in the form of a working paper. From the results of this research, it can be concluded that Indonesian governmental policy regarding forest utilization has wide potential mitigations, and it is absolutely necessary to consistently implement a number of such programs related to climate change.

INTRODUCTION Indonesia has rich forest resources. From the aspect of biological resources, it is known as one of seven countries in the world which has mega-biodiversity, and as a state which has the third largest tropical forest after Brazil and Zaire. Indonesia has a wealth of biological resources, owning 10% of flowering plants, 12% of mammals, 16% of reptiles and amphibians, 17% of birds, and 25% of fish 1. Forests serve as carbon sinks (CO2) from the atmosphere and convert it into organic carbon (carbohydrate), and keep it in the overall form of body volume tree (biomass). Biomass plays an important role in the carbon cycle. From the total forest carbon, about 47% of this is stored in the vegetation of forests 2. Therefore, the biomass of forests is very relevant to the issue of climate change, as it is caused by carbon emissions in cases of forest damage, fires, logging, deforestation, and degradation. In Indonesia, deforestation, forest degradation, and peat are some of the major contributors to greenhouse gases (GRK). From total GRK emissions of about 2,250,000 metric tons, the forestry sector and peat accounted for 84% of total GRK emissions. As a sector in a developing country, the Indonesian Government greatly depends on forestry and forestry-related industries such as agriculture and mining. For national development, forests are continuously exploited. This means that deforestation and forest degradation will continue and cannot be avoided. The extensive forest area was originally approximately 144 million hectares, but now only 130.68 million hectares remains 3. Each year, the extent of forest cover decreases with deforestation and forest degradation and accompanying forest fires during the dry season. According to data from the statistical sources of the Ministry of Environment in 2015, it clearly appeared that vast forest and water areas of Indonesia in every province were addressed in the decision letters of Ministry of Forestry from 1999–2015 (Table 1). This indicated that it was not very relevant if carbon emissions into the atmosphere were predicted according to the data of the decision letters, but 8th International Conference on Global Resource Conservation (ICGRC 2017) AIP Conf. Proc. 1908, 030006-1–030006-8; https://doi.org/10.1063/1.5012706 Published by AIP Publishing. 978-0-7354-1600-0/$30.00

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that it remained extensive from 1999-2015, with the vast majority of carbon emissions being released as a result of deforestation, degradation, and forest fires. Thus, the data before 2015 could account for the extent. Of course, every year every province should have the data for forest areas periodically reported to the Minister of Environment and Forestry. Such conditions would make it difficult to predict a number of carbon emissions released into the atmosphere due to extensive depreciation, and the forest may also increase as a result of activities in critical land rehabilitation and rehabilitation around watersheds (Table 2). This can be evidenced by the vast forest comparison data according to its function in 2005, 2008, and 2015. This condition also shows that extensive forest production exceeded that allocated by vast forest conservation and protected forest. It indicates that the utilization of forest as a development resource will continue to result in deforestation and forest degradation. Based on the results of the analysis of forest cover from 2000 to 2009, which was gathered by FWI 4, Indonesia has suffered deforestation of around 15 158 926.59 hectares with the rate of deforestation of about 1 515 892.66 hectares per year. The rate of deforestation increases due to utilization for non-food agriculture purposes such as forestry, fisheries, animal husbandry, mining, and a resettlement area of 13 025–053 hectares (Bureau of Statistics, the Ministry of Forestry, 1986). Spatially, in a number of the large islands in Indonesia such as Borneo, Sumatra, Java, Sulawesi, and Papua, the estimate of damage due to fire in 1997–1998, which spread in the jungle lowlands, mountain forests, peat swamps, dry shrubs, grass, forest industry plants, farms, and plantations was a total of about 9 745.00 hectares 5. The high rate of deforestation and forest degradation has reduced the ability of forests in Indonesia to absorb carbon. According to gather data and information, about 21 million hectares of peat could potentially unleash a huge volume of carbon and GRK. This shows that Indonesia can provide a large donation to restraining the rate of GRK emissions due to deforestation, forest degradation, and forest fires. In addition, the government has committed to lowering emissions by as much as 26% in the year 2020, with the support of 41% of international commitment. It poured over presidential Regulation No. 61 in 2011 about the national action plan for decreasing GRK emissions (RAN-GRK) and in new developments it was mentioned that the Indonesian Government is committed to lowering emissions by 29% in the year 2030 with the international support of 41% 6 of the results of the meeting of the COP (Conference of the Parties). From the above background, the author wants to review government policy in terms of forest utilization aspects of climate change.

EXPERIMENTAL DETAILS The method of this research involves a descriptive approach to regulation and legislation and the libraries are sourced regarding the results of research and analysis of the literature reports that relate to the research objectives. The results of this study have been described in the narrative as well as interpretative style and compiled in the form of a working paper.

RESULT AND DISCUSSION The author collected the statistical data sources from the Ministry of Environment and Forestry (2015) regarding vast forest conservation areas and water areas in Indonesia, according to the provincial decision letters of different years from 1999–2015, which can be seen in Table 1. These data are not so relevant if used to predict carbon emissions into the atmosphere, as the data are based on different decision letters of the Ministry in each province. Forest and water areas in each province have remained the same from 1999–2015, and these conditions will create an error result if used to predict emissions into the atmosphere, or of carbon absorption by forests. The years of each decision letter of the Ministry must be the same in order to determine the starting point of the basic conditions or standard lines which must be consistent with GRK emissions that can be saved. This is known as the Forest Reference Emission Level (REL) and is derived from the average of historical emissions in a specific period, so we must use a careful approach to assess the reduction of carbon emissions.

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TABLE 1. Area of forest and Conservation Area of Indonesian Waters by Provinces Based on Ministry of Forestry Decree Conservation

Decree

Province

Waters

Year

Mainland

(Hectare) Aceh

1

(Hectare)

Limited Permanent Convertible Total Area Land Total Area of Protection Production Production Production Area Forest Land and Forest Conservation Total Forest (Hectare) Forest (Hectare) Forest (Hectare) Forest (Hectare) (Hectare) Area (Hectare) (Hectare)

2015

-

1.058.144,00

1.058.144,00

1.788.265,00

141.771,00

554.339,00

15.409,00

3.557.928,00

3.557.928,00

2014

-

427.008,00

427.008,00

1.206.881,00

641.769,00

704.452,00

75.684,00

3.055.794,00

3.055.794,00

Sumatera Barat

2013

37.164,00

769.775,00

806.939,00

791.671,00

233.211,00

360.608,00

187.629,00

2.342.894,00

2.380.058,00

Riau

2014

-

633.420,00

633.420,00

234.015,00

1.031.600,00

2.331.891,00

1.268.767,00

5.499.693,00

5.499.693,00

Jambi

2014

-

685.471,00

685.471,00

179.588,00

258.285,00

963.792,00

11.399,00

2.098.535,00

2.098.535,00

Sumatera Selatan

2014

48.707,00

741.918,00

790.625,00

577.327,00

208.724,00

1.713.531,00

176.694,00

3.418.194,00

3.466.901,00

Bengkulu

2012

-

462.965,00

462.965,00

250.750,00

173.280,00

25.873,00

11.763,00

924.631,00

924.631,00

2000

-

462.030,00

462.030,00

317.615,00

33.358,00

191.732,00

-

1.004.735,00

1.004.735,00 654.562,00

Sumatera Utara

Lampung

1

Kepulauan Bangka Belitung Kepulauan Riau

2

DKI Jakarta Jawa Barat

1

2012

-

35.454,00

35.454,00

185.531,00

-

432.884,00

693,00

654.562,00

2015

-

12.294,72

12.294,72

97.662,65

118.833,75

78.830,37

74.510,24

382.131,73

382.131,73

2000

108.000,00

272,34

108.272,34

44,76

-

158,35

-

475,45

108.475,45

2003

-

132.180,00

132.180,00

291.306,00

190.152,00

202.965,00

-

816.603,00

816.603,00

Jawa Tengah

2004

110.117,00

16.413,00

126.530,00

84.430,00

183.930,00

362.360,00

-

647.133,00

757.250,00

DI Yogyakarta

2000

-

910,34

910,34

2.057,90

-

13.851,28

-

16.819,52

16.819,52

Jawa Timur

2011

3.506,00

230.126,00

233.632,00

344.742,00

-

782.772,00

-

1.357.640,00

1.361.146,00

Banten

3

1999

51.467,00

112.991,00

164.458,00

12.359,00

49.439,00

26.998,00

-

201.787,00

253.254,00

Bali

1999

3.415,00

22.878,59

26.293,59

95.766,06

6.719,26

1.907,10

-

127.271,01

130.686,01

Nusa Tenggara Barat

2009

11.121,00

168.044,00

179.165,00

430.485,00

286.700,00

150.609,00

-

1.035.838,00

1.046.959,00

Nusa Tenggara Timur

2014

256.482,00

260.219,00

516.701,00

684.403,00

173.979,00

296.064,00

113.604,00

1.528.269,00

1.784.751,00

Kalimantan Barat

2014

190.945,00

1.430.101,00

1.621.046,00

2.310.874,00

2.132.398,00

2.127.365,00

197.918,00

8.198.656,00

8.389.601,00

Kalimantan Tengah

2012

22.542,00

1.608.286,00

1.630.828,00

1.346.066,00

3.317.461,00

3.881.817,00

2.543.535,00

12.697.165,00

12.719.707,00 13.855.833,00

Kalimantan Timur dan Kalimantan Utara

4

2014

-

1.704.666,00

1.704.666,00

2.848.243,00

5.045.879,00

4.077.346,00

179.699,00

13.855.833,00

Sulawesi Utara

2014

69.800,00

245.165,00

314.965,00

161.784,00

208.927,00

64.367,00

14.696,00

694.939,00

764.739,00

Sulawesi Tengah

2014

340.119,00

648.374,00

988.493,00

1.276.087,00

1.390.971,00

401.814,00

217.322,00

3.934.568,00

4.274.687,00

Sulawesi Selatan

2009

606.804,00

244.463,00

851.267,00

1.232.683,00

494.846,00

124.024,00

22.976,00

2.118.992,00

2.725.796,00

Sulawesi Tenggara

2011

1.504.160,00

282.924,00

1.787.084,00

1.081.489,00

466.854,00

401.581,00

93.571,00

2.326.419,00

3.830.579,00

Gorontalo

2010

-

196.653,00

196.653,00

204.608,00

251.097,00

89.879,00

82.431,00

824.668,00

824.668,00

Sulawesi Barat

2014

-

215.190,00

215.190,00

452.030,00

330.700,00

71.859,00

22.597,00

1.092.376,00

1.092.376,00

Maluku

2014

9.208,00

420.330,00

429.538,00

627.256,00

894.258,00

643.699,00

1.324.866,00

3.910.409,00

3.919.617,00

Maluku Utara

2013

-

218.499,00

218.499,00

584.058,00

666.851,00

481.730,00

564.082,00

2.515.220,00

2.515.220,00

Papua Barat

2014

928.350,00

1.711.908,00

2.640.258,00

1.631.589,00

1.778.480,00

2.188.160,00

1.474.650,00

8.784.787,00

9.713.137,00

Papua

2012

1.019.017,00

6.736.267,00

7.755.284,00

7.815.283,00

5.961.240,00

4.739.327,00

4.116.365,00

29.368.482,00

30.387.499,00

5.320.929,00

22.108.630,99

27.429.555,99

29.673.382,37

26.798.382,01

29.250.783,10

12.942.295,24

120.773.441,71

126.094.366,71

Indonesia

Source: Statistics Ministry of Environment and Forestry 2015

Additionally, the possibility of shrinking area spaces will occur if deforestation and forest or land degradation increase, and rehabilitation activities for crisis land like surrounding watershed areas and extensive forest areas will also increase. Government efforts to recover forest in the critical land is aimed at about 1,221,814 hectares, but yet remains at about 5,830,200 hectares, and government efforts to carry out rehabilitation and greening in the critical land is 5,814,545 hectares outside national forests, but yet remains at 7,269,700 hectares. These activities are carried out in some islands of Indonesia to execute government commitment for dealing with climate change 7, as a manifestation of the government's commitment to addressing climate change. In addition, the government also carried out forest rehabilitation from 2010–2014 and until 2014, approximately 2.5 million hectares was identified as a target for forest rehabilitation by the government for necessary conservation of watersheds, city forests, mangrove forests, cities, and swamps 8. It has also shown the commitment of the government to deal with climate change. From the activities of critical land forest rehabilitation, extensive areas come back to coverage with forest. Therefore, it can be concluded that the statistical data of the Ministry of Environment and Forestry in 2015 cannot

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be used to predict forest carbon, as the carbon emissions will create an error. This can be evidenced by the vast forest comparison data according to its function in 2005, 2008, and 2015, as shown in Table 2 below. TABLE 2. Comparison of vast forest kind in 2005, 2008 and 20015

Types of forest

2005

2008

2015

(million hectares)

(million hectares)

(million hectares)

Conservation forest

20.080

19.908

22.109a

Protected forest Limited productions forest

31.782 21.717

31.604 22.502

29.673 26.798

Permanent productions forest Convention production forest

35.813 14.057

36.649 22.795

29.251a 12.942

The function has been defined Total Figures are rounded

0.007 123.459a

0.233 133.694a

0 120.773

Source :Kemenhut (2006, 2009a, KemenLHK, 2015)

Table 2 shows that up to 2005, 2008, and 2015, extensive forest areas have decreased as widespread episodes of shrinkage have occurred. These conditions have also been shown to exceed forest production, which involved vast conserved and protected forests. It indicated that the utilization of forests as a development resource will continue to result in deforestation and forest degradation. Deforestation can be interpreted as the change in forest cover due to governmental policies for the utilization of the forest, both planned and unplanned, legitimate or illegal activities. Forest degradation can also be caused by legitimate or illegal activities, for example taking of legal forest products and seeking illegal woods. The allocation of forests occurs when forests are converted from a non-forest area, such as plantation of agricultural land, mining, and settlement. Extensive forest areas were converted to 4.5 million hectares in 2007 and increased in 2010 to about 4.9 million hectares 9. Up to mid-2010, the government provincial in Sumatra, Kalimantan, and Sulawesi proposed to convert use of an area of 6.5 million (Ministry of Forestry, 2010). The conversion of forests to palm oil plantations is the dominant reason for the forest decrease. According to the data of Palm Watch 11, palm oil plantations reached 1,652,301 hectares in 1989. During 1993-1994 these increased to 3,805,113 hectares and more and in 1998 increased again to 8,204,524 hectares. The increase in conversion of forests to palm oil plantations due to the interconnectedness with renewable energy in the world and vegetable ingredients as a source of alternative energy meant that as much as 10.25 million hectares of land were needed for national vegetable materials in 2015 (Minister for Research, 2007). According to the data of the Ministry, the palm oil plantations, both large and small, increased every year and reached 7 007 867 million hectares in 2008 and 8 430 026 million hectares in 2010 10. Within both the Palm Center of Borneo and Sumatra the limitation of land availability has led the government to plan an expansion in Papua (AFP, 2008). The government has published several regulations and policies, enacted as Act No. 18 of 2004, regarding plantations that introduce the right to attempt (HGU) for 35 years for the plantation owners. An accelerated government program was published as Presidential Decree No. 5 of 2006, regarding the national energy policy, and Presidential Instruction No. 1 of 2006 about provision use of biofuels as an alternative fuel. In 2007, the regulations of the Minister of Agriculture No. 26/Permentan/OT/140/2/2007 was published, which stated that lands for palm oil plantations in Papua were provided twice the normal broad 100,000 hectares. Then, followed the regulation of the Minister of Forestry No. P22/Menhut-II/2009 as the legal basis for the palm oil companies to possess estates of 100,000 and 200,000 hectares in Papua. Next, the national government published principle permits and decisions to convert forest areas to palm oil plantations, with a total area of 9.13 million hectares (AFP, 2008). According to Palm Watch 11, in 2009 these areas almost tripled at 26.7 million hectares and were planning to convert about 2.8 million hectares areas in the following years to palm oil in Papua. The mining sectors also require development for the conversion of forests, often compared with agriculture and plantations. According to the data of the Ministry of Forestry (2009a), lease licenses were used to cover 344,000 hectares of mining areas until 2008. However, many mining activities including the licenses which were issued by the district government really did not operate based on the licenses’ 12-14. These two factors and small mining activities were carried out to cover the real impact of mining on forests mining. In addition, many mining activities

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are carried out in conservation areas and protected forests which should be protected if many mineral ores are found in these areas. Although the laws prohibit opencast mining in the protected forest, at least 13 companies acquired operational licenses for nearly 850,000 hectares of protected forest areas before the laws were enacted, and were considered as exceptions, so these companies continued their activities listed in the attachment decision President no. 41 of 20046. Governmental policy regarding utilization of forest for mining plans within protected forest and forest conservation areas has been running since 2000, with a total of about 11 441 852 hectares (Department of Forestry, 2000). These conditions increased forest destruction from deforestation and forest degradation. Forest utilization plans like these should not actually happen because forest conservation and forest protection actually aim to protect and preserve ecosystems and their ecological functions. However, in practice, other activities like mining will be able to be carried out to the detriment of these forest types. The Ministry of Forestry has the authority to decide where national forest areas are, and their use for non-forestry activities such as mining to forest conservation and protected areas. The lease licenses for forest areas are regulated by Government Ordinance No. 24 in 2010, regarding the utilization of forest areas that replaced the guidelines for issue of lease licenses for utilization permits in the regulation of the Minister of Forestry No. P. 64/Menhut-II/2006. This ordinance is one of the contributing factors to deforestation and forest degradation in Indonesia. In the laws and regulations which are outlined in the regulation of the Minister of Forestry No. P. 30/Menhut/II/2009, regarding the procedures for reducing emissions from deforestation and forest degradation, it is defined that "deforestation means the permanent change of forest areas from forest not because of human activities” (article 1 (10)). In this regulation, "degradation" is defined as meaning to decrease the amount of forest cover and carbon reserves during a certain period due to human activities (article 1 (11)). Here, according to the government, human activity relates to perpetrators of illegal logging and deforestation to perpetrators of forest degradation. Illegal logging is also one of the biggest threats of deforestation through forest degradation. The damaged forest is easier to open, so that forest degradation activities occur easily, such as forests being cut down and not been cared for by concessionaire holders who rarely leave trees in the forest, so allowing easy conversion to agricultural lands and plantations. In Indonesia, the high rate of deforestation and forest degradation areas occur in Sumatra and Kalimantan with illegal logging in all types of forests, such as fixed production forests, conserved, production forest, forests conserved and non-forest protected areas from logging, hauling, and distribution of the wood, until the implementation of the rule of law 15. The issue of licenses for the forest plant industry (HTI) in old-growth forests also became a trigger for forest degradation, after the government started the program of HTI for pulp and paper in the 1990s. The government developed the HTI in 1995, covering 1.4 million hectares, then in 2000 this increased to approximately 1.8 million hectares, and 2.3 million hectares in 2000, with plans to increase to 10.5 million hectares of HTI by 2030 (Resosudarmo et al., 2003). The opening of forest areas occurred not only in national forest areas but also outside national forest areas, known as areas for other utilization (APL). According to a circular letter to the Minister of Forestry No. SE 9/Menhut-VI/2009, regarding volume of economic timber in areas of lease utilization forest for other utilization (APL), licenses has been issued to permit allocation which expressed permission to use wood not necessary for production capacity to less than 50 m3 of wood volume with up to 30 cm in diameter, with intensity of 100% in one candidate's permission to use the wood. It was shown that the activities in forest areas to open the APL will also increase deforestation and forest degradation. With a delay in the enactment of regulations, "blanks" often occur in the new legislation or change of law, as the old legislation continues to apply. These blanks generate uncertainty about more specific guidelines that must be adhered to and referenced by the projects or programs carrying out these activities, such as REDD+ as in Government Ordinance No. 27 in 2012 about the environmental license of environmental legislation for the protection and management no. 32 in 2009. Presidential Instruction No. 10 in 2011, which was issued on 20 May 2011, announced the postponement of the issue of new forest concession licenses. This instruction aimed to suspend new licenses for the cutting down of forests for two years. Its enforcement involved points that influenced the issue of new licenses. These happened five months before the Presidential Instruction was proclaimed. In the next 11 days after its delay was enforced, the Ministry of Forestry announced the Minister's decision 292/Menhut II/2011 regarding the change in the status of forest areas and made a non-forest area of almost 1.2 million hectares in Central Kalimantan. This resulted in unresolved matters of forest areas and the status of the land affected by the postponement 16.

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In addition, there is the exception with delay activities related to food security and energy (Ministry of Agriculture and the Ministry of Energy and Mineral Resources is not covered in the Presidential Instruction), which has opened new divisions that can stimulate new delays in licenses, although the moratorium map of indication which has been announced continually was renewed in 2011, and will be kept updated by the Ministry of Forestry, becoming the tool of supervision for the public to secure and perhaps even to add the extents covered by such delays (Cifor, 2013), so that deforestation and forest degradation are unavoidable. This moratorium, which was entered in Presidential Instruction No. 10, delayed the issue of new licenses for the primary natural forests and peatlands, located in conservation protected forests, forest production and other utilizations stated in the indicative map of the new delay of licenses (PIPIB). In practice, the moratorium map was not a map which was dead, and did not change, as it was revised every six months, or when complaints arose from license holders who got licenses before Presidential Instruction No. 10 in 2011, it could just be changed because there was an announcement of an exception delay with related energy and food security activities, as described above. The actual data for deforestation has been running since 1986 covering 13,025,053 hectares, which are utilized for non-food purposes such as agriculture forestry, fishing, stock farming, mining, and transmigration (Bureau of Statistics Department of Forestry, 1986). Until 1987, HPH reached 19 HPH with 564 units with about 55,468.35 million hectares of forest area (Bureau of Planning and Food Department of Forestry, 1986). Based on the results of the analysis of forest cover from 2000 to 2009, which were gathered by FWI 4, Indonesia has suffered deforestation of around 15,158,926.59 hectares, with a rate of deforestation of about 1,515,892.66 hectares per year. Forest fires are also triggers for deforestation and forest degradation, which result in the destruction of forests. Indonesia suffered serious forest fires in 1997–1998. These fires resulted from the utilization of forest land by careless conversion or improper combustion of land, as well as the deliberate burning of wild activities by people who were harmed, and usually due to disputes about the change of traditional rights over land utilization. In 1997– 1998, fires occurred in the Sumatra district covering 1.7 million hectares, 6.5 million hectares in Borneo, 0.1 million hectares in Java, 0.4 million hectares in Sulawesi, and in Papua about 1 million hectares of forest categories, which involved utilization lands such as mountain forest in 0.1 million hectares, lowland forests of 3.3 million hectares, 1.45 million hectares of peat forest, open grassland and farmland of 4.6 million hectares, and plantation and HTI of 0.3 million hectares with total burned areas in 1997-1998 of about 9.7 million hectares. The large-scale cultivation of land was pushed with governmental policies announced early in the 1980s, especially, Kepmentan No. 764/Kpts/Um/1980, regarding the release of forest for plantations, agriculture, fisheries, and food security, and the Ministry of Forestry No. 417/II/1986 regarding the plantation timber industry (Indranto et al., 2003). Forest fire areas continued every year from 1999–2015 (Kemenhut, 200917, 2015) as shown in Table 3. TABLE 3. Extensive forest fires in Indonesia, 1999-2015

No. 1 2 3 4 5 6 7 8

Year

Extensive forest fire (hectares)

1999 2000 2001 2002 2003

44.090 3.016 14.329 35.496 3.545

2004

3.343

13 14

5.501 4.140

15 16

2005 2006

No. 9 10 11 12

Year

Extensive forest fire (hectares)

2007 2008 2010 2011 2012

6.974 6.793 3.500 2.011 9.606

2013

4.918

2014 2015

44.411 11.226

Sumber: Kementerian Kehutanan (2009b) (data 1999-2008) dan http://sipongi.menlhk.go.id/hotspot/luas_kebakaran (data 2010-2015)

Table 3 shows that the extensive forest fires did not decrease from 1999–2015 in Indonesia. The government had established the center for the control of forest fires which is charged with the prevention of the occurrence of fires. This was implemented with the National Coordination Team for controlling forest land fires. However, forest and land fires continued due to the inadequacy of prevention plans, management, budget, equipment, and human resources.

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In addition, most of the land and forest fires occurred in areas of peat during the period 2001–2012 as shown in Table 4. TABLE 4. Extensive peat cumulative annual school of biological oxidation and fires in Indonesia during the period from 2001 to 2012

Year10

Extensive peat who experience biological

First

Second

Third

oxidation (hectares)

Fire

Fire

And subsequent Fire

2001 2002 2003

8.788.942 9.027.177 9.255.687

69 9.544 2.452

109.569 558.328 174.069

0 45.431 72.525

2004 2005

9.540.238 9.890.367

6.768 16.720

252.339 168.521

151.882 158.664

2006 2007

10.414.498 10.677.356

22.462 3.625

441.647 43.080

332.452 66.613

2008 2009 2010 2011

10.952.204 11.361.302 11.563.432 11.821.646

7.882 17.664 2.008 5.455

39.179 166.760 20.783 95.383

80.587 299.092 66.490 230.646

2012

12.083.405

947

89.032

262.522

11

10

Indicate the year of the occurrence of extensive peat changes from the previous year. For example, the year 2001 shows the changes in the area of peat from 2000 to 2001 11 This shows the cumulative vast peat oxidation in school annual biological including extensive peat was broken in the previous year (including land deforestation prior to 2001), which contributes towards emissions run from the biological oxidation of peat. Source: KemenLHK (Badan Penelitian, Pengembangan dan Inovasi, 2015)

Table 4 shows that the extensive peat areas suffered biological oxidation every year including extensively damaged peat which increased from 2001–2012. These conditions were caused by the need for biological oxygen in damaged peatlands increasing from year to year due to peat soil conditions which were dry. Peat fires from 2001– 2012 demonstrated variations of the first fire, a second fire, third fire and so on. However, the most extensive first fires occurred in 2006 and 2009 respectively, involving 22,462 hectares and 17,664 hectares. The second fires occurred in 2002, reaching 558,328 hectares and 2006 reaching 441,674 hectares. Third and subsequent fires occurred in 2006 reaching 332,452 hectares and 299,092 hectares in 2009. These conditions indicated that in 2002, 2006, and 2009, the content of the coals were still burning from the previous year, as the peat became dry due to careless utilization of forest policies that actually should be protected and preserved, as it serves as a buffer system for the protection of life in the forest and peat ecosystems. These conditions coupled with high temperatures and strong winds in the dry season mean that forest fires are easily spread. GRK emissions from deforestation are mainly caused by land utilization activities related to the conversion of forest land into the non-forest land of around 95%, and with great intensive fires that also cause deforestation events accounting for 5% of emissions from deforestation GRK. The occurrences of deforestation in the productive forest could account for 44%, and the occurrences of deforestation in forest lands allocated to APL accounted for 43% GRK. High emissions from forest degradation that appear to be caused by conventional loggings account for 62% and 38% of forest fires. Forest degradation often results in broken remnants of trees that are prone to further degradation, which will cause fires during the dry season. Degradation also occurs in conservative forest and protected forest areas, which reaches 13% with logging activities and forest encroachments. Forest degradation in productive forests reaches 66% with conventional logging and forest fires. Forest degradation occurs in conservative forests and protective forests at around 20%, as well as in the APL at about 15%, which are both caused by illegal logging and forest fires 18.

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The high rate of deforestation and forest degradation have reduced the forests’ abilities to absorb carbon in Indonesia. Obviously, Indonesian forestry sectors have potential mitigations or preventive activities for deforestation and forest degradation, so the management of forest resources which deal with climate change are thoroughly required.

ACKNOWLEDGMENTS Thank you, I convey to the Committee and ICGRC 8th which was to provide the opportunity and keep participation on Seminar Internasional "Green Movement for Global Conservation" in Malang, 19-20 Juli 2017.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18.

http://sipongi.menlhk.go.id/hotspot/luas_kebakaran Badan Standarisasi Nasional, Pengukuran dan Penghitungan Cadangan Karbon Pengukuran Lapangan untuk Penaksiran Cadangan Karbon Hutan (Ground based forest carbon accounting) (Badan Standarisasi Nasional, Jakarta, 2011). Ministry of Forestry, Statistik Kehutanan Indonesia Tahun 2010. Kementrian Kehutanan Republik Indonesia (2011). www. dephut.go.id/index.php?q=id/node/8347 [04 Juni 2017] Forest Watch Indonesia (FWI), Potret Keadaan hutan Indonesia, periode Tahun 2000–2009 (FWI, Bogor, Indonesia, 2011). G. Applegate, R. Smith, J. J. Fox, A. Mitchell, D. Packham, N. Tapper dan G. Baines, Kebakaran Hutan di Indonesia Dampak dan Pemecahannya “Ke Mana Harus Melangkah”. (Yayasan Obor Indonesia, Jakarta, 2003). I. P. G. Ardhana. 2012. Orasi Ilmiah “Kajian Kerusakan Sumberdaya Hutan Akibat Kegiatan Pertambangan” FMIPA Universitas Udayana. Denpasar Anon. The Land Resources of Indonesia. A. National Review (Ministry of Transmigration. Jakarta, 1990), pp. 294. Pusat Humas Kementerian Kehutanan. 2011. Kehutanan Indonesia. GIZ, Kemeterian Kehutanan (2009). Ministry of Forestry, Statistik Kehutanan Indonesia Tahun 2008. Kementrian Kehutanan Republik Indonesia, Jakarta, Indonesia. www. dephut.go.id/index.php?q=id/node/6122 [04 Juni 2017]. Ministry of Forestry, Ministry of Forestry Working Group baseline and mitigation scenarios, Kementrian Kehutanan (2009). Sawit Watch, Peta investigasi Sawit Watch (Sawit Watch, Bogor, 2009). I. A. P. Resosudarmo, J. Carol and P. Colfer, Ke Mana Harus Melangkah? “Masyarakat, Hutan, dan Perumusan Kebijakan di Indonesia” (Yayasan Obor Indonesia, Jakarta, 2009). I. A. P. Resosudarmo, S. Mardiah and N. A. Utomo, “Extractive land use, spatial planning, and their implications for REDD+ in Indonesia: A preliminary analysis”. Paper to 3rd IRSA International Institute Conference (Padang, Indonesia, 2011). I. A. P. Resosudarmo, G. B. Indrarto, P. Murharjanti, J. Khatarina, I. Pulungan, F. Ivalerina, J. Rahman, M. N. Prana and E. Muharrom, Konteks REDD+ di Indonesia “Pemicu, pelaku, dan lembaganya” (CIFOR & ICEL, Jakarta, 2013). Indonesian Center for Environmental Law (ICEL). 2006. Manual investigasi illegal logging dengan pendekatan UU hehutanan, UU tindak pidana pencucian uang, UU pemberantasan tindak pidana korupsi. ICEL, Jakarta, Indonesia. www. icel. or.id/manual-investigasi-illegal-logging-denganpendekatan-uu-kehutanan-uu-tindakpidanapencucian-uang/ [06 Juni 2072]. Murdiyarso, REDD+ Realities in Indonesia. Dalam: Angelsen, A. (ed.) Realising REDD+: National Strategy and Policy options (CIFOR, Bogor, 2009), pp. 32-33 Ministry of Forestry, Eksekutif data strategis kehutanan 2009. MoF (2009). INCAS, Inventarisasi Nasional Emisi dan Serapan Gas Rumah Kaca di Hutan dan Lahan gambut Indonesia (Kementerian Lingkungan Hidup dan Kehutanan, Badan Penelitian, Pengembangan dan Inovasi, Jakarta, 2015).

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The Implementation of Biological Monitoring Working Party Average Score Per Taxon (BMWP-ASPT) in a Water Quality Analysis at Kalibokor Drainage in Surabaya Region Kristiandita Ariella1, a) and Atiek Moesriati1, b) 1

Environmental Engineering Department, Sepuluh Nopember Institute of Technology, Arif Rahman Hakim Street, PO BOX 60111, Indonesia a)

Corresponding author: akristiandita@yahoo.co.id b) atiek@enviro.its.ac.id

Abstract. This study was carried out in March 2017 at Kalibokor drainage in Surabaya. The aims of the study are to determine the water quality and to investigate the environmental quality using the Biological Monitoring Working Party Average Score Per Taxon (BMWP-ASPT) indices. Water quality was identified using the BMWP-ASPT method. Its score was calculated based on determining the number of macroinvertebrate taxa found. The result shows that ten taxa were detected at Kalibokor drainage, i.e., seven taxa from Mollusca, one taxon from Coleoptera, one taxon from Oligochaeta, and one taxon from Crustacea. Our study indicated that the water quality at Kalibokor drainage is poor and heavily polluted. However, this result is obtained from a particular sampling point. At other sampling points, we found an average level of polluted water quality. This finding also strongly demonstrates that these indices should be adjustable based on the geomorphological and environmental features of Surabaya. Subsequent research should be performed intensively to recognize the effectiveness of the BMWP-ASPT indices for measuring water quality.

INTRODUCTION Kalibokor Channel is one of the primary drainage systems in Surabaya and is classified as water class III based on Perda Surabaya number 2 (2004). The environmental council (BLH) of Surabaya has stated that the water quality level at the Kalibokor drainage has already been degraded. Since it passes some area activities such as domestic, educational, and commercial activities, their wastewater has spread many water pollutants into the Kalibokor Channel. Even though the council has undertaken physicochemical monitoring, biological methods using macroinvertebrates have not been applied specifically to this drainage. The aim of this study is to monitor the Kalibokor drainage’s water quality. In this study, a BMWP-ASPT method is used to determine the water quality based on its pollution level. The biomonitoring method utilizes the presence of bioindicators as a water quality indicator. Bioindicators are groups of organisms that are sensitive to changes in environmental conditions. Changes in environmental conditions will affect the life and existence of the organism so that it can be used as a water quality guide.1 The bioindicator used is a non-invertebrate organism (macroinvertebrate). Macroinvertebrates play an important role in mineralization processes and maintain the stability of the bottom substrate of the water. Consequently, their existence plays a role in the chemical processes that occur in the water.2 This biomonitoring method is used for research, as it does not take a lot of time, is low cost as the equipment is easy to use and self-made, and the sampling process does not require special skills so that the public can participate in monitoring the cleanliness of the river and its surrounding environment 3. However, the lack of biomonitoring methods is unknown specifically for pollutants affecting environmental quality and the results of the study are annual. The chemical method has the advantage that it can identify certain pollutants that affect the quality of water


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bodies and is temporary. Therefore, the determination of water quality using macroinvertebrates can be used as an alternative method of water quality monitoring to overcome the water quality problem.

EXPERIMENTAL DETAILS Our study started by determining sampling locations by considering some factors such as the dimension of the drainage system, hydraulic aspects, and the river bends or creeks. We selected eight sampling points along the channel and the samples were taken over a month with four repetitions to get the accurate data.

P1

P2

P3 P4

(a)

P5

(b) P8 P7 P6

(c) FIGURE 1. Sampling Location (a) first segment, (b) second segment, and (c) third segment

Sampling is done by placing nets on the river bed. The place around the mesh frames is stirred so that the macroinvertebrates that exist between the rocks are carried away by the water and caught in the net. The method can be repeated at least four times per sample location. After sampling, there is an advanced stage before the sample is identified as follows 4: 1. Separation of macroinvertebrates The raised substrate is separated from the macroinvertebrates using a 500μm net. Samples are separated from mud, twigs, or stones carried during sampling using tweezers and sorting containers. 2. Preservation of macroinvertebrates Preservation of the sample is done using 70% alcohol to keep the sample in good condition until it is analyzed in the laboratory. 3. Identification of macroinvertebrates The principle of assessment of the biotic index using BMWP-ASPT method is to identify the most tolerant macroinvertebrate families using the BMWP-ASPT table. Once the most tolerant macroinvertebrates are identified, a water biotic index is determined based on the values listed in the BMWP-ASPT table. The calculation phase in detail is listed on the following points 5: 1. Identify the macroinvertebrates at the point to be identified with its tolerance level against contamination based on the BMWP-ASPT interpretation table score. 2. Thereafter, the score for each of the macroinvertebrate taxa found in the BMWP-ASPT interpretation table score is used. 3. Scores gained for each type of macroinvertebrate in a given location are summed and divided by the number of taxa found.

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Once the biotic index value is known, it is interpreted using the pollutant index table of the BMWP-ASPT method. TABLE 1. BMWP ASPT Interpretation Score Table

Famili Siphlonuridae Heptageniidae Leptophlebiidae Ephemerellidae Potamanthidae Ephemeridae Taeniopterygidae Leuctridae Capniidae Perlodidae Perlidae Chloroperlidae Aphelocheiridae Phryaneidae Molannidae Beraidae Odontoceridae Leptoceridae Goeridae Lepiddostomatidae Brachycentridae Sericostomatidae Astacidae Lestidae Agriidae Gomphidae Cordulegastridae Aeshnidae Corduliidae Libellulidae Psychomyiidae Philopotamidae Caenidae Nemouridae Rhyacophilidae Polycentropodidae Limnephilidae Neritidae Viviparidae Ancylidae Hydroptilidae Unionidae Corophiidae Gammaridae Platycnemididae Coenagriidae Mesoveliidae Hydrometridae Gerridae Nepidae Naucoridae Notonectidae Pleidae Corixidae Haliplidae Hygrobiidae Dytiscidae Gyrinidae Elminthidae Hydropsychidae Tipulidae Simuliidae Planariidae Dendrocoelidae Baetidae Sialidae Piscicolidae Viviparidae Hydropbiidae Lymnaeidae Physidae Planorbidae Sphaeriidae Glossosomatidae Hirudidae Erpobdellidae Asellidae Chironomidae Oligochaeta (all classes)

Skor 10

8

7 6 5

4 3

2 1

Source : Armitage et al., (1983)

Based on its score in the BMWP-ASPT table, we can determine the pollution level score using the BMWP-ASPT. Table 2 shows the range of scores for each pollution class level.

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No. 1 2

Pollution Level Excellent Good

3

Moderate

4 5

Fair Poor

TABLE 2. BMWP ASPT Interpretation Score Table for Each Class Macroinvertebrate Indicator Trichoptera (Sericosmatidae, Lepidosmatidae, Glossosomatidae); Planaria Plecoptera (Perlidae, Peleodidae); Ephemeroptera (Leptophlebiidae, Pseudocloeon, Ecdyonuridae, Caebidae); Trichoptera (Hydropschydae, Psychomyidae); Odonata (Gomphidae, Plarycnematidae, Agriidae, Aeshnidae); Coleoptera (Elminthidae) Mollusca (Pulmonata, Bivalvia); Crustacea (Gammaridae); Odonanta(Libellulidae, Cordulidae); Hirudinea (Glossiphonidae, Hirudidae); Hemiptera Oligochaeta (ubificidae); Diptera (Chironomus thummi-plumosus); Syrphidae There’s no benthos in the river

Source: Trihadiningrum and Tjondronegoro, 1998

RESULT AND DISCUSSION 10 9 8 7 6 5 4 3 2 1 0 T1

T2

T3

1st Sampling

T4

T5

T6

T7

T8

2nd Sampling

FIGURE 2. Observation result for four times sampling

Based on the data presented in Table 2, the results of the macroinvertebrate analysis with the BMWP-ASPT method indicates that points 2, 4, and 6 have a medium level of contamination. While points 1, 3, 5, 7, and 8 pertained to heavy pollution. This is because along the Kalibokor Channel it was found that many domestic waste disposal pipes coming from residential areas flow directly flow into the water body as you can see in Figure 3.

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FIGURE 3. Sampling Location Existing Condition

Visually, the color of the water, which is dominated by green and turbid brown with a pungent smell, is clear proof that the water in the Kalibokor Channel is contaminated. The habit of the surrounding people who throw garbage into the river is also one of the factors contributing to the high pollution level in the Kalibokor Channel. Based on the environmental conditions in the Kalibokor Channel, the type of macroinvertebrates that can live there is very limited. Poor environmental conditions are reinforced by laboratory analysis results showing some chemical parameters that do not meet quality standards. The chemical parameters tested are also factors that affect the presence of macroinvertebrates. Physicochemical monitoring of water quality has been undertaken by the environmental council (BLH) of Surabaya. The result of their monitoring is that the Kalibokor drainage is highly polluted by organic pollutants. Organic pollutants come from domestic wastewater that flows directly from residences to the Kalibokor drainage. Organic pollutants also affect the macroinvertebrates because the oxygen they need to live has been consumed by microorganisms who decompose the organic compounds in the water. Consequently, aquatic biota is decreased because they do not get the nutrients they need to survive. 6 In addition, hydraulic factors can also affect the presence of macroinvertebrates. River flow affects the presence of macroinvertebrates on the river bed. The large current velocity can sweep the macroinvertebrates to another place so that it can affect the benthos found during sampling.7 This also results in the findings from physicochemical and biological methods being the same.

SUMMARY The conclusion of this research, determining water quality through the BMWP-ASPT method as a biotic index by using macroinvertebrates of the Kalibokor Channel, is that the water of the channel is heavily polluted. Macroinvertebrates found along the canal consist of ten different families: Elmidae, Thyariidae, Planorbidae, Corbiculidae, Lymnaeidae, Viviparidae, Anodontidae, Sphaeriidae, Sundathelpusidae, and Tubificidae. Suggestions for this research include: 1) similar research is needed to determine water quality in the dry season to compare the presence of macroinvertebrates in two different seasons; 2) there needs to be a study comparing the condition of polluted and unpolluted rivers, as the presence of macroinvertebrates is strongly influenced by the physical and chemical conditions of the river; 3) monitoring with macroinvertebrates should consider hydraulic factors such as current velocity, water depth, and channel dimensions.

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ACKNOWLEDGMENTS I would like to convey my sincere gratitude to ICGRC 2017 committees who have pointed my research paper to be published in this proceeding. I would also thank the proceeding editors for the language editing and proofreading of the manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

C. Brito, Biological Monitoring Using Macroinvertebrates as Bioindicators of Water Quality of Maroaga Stream in the Maroaga Cave System, (Presidente Figueiredo, Amazon, 2014) B. Thompson and S. Lowe, J Environ Toxico, 127 (2004). S. Tjokrokusumo, J Hidrosfir 1, 8-20 (2006). S. Rahayu, R. H. Widodo, M. van Noordwijk, I. Suryadi and B. Verbist, Monitoring Air di Daerah Aliran Sungai, (World Agroforestry Centre - Southeast Asia Regional Office, Bogor, 2009). Y. Trihadiningrum and I. Tjondronegoro, Bioindikator Pencemaran Badan Air Tawar di Indonesia : Siapakah Kita, Lingkungan dan Pembangunan (1998). Risamasu, J. L. Fonny and H. B. Prayitno, Jurnal Ilmu Kelautan, 16, 135-142 (2011). I. P. M. Unggul, “Biomonitoring Kualitas Air Sungai Sampeyan Bondowoso dilihat dari Keanekaragaman Makroinvertebrata dan Kualitas Ekologi Struktur Sungai”, Bachelor Thesis, Jurusan Teknik Lingkungan ITS, Surabaya, 2006.

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Managing Biodiversity for a Competitive Ecotourism Industry in Tropical Developing Countries: New Opportunities in Biological Fields Luchman Hakim1, 2, a) 1

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 2 Brawijaya Tourism Research Centre - University of Brawijaya, Malang East Java, Indonesia 65145 a)

Corresponding author: luchman@ub.ac.id

Abstract. Managing biodiversity for sustainable and competitive ecotourism destinations requires a basic understanding of the principles of biology, which are poorly understood in tropical developing countries, including Indonesia. This paper describes the current status of tourism in Indonesia, identifies environment and biodiversity vulnerability in tourism destinations, and explores the challenges of the biological field in supporting ecotourism development. This review found that tourism, especially nature-based and ecotourism, has grown significantly in Indonesia, and the contribution of Indonesian biodiversity has been identified as significant. Threats to biodiversity, however, are found in nature-based tourism destinations. Issues related to pollution, exotic plant species invasion, habitat changes and degradation, habitat loss, and wildlife disturbance are widely reported, indicating the importance of such issues in destination management. Pollution is found in both terrestrial and aquatic ecosystems. Water pollution is an important issue among lakes and rivers. To date, there are few assessments of the impact of tourism activities on aquatic ecosystems, resulting in the management of aquatic ecosystems facing numerous difficulties. These studies identify the invasive plants found, which become a crucial problem in many nature-based tourism destinations, and which significantly contribute to a reduction in the existence of many flora-fauna in a wild habitat. Habitat changes and degradation are mostly influenced by tourism infrastructure development. Massive infrastructure development often leads to habitat loss, which is a crucial step in local biodiversity extinction. Increasing and uncontrolled visitor behaviors influence animal behavior changes, which is recognized as a dangerous phenomenon affecting animal survival in the future. An agenda for future integrative biological research is needed to improve resource management, to increase sustainability and the competitiveness of the tourism industry in Indonesia.

INTRODUCTION Recently, ecotourism has grown as a significant sub-sector of the tourism industry in Indonesia.1-3 Ecotourism involves responsible travel to a natural environment, which positively contributes to environment and biodiversity conservation, local economic growth and development, and strengthening of the socio-cultural aspects of the local community. Global awareness regarding environmental degradation and biodiversity extinction has undoubtedly increased in many places, and numerous strategies to countermeasure environmental degradation have been promoted.4-5 In such cases, ecotourism has been considered as a tourism industry, which passively supports environmental conservation. Globally, ecotourism has received considerable support from environmentalists and travelers who are interested in the environment, local development, and social issues.6-7 Indonesia is a region of great biological diversity, where the forces of the tourism industry have contributed to environmental degradation. Tourist visitation growth and infrastructure development in natural areas raise a number of interesting socio-ecological issues. The most significant issues regarding the environment and tourism, however, are the unsustainable uses of natural resources.8 Nature-based tourism destinations are naturally dynamic systems and ecologically represent a living system where many living creatures (biodiversity) interact and create numerous


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biological phenomena and an outstanding landscape, which is a basic resource for tourism attraction development. Many ecosystems and living creatures, however, are susceptible to environmental changes. Environmental integrity and quality may decline as a result of increased tourist visitation beyond the carrying capacity of a destination. Environmental resources are vulnerable to a wide range of tourist activities that can damage or destroy the natural capital. Environmental degradation as a consequence of tourist visitation and natural resource consumption in tourism destination areas occurs mainly in areas with less environmental management 9-10. Biodiversity is a backbone of the tourism industry. The role of biodiversity in tourism ranges from biodiversity as an attraction (i.e., many wildlife are focal point species), resources for consumptive goods (i.e., culinary), natural components to support environmental survival (i.e., pollination), to aesthetics (i.e., ornamental plants).11-12 Biodiversity, however, is not a tourist attraction unless its tourism potential value is converted and actualized as objects which are able to attract tourists. The success of tourism attraction, therefore, depends on the ability of tourism planners and managers to actualize the potential value of biodiversity as a tourist attraction. Managing biodiversity to increase destination performance is important, especially in terms of destination sustainability and competitiveness. In such a case, the use of biological sciences for supporting sustainable destination management continues to increase. Through a range of biological research, many countries seek to inventory, design, and develop biological resources which can lead to more interesting and competitive tourism destinations. Biology serves primarily to help planners and destination managers understand the biological characteristics of sites to minimize the environmental impact caused by tourism activity. There has been a growing concern in recent years around planners and destination managers in developed countries using biological principles to enhance the sustainability and competitiveness of tourism destinations.11-13 In developing countries, however, biological fields were rarely used in research and investigations related to the sustainability and competitiveness of tourism destinations. Biology principles have been applied in agriculture, fisheries, animal husbandry, food technology, and medicine. Biology has also become a fundamental science in environmental conservation. The use of biological principles in the tourism industry, however, is still limited. There are few empirical case studies on the use of biological principles in tourism studies. The aims of this paper are to describe the potential value of biodiversity in the Indonesian tourism sector, especially in ecotourism, review environmental degradation in tourism destination areas, and discuss the challenges for ecotourism development and biodiversity conservation. Through this review, we hope to provide an insight into the fundamental aspect of biology as a key for sustainable tourism development.

Biodiversity in the Indonesian Tourism Industry Known as a mega-biodiverse country, Indonesia is home to numerous living creatures which are important to the tourism industry. This archipelago contains 17 500 islands (990 inhabited permanently), whose terrestrial and aquatic ecosystems are a habitat of algae (1 500 species), fungi (80 000 species), lichens (595 species), fern (2 197 species), spermatophytes (30–40 000 species), and fauna species of mammals, birds, reptiles, fish (ca. 8 157 species). Many living creatures are endemic and local to particular regions and cannot found in other places. About 270 species of mammal, 386 species of bird, 328 species of reptile, 204 species of amphibian, and 280 species of fish are present. The uniqueness of the fauna diversity, form, and distribution especially follows the Wallace line pattern. The western part of Indonesia is influenced by Asian fauna (i.e., Sumatran Tiger, Javan Rhino, Sumatera Rhino), while the eastern part is influenced by Australia fauna (i.e., Marsupials). In Indonesia, the climate affects the spatial pattern of vegetation and animal distribution. These facts contribute to the attractiveness of many places in the Indonesian archipelago. 14-17 Many areas with a potential biodiversity value and outstanding landscapes have been protected by the Indonesian government. These areas are essential for Indonesian mega-biodiversity conservation (Table 1). Tourism has grown significantly in some protected areas, especially national parks. In Indonesia, there is a relationship between tourist visitation and biodiversity hot spot areas. Areas with high visitation levels coincide with those areas of endemic biodiversity levels and outstanding landscapes.

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TABLE 1. The conservation area of Indonesia as potential sites for ecotourism development

Types of conservation area Strict Nature Reserve (Cagar Alam) Wildlife sanctuary (Suaka Margasatwa) National Park (Taman Nasional) Nature Recreation Park (Taman Wisata Alam) Grand Forest (Taman Hutan Raya) Hunting Park (Taman Buru)

Number (unit) 249

Area (ha)

Notes

4.928.928,92

Tourism not allowed

77

5.342.379,74

50

16.375.253,31

124

1.041.345,21

21

347.427,34

14

224.816,04

Famous tourism destintion includes Bromo Tengger Semeru NP, Komodo, Most visited by tourism includes Ijen Crates

In Indonesia, three forms of biodiversity – genes, species, and ecosystems – are widely involved in tourism. Low genetic diversity has a number of consequences. Genetic diversity is ecologically important as it generates population fitness and sustainability. It is important as an adaptation mechanism to a changing environment. Genetic diversity also contributes to the diverse morphology of flowers and leaves of numerous plants. This diversity also contributes to the numerous fruit tastes. When a population undergoes a mechanism of genetic diversity pressure (i.e., inbreeding, habitat fragmentation), this is the entry point to the loss and extinction of that population.17 This is disadvantageous for tourism attraction development. Indonesian conservation and protection, both of terrestrial and aquatic species, is required for biodiversity and outstanding landscapes. It is not surprising that conservation areas, especially national parks, are the main contributors attracting tourists to enjoy biodiversity in the wild. Statistical data show that the number of tourists in national parks has grown significantly (Table 2). TABLE 2. Number of tourist in conservation area in 2013-2015

Destinations National Park Nature Recreation park

2013 1,965,215 2,768,111

2014 2,440,071 3,589,485

2015 2,168,480 3,337,219

Species and ecosystem diversity are clearly important in tourism. Many animals have become famous tourist attractions. Throughout the Indonesian archipelago, there are mammal, bird, and reptile species that area highly valuable for ecotourism programs, like Elephas maximus (Elephant safari in Tangkahan North Sumatra, Way Kambas National Park, Lampung), Pongo pygmaeus (Tanjung Puting National Park), Varanus komodoensis (Komodo National Park), Bos javanicus (Alas Purwo National Park), and the diverse butterfly species in Bantimurung-Bulusaraung National Park (Table 3). Tourists can focus on a single object such as bird-watching and mammal watching or on multiple animal objects. Depending on the area richness, fauna endemism, and season, there may be a range of wildlife programs. Indonesia has many outstanding ecosystems and landscapes, which are ecologically and sociologically important, including the complex of Mt. Bromo Tengger Caldera, Mt. Semeru in East Java, Mt. Batur-Caldera Batur (Bali), and the three colored lakes in Mt. Kelimutu (Flores). These areas have received many tourists from numerous countries. The marine and coral reefs are rich in terms of marine biota, leading to many places being favorites for diving and snorkeling. TABLE 3. The landscapes and focal point species of ecotourism in some Indonesian national park

Conservation area Berbak NP.

Global status Ramsar

Landscapes Peat swamp forest; the largest wet land conservation area in South East Asia

Bukit Barisan Selatan NP

World Heritage

Tropical rain forest

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Flagship species Sumatran Rhinoceros (Dicerorhinus sumatrensis) Sumatran Tiger (Panthera tigris ssp. sumatrae), Asian Tapir (Tapirus indicus ) Malayan sun bear (Helarctos malayanus), Dark-handed Gibbon (Hylobates agilis), Siamang (Symphalangus syndactylus),

Sumatran Surili (Presbytis melalophos) Sumatran Orangutan (Pongo abelii) Sumatran Rhinoceros (Dicerorhinus sumatrensis) Sumatran Tiger (Panthera tigris ssp. sumatrae), Sumatran Elephant (Elephas maximus ssp. Sumatranus) Sumatran Rhinoceros (Dicerorhinus sumatrensis) Sumatran Tiger (Panthera tigris ssp. sumatrae), Sumatran Elephant (Elephas maximus ssp. Sumatranus)

Gunung Leuser NP

World Heritage, Biosphere Reserve

Tropical rain forest

Kerinci Seblat NP

World Heritage

Mountainous forest with rivers, scenic waterfalls and the Lake Gunung Tujuh (the highest caldera lake in Southeast Asia )

Gede Pangrango NP Ujung Kulon NP

Biosphere Reserve World Heritage Biosphere Reserve

Mountain landscapes

Javan Gibbon (Hylobates moloch)

Lowland tropical forest

Javan Rhinoceros (Rhinoceros sondaicus), Javan Gibbon (Hylobates moloch) Javan Hawk-eagle (Nisaetus bartelsi)

Bromo Tengger Semeru NP Alas Purwo NP

Biosphere Reserve

Komodo NP

World Heritage, Biosphere Reserve -

Gunung Rinjani NP Kelimutu NP

-

Danau Sentarum NP Tanjung Puting NP Kayan Mentarang NP

Ramsar World Heritage -

Tropical mountain forest with spectacular wide sand caldera and activate volcanoes Lowland tropical forest, savannah, white sandy coastal

Savannas and tropical deciduous forest Mountain forest and mountain lake (Segara Anak) Mountain semi-arid forest with three color lakes Wetlands dominated by lakes Tropical swamp ecosystems Luxurious tropical rainforest

Banteng (Bos javanicus) Green turtle (Chelonia mydas) Olive Ridley (Lepidochelys olivacea) Hawksbill Turtle (Eretmochelys imbricata) Leatherback Sea Turtle (Dermochelys coriacea) Komodo dragon (Varanus komodoensis)

Barking Deer (Muntiacus muntjak) Numerous birds Bornean Orangutan (Pongo pygmaeus) Proboscis Monkey (Nasalis larvatus) Bornean Orangutan (Pongo pygmaeus) Proboscis Monkey (Nasalis larvatus), Clouded leopard (Neofelis nebulosa), Horsfield’s Tarsier (Tarsius bancanus) several hornbill species (Buceros)

Beyond the national parks, six places with valuable bio-geological aspects include Batur Geopark, Gunung Sewu Geopark, Caldera Toba, Geopark Merangin, Geopark Rinjdani, and Geopark Ciletuh. The Batur Geopark and Gunung Sewu Geopark have been included in the UNESCO Global Geopark, representing the bio-geological value of such ecosystems in the global context. The biodiversity of the Geopark is high but few comprehensive biodiversity data are available. This problem results from the small number of taxonomists working in such parks. The Indonesian culture has been identified as rich, and the cultural background influences numerous outstanding cultural landscapes which are unique and beautiful. Cultural landscapes are home to numerous biodiversity, and

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some of these have been visited by international tourists, i.e., the paddy terrace in Ubud-Bali.18 Many cultural landscapes, however, are still unexplored by tourism development. Environmental issues and natural resource management are crucial issues in the global tourism competitiveness index. Indonesia is competitive in terms of natural resources but faces problems in natural resource management, especially in issues of environmental sustainability and health-hygiene (Fig.1). From 141 evaluated countries, the rank for environmental sustainability for Indonesia was 134, lower than Singapore (global rank 51), Thailand (global rank 116), and Malaysia (global rank 119)18 . These data are relevant and consistent with many reports on the problems of environmental management, especially in tourism destinations in Indonesia. These data show that future management of natural resources is important.

FIGURE 1. Indonesian competitiveness index in tourism industry (Crotti and Misrahi, 2015)

Environment and Biodiversity Vulnerability in Tourism Destinations In the beginning of attraction and destination development, most of the “virgin” sites, biologically represent integrated biological systems which are supported in their functions by numerous organisms. A place with high biodiversity will produce attractive and beautiful landscapes more than the same place with low biodiversity. The “virgin” sites often show beautiful landscapes, uniqueness, and attractiveness of nature. These features were gradually recognized and transformed into tourist attractions and destinations. The transformation of integrated biological systems into attractions and destinations is a critical point for many changes that have serious future ecological consequences. Conceptually, the recreational value of landscape is greatly reduced if the biodiversity level is disturbed and decreased. Biological system-based problems in tourism destinations have been identified as numerous. These include: 1. Pollution. Increased tourism usually produces higher pollutants. Pollution can cause an increase of attraction destination competitiveness through some mechanisms (i.e., decrease of visual quality, hygiene and health issues). In many tourist destinations, pollution arises as a result of an increase of any substance (i.e. liquid, solid, gas) and sound (machine, tourist noise) at the site in the destination system at a faster rate than it can be decomposed and recycled naturally (Sun & Walsh, 1998; Katircioglu, 2014). Some mechanisms of pollution incidents in tourism destinations are: x Abundant tourist vehicles sand mismanagement of parking x Noise x Waste x Poor sanitation 2.

Exotic plant species invasion. Recently, the invasion of exotic plant species, both in terrestrial and aquatic ecosystems is a crucial environmental problem, including many tourism destinations in

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Indonesia.19-21 Exotic plant species invasion is often exacerbated both by natural and anthropogenic factors, including nutrient availability in water and soil, and forest fires. The typical process of exotic plant species invasion includes: x Introduction of ornamental plant species x Introduction of plants against forest fires x Introduction of economical fish in lakes x Biomass waste from agricultural activity x Introduction of materials for tourism infrastructure development which contain weed seeds x Misconception of multipurpose tree species (MPTS) as plant species in forest rehabilitation programs x Introduction of spices and vegetable species 3.

4.

5.

Habitat changes and degradation. Development of attractions and changes of wild habitats are inextricably linked, causing habitat changes in most nature-based tourism destinations. The nutrient status of soils is potentially altered, and many key species may disappear. Habitat changes and degradation generated by tourism development causes many wildlife, especially small mammals, reptiles, and birds, to seek refuge and new habitat.22-23 This process is often dangerous for wildlife. The mechanism of habitat change includes: x Modifying habitat x Introducing exotic species x Establishing tourist tracks and corridors x Habitat loss. The most important form of habitat loss is the permanent loss of wildlife habitat due to the conversion of habitats for other purposes of land use. This loss of habitat has ecological consequences, leading to reduced reproduction success and decreased population survival which may lead to species and population extinction.24 Tourism potentially permanently removes biodiverse habitats though: x Destruction of coral reef ecosystems x Forest fires x Developing tourism infrastructure and complexes by clearing wild habitats and building new ecosystems Wildlife disturbance. Tourists may impact directly and indirectly on wildlife. Scholars point out that impacts can be numerous, including changes in population structure, changes in behavior, changes in reproductive patterns, increase of predators, and disease vulnerability. The typical process of wildlife disturbance includes: x Food provisioning x Intensive contact between tourists and wildlife x Lighting x Unsustainable animal food in the wild x Noise x Illegal hunting, illegal species collection

Biology and The Challenges for Ecotourism Development and Biodiversity Conservation Ecotourism continuously grows as a significant form of tourism in Indonesia. Problems, however, are related to the ability of resource management regarding sustainability and competitiveness in the tourism industry.24 Principally, biology is more than understanding the basic principles of living systems on earth; it also includes contributions to a recent wide range of industry, including eco-industries such as ecotourism. The principal contribution of biological knowledge is fundamental from planning, implementation, and development to monitoring.

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Tourism destinations, ecologically, comprise a complex of biotic and abiotic components, providing a space for recreational activity. Tourism destinations as recreational sites whose areas consist of gateways, corridors, communities, and attraction complexes.25 Attraction is the heart of a destination, in which it is key to attract tourists to come to destinations. In nature-based tourism destinations, the quality of the attraction depends on the biological integrity of the destination’s systems. Biological integrity is a key issue in tourism attraction and destination management. Biodiversity and biological system integrity play a fundamental role in influencing destination properties and processes that affect destination sustainability. Effective management, therefore, depends on the understanding of key bio-ecological processes of attractions and destinations (Fig. 2). Destination sustainabilitycompetitiveness

_ + Biological integrity

PERCEIVED by human: ¾ beauty nature ¾ fresh environment ¾ unique landscape ¾ entity to reduce physiological stress ¾ media for education

Function

¾ Modification ¾ over consumption

Mismanagement

Management guided by involving biological principles

Changes in Biological integrity

Maintaining-enhancing biological process

Recreational Developers activity

Tourism activity

FIGURE 2. Biological integration is the key for sustainable tourism resources management

A harmonious nature provides substantial aesthetic benefits for the tourism industry. Tourism destination management may include a variety of ecosystem components, ranging from abiotic to biotic factors. The waterfall basically is a natural feature which is related to hydrological processes, whose quality depends on the forest quality, especially the diversity, structure, and composition of the vegetation.26 Many animal species involved in tourism as attractions also have special characteristics which are important for the population survival.27 These suggest that factors such as climate, food availability, and demography might also affect the survival of many fauna objects of tourism. The biological field is important as the first step of tourism destination management. Among the first aspects that biology can contribute to includes identification, description and mapping of bio-resources. Identifying and mapping biodiversity is an important part of developing tourism attractions and destinations in the new vision of sustainable tourism. The overall goal of identifying and mapping biodiversity is that it generates a vivid picture of bio-resources. A failure of biodiversity database mapping might lead to the mismanagement of biodiversity. This can be found in the case of birds as potential resources for tourism development. In Indonesia, birds for bird-watching activities are in abundance, both in term is bird diversity and abundance, but there are few numbers of tourism operators and people participating. In many conservation areas, birds dominate a considerable proportion of fauna diversity and provide opportunities for bird-watching programs. However, the number of bird-watching operators is limited, and the number of Indonesian tourists involved in birding is low. It becomes a challenge for biologists to identify and map bird diversity and distribution and establish a design for bird-watching activities based on biological information. In Indonesia, many aquatic-based tourism destinations are especially prone to tourist disturbance such as pollution, riparian vegetation disruption, and exotic aquatic biota invasion. This can be found in Lake Ranupani, Lake Toba, and Lake Tondano.28-29 Without proper management, these lakes and their biological richness will become extinct. Identifying and describing ecosystems is crucial to determine the relationship of ecosystem components. Much environmental deteriorations is caused by changes of ecosystem components, loss of species in the food web, and

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the introduction of new species. Describing ecosystems fundamentally is important to define destination carrying capacity. In fact, many nature-based tourism destinations are facing serious problems regarding visitor numbers. The case of visitor abundance in Penanjakan point in Bromo Tengger Semeru is a classical problem related to carrying capacity issues and visitor management. Carrying capacity is becoming an increasingly important issue in the environmental management of nature-based tourism destinations.30 Carrying capacity has been initiated and encouraged in some nature-based tourism destinations, but most places still face difficulties in defining the carrying capacity of the area. Another strategy may, therefore, be to focus on developing new sites beyond the main sites which are highly visited. In Indonesia, the biophysical environment of tourism destinations needs to be evaluated. The importance of evaluating tourism destinations, especially in the perspective of ecosystem biodiversity and integrity of creating sustainable nature-based tourism destinations, is also dealt with in the competitiveness of the national tourism industry. Scholars point out that one of the fundamental roles of ecotourism development is supporting biodiversity conservation in destinations. In Indonesia, this can be implemented through promoting flagship species and charismatic mega-fauna species as components to attract tourists.1, 29-31 Flagship species are important in tourism and many destinations seek potential candidates for flagship species. There are, however, no flagship species that can be found and promoted without a detailed biological survey of the biological and ecological aspects of the targeted flagship species. When a species is found and promoted as a flagship species, interpretation about such species requires biological information of the species. There is no ecotourism without nature interpretation, as education is a crucial part of ecotourism. 7, 32 highlights the importance of the biological field in the ecotourism industry. Ecological theory Tourism uses natural resources Biodiversity

concept, prediction models to describes pattern and process

Tourism influence environmentbiodiversity

Natural resourcesbiodiversity management

Biology (i.e. Biodiversity, evolution, genetics, reproductiondevelopment, etc.

Linkages

Tourism destination ecology Scientific understanding and approach in natural resources-biodiversity uses in tourism industryfrom planning, implementation, development, evaluation- to meet destination sustainability and competitiveness

Applied ecology (i.e.: Human ecology, wildlife ecology, plant ecology, landscape ecology, aquatic ecology, etc.)

FIGURE 3. Conceptual models of tourism destination ecology

Linking ecotourism development and biodiversity conservation require many different kinds of data, especially biological data. Basically, the data include species diversity, species characteristics, population structure, and how species and their environment interact. Further potential data should be explored including genetic diversity. Understanding genetic diversity leads a destination planner and manager to optimize germ plasma potential for numerous tourism product developments. It also contributes significantly to biodiversity conservation design and consideration in tourism destination areas, especially in conservation that is involved in the tourism industry. The impressive growth of tourism in biodiversity-rich areas will have a major impact on the future of biodiversity and landscapes. Tourism is a strong driver of landscape patterns. Tourism destination ecology requires basic and applied ecology (Fig.3).

SUMMARY There is a significant role for the biological field in tourism industry development. First, biology provides basic data related to the characteristics of bio-resource attractions. Tourism products, especially biodiversity-based

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products, should be shaped to satisfy the tourist. So far, economic and physical aspects have been involved as a consideration of actualized biodiversity potential as attractions, and few biological considerations are involved. Second, biology provides basic principles of species-environment relationships. Biology serves to primarily help destination planners and managers understand the consequences of a biological system disturbance. Biological approaches are important tools in the assessment and evaluation of the impact of tourism on the environment. Third, biology should be involved in tourism destination and attraction planning. Sustainable destinations require planning systems with carefully organized biological information of sites for use in destination planning.

ACKNOWLEDGMENTS I would like to express my deepest gratitude to the ICGRC 2017 committees for accepting this manuscript to be published in this proceeding.

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M. F. Kinnaird and T. G. O'Brien, Oryx, 30, 65-73 (1996). M. J. Walpole and H. J. Goodwin, Annals of tourism research 27, 559-576 (2000). L. Hakim, M. Soemarno and S. K. Hong, Journal of Ecology and Environment 35, 61-71 (2012). V. May, Tourism Management 12, 112-118 (1991). C. A. Gunn and T. Var, Tourism planning: Basics, concepts, cases (Psychology Press, 2002). S. Gössling, Ecological Economics 29, 303-320 (1999). O. Krüger, Biodiversity and Conservation 14, 579-600 (2005). J. L. Hill and T. Gale, Ecotourism and environmental sustainability: principles and practice (Ashgate Publishing, Ltd, 2009). L. Hakim, “Planning and Design for Ecotourism and Nature Conservation in Java and Bali”. Ph.D. Thesis. Hiroshima University, Japan, 2008. R. Van der Duim, and J. Caalders, Annals of tourism research 29, 743-761 (2002). D. Newsome, S. A. Moore and R. K. Dowling, Natural area tourism: Ecology, impacts, and management (Vol. 58). Channel view publications (2012). K. Higginbottom, Wildlife tourism. (Common Ground, Altona, 2004). K. Sukenti, L. Hakim, S. Indriyani, Y. Purwanto and P. J. Matthews, Journal of Ethnic Foods 3, 189-200 (2016). D. Tyler and J. M. Dangerfield, Journal of sustainable tourism 7, 146-158 (1999). V. H. Heywood and R. T. Watson, Global biodiversity assessment (Vol. 1140) (Cambridge University Press, Cambridge, 1995). S. Ross and G. Wall, Wallace’s line: Implications for conservation and ecotourism in Indonesia. Tourism and the less developed world: Issues and case studies, 223-234 (2001). L. Hakim, J. E. Kim and S. K. Hong, Journal of Ecology and environment 32, 1-8 (2009). R. Crotti and T. Misrahi, The Travel & Tourism Competitiveness Report 2015 (World Economic Forum, 2015). D. Sun and D. Walsh, Journal of Environmental Management 53, 323-338 (1998). S. T. Katircioglu, Renewable and Sustainable Energy Reviews 36, 180-187 (2014). T. Whitten, R. E. Soeriaatmadja and S. A. Afiff, Ecology of Java & Bali (Vol. 2) (Oxford University Press, 1996) L. Hakim, A. S. Leksono, D. Purwaningtyas and N. Nakagoshi, Journal of International Development and Cooperation 12, 35-45 (2005). L. Hakim and H. Miyakawa, Biodiversity Journal 6, 831-836 (2015). P. C. Reynolds and D. Braithwaite, Tourism Management 22, 31-42 (2001). M. Marzano and N. Dandy, Biodiversity and Conservation 21, 2967-2986 (2012). T. M. Brooks, R. A. Mittermeier, C. G. Mittermeier, G. A. Da Fonseca, A. B. Rylands, W. R. Konstant, P. Flick, J. Pilgrim, S. Oldfield, G. Magin and C. HiltonǦTaylor, Conservation biology 16, .909-923 (2002). B. J. Hudson, Annals of Tourism Research 25, 958-973 (1998). A. M. O'Reilly, Tourism Management 7, 254-258 (1986). M. J. Walpole and N. Leader-Williams, Biodiversity & Conservation 11, 543-547 (2002).

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30. R. R. Butarbutar, L. Hakim and I. R. Ssastrahidayat, International Journal of Conservation Science 6, (2015). 31. L. Hakim, D. A. Guntoro, J. Waluyo, D. Sulastini, L. Hartanto and N. Nakagoshi, Journal of Tropical Life Science 5, 152-157 (2015). 32. S. K. Jacobson and R. Robles, Environmental Management 16, 701-713 (1992).

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Skeleton Microstructure of Porites lutea in Kondang Merak, Malang, East Java Oktiyas Muzaky Luthfi1, a), RM. Agung M. Rizqon Sontodipoero1,b), Andik Isdianto1,c), Daduk Setyohadi2,d), Alfan Jauhari2,e), I Nyoman Januarsa1,f) 1

Department of Marine Sciences, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Veteran Street, Malang, Indonesia 65145 2 Department of Fisheries Resources Utilization, Faculty of Fisheries and Marine Sciences, University of Brawijaya Veteran Street, Malang, Indonesia 65145 a)

Corresponding author: omuzakyl@ub.ac.id b) agungrizqon18@gmail.com; c) andik.isdianto@ub.ac.id; d) dadukdaduk@gmail.com; e) jauharialfan@yahoo.com; f) rethugjr@gmail.com

Abstract. Research on coral microstructure in Indonesia, especially in East Java is rarely done. Therefore, this study aims to examine the shape of Aragonite Crystal coral Porites lutea in Pantai Kondak Merak, East Java, especially in 1998 which is the time of El Nino and has a global impact on coral growth. The shape of the aragonite crystal on the reef can be seen using the Scanning Electron Microscopy-Energy Dispersion X-Ray (SEM – EDX). Based on the coral aragonite crystal form, the increasing temperature in 1998 was not proven to have a devastating effect on the growth of corals of Pantai Kondang Merak. In contrast, the temperature at this site should support corals in order to grow rapidly, but there are other environmental factors that ultimately inhibit the growth of the coral.

INTRODUCTION The coral growth rate is influenced by various factors such as pH1, light, temperature, sedimentation2, depth3 and hydro-oceanographic parameters such as currents and waves.4 Sedimentation and seawater temperature is the most influential factors. Sedimentation will inhibit zooxanthellae for photosynthesis that will inhibit the growth of the coral.5 The phenomenon of increase of sea surface temperature because of the El-Nino event also can lead to coral growth being inhibited.6 Other information that can be recorded by a coral skeleton is CO2 emission in the air and seawater, which can result in global warming and ocean acidification. Acidification leads to a decrease in coral growth rate while an increased sea level temperature increases coral bleaching. A study of the microstructure of a coral skeleton actually studied coral biomineralization that occurred in the fibers and center of calcification. The coral skeleton was aragonite crystal (CaCO 3) under the organic layer in the ectoderm. The crystal then is known as biocrystal composed from organic compound and mineral ions. An individual aragonite crystal will growth into other elements such as wall and septa. Crystallite or fiber in a coral skeleton is sub-micrometer to molecular in size. Skeletal fiber has 10 micrometers in length and has an increment of aragonite of about 3–5 μm.7


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The means of coral growth is a linear increase in length, weight, volume or area of the skeleton interpreted as an increase in the weight of the coral skeleton composed of calcium carbonate in the form of aragonite crystals and calcite.4 Measuring coral growth usually takes a long time, therefore researchers generally use X-rays to view the annual band and calculate growth rate.6. Measurements using the X-ray method have a weakness; they cannot calculate the increase of aragonite crystals on the coral. The aims of this study were to learn the coral microstructure of P. lutea that was taken from high and density band. Coral P. lutea has annual banding, dark and light, that was representing high or low density of aragonite crystal that build them. For example, the dark band in P. lutea has meaning that low density of aragonite crystal in its skeleton structure

EXPERIMENTAL DETAILS Porites lutea Sampling Sampling was conducted in Pantai Kondak Merak in 2015. The sample was taken at ± 2 m with characteristics of the sandy substrate. Samples taken at coordinates –8° 39' 7" S; 112° 51' 8" E (Figure 1). The size of the sample was ± 30 cm in diameter and height ± 15 cm. This size may be considered to 10 years old and have matured gonads because according to8, a coral massive aged 4–7 years is a mature colony. The samples obtained were then stored in cool boxes filled with seawater to minimize polyp specimens. Coral samples were then soaked in chlorine solution for 2 weeks and dried in an oven at 60oC for 4 days before cutting.

FIGURE 1. Sampling Location Maps

Sample Selection Cutting and Preparing Scanning Electron Microscopy The clean and dry specimen was then cut into a slab of 1 cm thickness and with a width of 10 cm. We chose the middle of the specimen that has the perfect growth axis. The sliced sample then was rinsed using tap water to remove organic matters or other contaminants. The sliced sample was rinsed in the ultrasonic bath. To know the average age the specimen was then photographed using X-ray at Higina Clinic with the exposure detail was 3.2 mAs in 56 kV and 200 mA. The sub-samples were obtained from the high-density band (HD) and low-density band (LD); the small pieces of sub-samples were coated using Au – Pt that was coated with conducting glue. For this analysis, we used SEM EDX JEOL Model JSM-7800F for 10 KeV.

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FIGURE 2. Slab Results in A: Annual Band; N: New Growth; B: Bottom; Q: Top; M: Microborer

RESULT AND DISCUSSION Age of Coral According to X-ray analysis, the P. lutea was 27 years old and had an average growth rate of 0.55 cm/ year. The banding pattern in P. lutea can recover 3 important data, which were skeletal density, linear extension rate, and calcification rate. The coral also recorded the environment across time from the beginning growth. The average growth rate of P. lutea in higher latitudes had a lower growth rate than lower latitudes.9 measured 400 colonies of Porites in the Great Barrier Reef (GBR) at high latitude and low latitude and concluded that lower coral in lower latitude has significant higher in their growth rate than coral in a higher latitude. Nutrients and sediment also influenced the growth rate of coral P. lutea, which is related to the availability of light.10 Another study reported that the extension rate was correlated to water clarity.11 P. lutea in Pantai Kondang Merak was in a reef flat area that has strong current and high waves, so coral in this location uses a lot of energy to adapt hydraulic energy from sea water. 11 reported that P. lutea from 11 fringing reefs in the Phuket, South Thailand, has an inverse relationship between skeletal density and extension rate; if the hydraulic energy increases, the extension rate of coral will decrease.

Element Content on Coral Skeleton Hight Density (HD) Figure 3 shows the observed sample in the rainy season (High Density) in 1998 showing that the outer shell of atoms (K) has a carbon (C) content percentage of 4.88% for the weight total (Wt%) and 8.80% for the atomic total value (At%); oxygen (O) of 48.11% for weight total (Wt%) and 65.17% for atomic total value (At%); sodium (Na) of 1.26% for weight total (Wt%) and 1.18% for atomic total value (At%); silica (Si) of 0.46% for weight total (Wt%) and 0.36% for atomic total value (At%); and calcium (Ca) of 45.29% for weight total (Wt%) and 24.49% for atomic total value (At%). From Figure 3 can also be found 3 elements of Ca it's due to Ca being embedded in CaCO3 compound. Ca content is found in some amount of energy fired, among others, at 0.3 KeV (1.8 Kcount); 3.8 KeV (9.7 Kcount); and 4 KeV (1.9 Kcount), the C content is found at 0.3 KeV (1.9 Kcount) energy, the O content at 0.6 KeV (3 Kcount) energy, Na content at 1.1 kV energy, and Si content at 1.8 K KeV (0.5 Kcount).

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FIGURE 3. Scanning Electron Microscope result on High Density

Low Density (LD) Figure 4 shows the observed sample in the dry season (Low Density) of 1998, showing that the outer shell of atoms (Shell K) has a carbon (C) content percentage of 10.18% for the total weight (Wt%) or 17.85 % For the atomic total value (At%); oxygen (O) of 44.01% for the weight total (Wt%) or 57.91% of the atomic total value (At%); silica (Si) of 0.82% for weight total (Wt%) or 0.62% for atomic total value (At%); and calcium (Ca) of 44.99% for the weight total (Wt%) or 23.63% of the atomic total value (At%). Ca content is found in some kind of energy fired, among others, at energy 0.3 KeV (0.6 Kcount); 3.8 KeV (5.7 Kcount); and 4 KeV (0.9 Kcount), the C content is found at 0.3 KeV (1 Kcount) energy, the O content at 0.6 KeV (1.8 Kcount) energy, Na content at 1.1 kV energy, and Si content at 1.8 K KeV (0.3 Kcount).

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FIGURE 4. Scanning Electron Microscope result on Low Density

Middle Side Figure 5 shows the sample observation on the transition between the dry season and the rainy season (between LD-HD) in 1998 showing that the outer shell of atoms (K Shell) has a carbon (C) content percentage of 10.18% for the weight total (Wt%) or 17.85% of the atomic total value (At%); oxygen (O) of 44.01% for the weight total (Wt%) or 57.91% of the atomic total value (At%); silica (Si) of 0.82% for weight total (Wt%) or 0.62% for atomic total value (At%); and calcium (Ca) of 44.99% for the weight total (Wt%) or 23.63% of the atomic total value (At%). Ca content is found in several kinds of energy fired, among others, at energy 3.8 KeV (6.9 Kcount); and 4 KeV (1.2 Kcount), the C content is found at 0.3 KeV (1.2 Kcount), O at 0.6 KeV (1.8 Kcount), and Si content at 1.8 KeV (0.5 Kcount) energy.

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FIGURE 5. Scanning Electron Microscope result on Middle Side

Figures 3–5 show the elemental content of three layers of coral (LD; between LD and HD; HD). It can be seen that there are 3 elements that dominate in P. lutea, they are C; Ca; and O. This is normal because these three elements are the main constituents of coral (CaCO3). If we look more closely among the three images, the difference in density is evident whereas in the HD section it has a third element content higher than LD or between LD and HD (the center). Therefore, it can be seen that during the dry season the coral will experience rapid growth, but has a low-density level. Conversely, during the rainy season, the coral will experience slow growth but has a high level of density. Furthermore, if we look at the table LD the middle has the percentage of the same element content but not with the number of elements contained. This can happen because the location of the shooting is not too far between the LD and the middle, and at that time changes in environmental conditions occur constantly. While the discovery of silica on coral samples derived from run-off (river flow) and Sodium comes from salt residues contained in the waters. Compared with another study [13], the graph pattern and the distribution of elements from coral samples can be said to be appropriate and in good condition.

Form of Deposited Particles and Aragonite Crystal Sample Condition The observations using Scanning Electron Microscopy on coral samples with 100× magnification and 20 kV energies (Figure 6) showed that vertically coral samples have many holes (± 150 holes in HD part). The size of the holes ranges between 100 and 200 μm. Figure 6 also shows fragments of organism shells, broken coral crystals from sampling and unknown material. Residual cut (RC) scattered almost on the entire surface of the sample (Figure 6), the residual cutting resulted from the previous sample cutting. In addition to the residual cut in the sample shell fragments and unidentified material were found, the existence of this material in the rest of the detritus results generally derive from coastal or riverine activities.12 Furthermore, if comparing the size of the hole there is a match where the hole size ranges from 100–200 μm. In another study13, hole size on Porites corals ranged from 50–200 μm. The size of the hole and the number of holes will be the location of deposits of dissolved particles, including heavy metals will be deposited on the coral holes.

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FIGURE 6. The High Density (HD), UM: Unidentified Material, SF: Shell Fragment, RC: Residual Cut

Aragonite Crystal The aragonite crystal type on the Porites lutea coral sample goes into the Spherulitic type (Figure 7).

FIGURE 7. Observation Results on Sample Tip Section. A: Magnification 250 x; B: Magnification 500 x; C: Magnification 2,000 x; D: Magnification 10,000 x; E: Magnification 25,000 x

Hard coral polyps (scleractinians) are formed by the exoskeleton of aragonite (CaCO 3). Each aragonite is in the form of a fiber or a crystal, generally called aragonite crystal. A set of aragonite crystals will then form the wall, septa, and dissepiment.14 While15, explains that the main constituent corals are sclerodermites which contained aragonite crystals shaped like a "3D fan", and the aragonite crystals have a size of ~ 0.05–4μm in diameter16. concluded that the construction and coral growth based on aragonite crystal shaped formations resembling a "3D fan" called sclerodermites, which is the main constituent of the coral structure.

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According to15, there were 5 stages of morphological change based on the aragonite crystal form; (i) equant; (ii) tabular; (iii) dendritic to open; (iv) coarse spherulitic; (v) closed fine spherulitic. If the aragonite crystal forms an equant crystal then it can be estimated that the coral has a slow aragonite crystal growth, whereas if the coral has a spherulitic aragonite crystal pattern (iv and v) it is expected that the aragonite crystal growth is fast. It was influenced by sea temperature changes. In this sample, the aragonite crystal categorized as “closed fine spherulitic” that means P. lutea in Pantai Kondang Merak has changed to grow fast, but because of environmental factors may inhibit coral growth in Pantai Kondang Merak area. According to15, submicron-sized granule formation will form aragonite crystals. The shape and size of the granule grains exhibit intracellular mineralization, whose process is adapted from unicellular organisms 17. mentioned that there are differences in the shape of granules in corals that have normal and low calcification of carbonate. It is uncertain whether coral samples have normal or low calcification of carbonate, as further research is needed.

SUMMARY Based on this research, these samples were from 1998 that El-Nino that caused the coral bleaching in around the world including Pantai Kondang Merak. Increased temperatures occurring in 1998 have not been shown to adversely affect the growth of corals in Pantai Kondak Merak. The increasing sea surface temperatures at Pantai Kondang Merak in 1998 should support for corals to grow well, but there were many environmental factors lead inhibit coral growth in this area.

ACKNOWLEDGMENTS This work was supported by the Directorate of Research and Community Service, Directorate General for Research and Development, Ministry of Research, Technology and Higher Education through DIPA Universitas Brawijaya Number: 063/SP2H/LT/DRPM/IV/2017. We thank Siddiq Pratomo Al-Idrus, Kahindra Donny Anggara, Sigit Rijatmoko, Saifur Rijal Fakri and Maulana Abdurrahman for helping take the samples and also for comments on this manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

N. C. S. Chan, S. R. Connolly, Global Change Biology (James Cook University, Townsville, 2013), pp. 2982 – 290. C. Rani, J. Jompa, Amiruddin, Tarani 4, 195–203 (2004). Susintowati. Jurnal Ilmiah Progresif 7, 21 (2010). N. P. Lalang, A. Zamani, A. Arman, Jurnal Teknologi Perikanan dan Kelautan 2, 111 – 116 (2014). M. J. M. Crabbe, D. J. Smith, Coral reefs 24(3), 437-441(2005). A. Arman, N. P. Zamani, T. Watanabe, Jurnal Ilmiah Aplikasi Isotop dan Radiasi. 1, 1 – 10 (2003). J. M. Lough, D. J. Barnes, Journal of Experimental Marine Biology and Ecology 245(2), 225-243 (2000). D. Barros, M. L. Monica, O. P. Debora, Bulletin of marine science 73(3), 713-724 (2003). P. J. Isdale, “Geographical patterns in coral growth rates on the Great Barrier Reef.” In J. T. Baker, R. M. Carter, P. W. Sammarco, K. P. Stark, (Eds.), Proc. Great Barrier Reef Conference, Townsville, (James Cook University Press, Townsville, Australia, 1983), pp. 327–330. M. J. Risk, P. W. Sammarco, Mar Ecol Prog Ser 69, 195–200 (1991). T. P. Scoffin, “ Banding in coral skeletons from Pulau Seribu as revealed by X-rays and U/V light analyses” In B. E. Brown, (Ed.), Human-induced Damage to Coral Reefs: Results of a Regional UNESCO (COMAR) Workshop with Advanced Training, (UNESCO Reports in Marine Science 1986), 40, pp. 126–134 S. A. S. Naqvi, Marine Geology 118, 198 – 194 (1994). Mansur, S. Herman, Mansur, A. P. Alexandra, Pereira, M. Marivalda, Key Engineering Materials 284-286, 43–46 (2005). Nothdruft, D. Luke, Webb, E. Gregory, Facies 53, 1 – 26 (2007).

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15. A. L. Cohen, T. A. McConnaughey, Reviews in mineralogy and geochemistry 54(1), 151-187 (2003). 16. K. P. Helmle, R. E. Dodge, Sclerochronology, In Encyclopedia of Modern Coral Reefs. (Springer Netherlands, 2011), pp. 958-966. 17. F. Marubini, C. Ferrier-Pages, Cuif, Jean-Pierre, Suppression of Skeletal Growth in Scleractinian Corals by Decreasing Ambient Carbonate-Ion Concentration: a Cross-Family Comparison (Proc. R.Soc. Land. B 270, The Royal Society, 2003), pp. 179-184.

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Copper (Cu) Content in Porites lutea at South Java Sea: Case Study at Pantai Kondang Merak, Malang, Indonesia Oktiyas Muzaky Luthfi1, a), Sigit Rijatmoko1, b), Andik Isdianto1, c), Daduk Setyohadi2, d), Alfan Jauhari2, e), and Ali Arman Lubis3, f) 1

Department of Marine Science, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 2 Department of Fisheries Resources Utilization, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 3 Marine and Environment Group Center for the Application of Isotopes and Radiation Technology National Nuclear Energy Agency, BATAN, Cinere Pasar Jumat Street, Jakarta, Indonesia 12440 a)

Corresponding author: omuzakyl@ub.ac.id b) sigit.rijatmoko@gmail.com c) andik.isdianto@ub.ac.id d) dadukdaduk@gmail.com e) jauharialfan@yahoo.com f) alilubis@batan.go.id

Abstract. The reef flat area in Pantai Kondang Merak is similar to others that are influenced by the high and low tide. Coral in this area should deal with high water temperature during low tide and should withstand exposure to air. These conditions lead to coral stress. Another stressor is heavy metals such copper. Annual band in Porites lutea can be a bioindicator to investigate the heavy metal contaminations as resulted from the human and natural activity. To measure heavy metal copper in the coral skeleton, we used ICP-OES (iCAP 7400 Series). Heavy metal was absorbed by coral tissue and is bind in their aragonite. This research showed 7 years of the heavy metal sample in coral banding were 9.3; 9.223; 10.09; 10.67; 8.7 and 10.37 mg/Kg (KM1). While in sample 2 (KM2) the concentration of copper was 12.98; 12.5; 15.023; 15.27; 19.811; 28.229 mg/kg. The average of heavy metal KM2 (19.157 mg/kg) is higher than KM1 (0.765 mg/kg). From these data, we can know the amount of heavy metal in South Java Sea from the past or sclerochronology.

INTRODUCTION Pantai Kondang Merak is a small bay in South of Java, directly facing the Indian Ocean. The type of coral reef in Pantai Kondang Merak is a reef flat that was dominated by slow growth massive coral such as Porites and Goniastrea.1 The coral existing in Pantai Kondang Merak became an important place for reef fish, invertebrate, and algae. The hard coral in this location also became a barrier to protect the coast from abrasion due to strong current and high wave from the Indian Ocean. The form of massive P. lutea that grows in Pantai Kondang Merak had two different life forms; they were massive coral and massive microatoll. Massive coral means it has the same growth in all direction or rounded like a ball2; while microatolls are defined as massive coral colonies with the dead area in the upper up area, some of the dead coral makes a hole due to eroded by invertebrate or algae.3-5 . The average size of massive P. lutea in Pantai Kondang Merak was 153.26 cm or categorized as medium size.1


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Almost all hard corals are very sensitive to physical and chemical changes of sea water, and the growth of coral was strongly influenced by their environment. The coral sensitivity can be proxies that serve to interpret the sea water conditions decades ago. Proxies can be defined as records taken from skeletons or fossils or corals to reconstruct past climatic conditions obtained during the surviving coral/ fossil.6-7 The internal growth band of a massive coral’s skeleton can record the physical-chemical sea water, the number of nutrient and pollution in seawater during its lifetime in continuous time series record, which then can be used as a coherent information of seawater condition year to year or chronological. Corals can also record heavy metals resulting from human activities such as dredging in the harbor area, reclamation and disposal domestic waste.8 The heavy metals then combine into the coral skeleton (aragonite), entering as particulates of the skeleton cavities, absorbed on the surface down into the skeleton, and attached to the coral skeleton matrix. These heavy metals will be permanently attached to the coral skeleton and covered by new aragonite layer. Copper (Cu) is an essential element for corals and needed in small amounts played a role as an enzyme catalyst in coral metabolism. Copper (Cu) plays in inhibiting the transport of electrons in the oxidation process of photosystem II.9 Some of the marine invertebrates such as anemones require compounds containing rubidium, vanadium, zinc, iron, cuprum, molybdenum, selenium, and iodine to activate enzymes associated with antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase and peroxydoxoxide.10 Copper will be biodegradable in total by about 600 years.8 Copper (Cu), mercury (Hg), tin (Pb), manganese (Mn), copper (Cu), zinc (Zn), and some other metals trapped in coral skeleton reflect the influence of humans and/ or land influences on marine ecosystems.11 Sources of heavy metals including copper (Cu) were coastal run-offs, offshore mining, anti-fouling paints, nuclear tests and domestic waste.9, 11 It was estimated that the amount of copper in the sea water reached 0.34 billion metric tons with an average concentration of 0.25 μg/L and the predicted total in the sea water was 1.338 x 109 km3. While the copper input from the anti-fouling paint can reach 15 × 106 kg/year.8 Potential sources of pollutants in the Sempu Island Nature Reserve are very large because in the same location used as a fishery port area there is fisheries vessel activity of up to 300 units per day. In this study, the massive coral P. lutea taken as the sample was 15–20 cm in diameter, thus it was expected the coral would record heavy metals from 2009 to 2015. The aim of this study was to determine the concentration of heavy metal copper (Cu) from year to year trapped in the coral skeleton of P. lutea by observed its internal growth band.

EXPERIMENTAL DETAILS The location of Research This research was conducted in the Pantai Kondang Merak (8°23'48.73"S, 112° 31'3.59" E), in April 2016 (Fig. 1). The local name of the P. lutea sampling is the west area (KM1) and east area (KM2), which has a depth of 1–4 m, depending on the tidal range. Both locations, KM1, and KM2 have a very strong current when ahead of the high or low tide. During the rainy season, the sea waters at the research sites can be very turbid due to a load of sediments from the mangrove and forest areas at the north of Pantai Kondang Merak.

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FIGURE 1. The research location indicates of red round tips in Pantai Kondang Merak, Malang

The Sampling of P. lutea The massive coral P. lutea is taken randomly at a depth of 1–4 m with the diameter of colonies ranging from 15–20 cm. The coral samples were taken for the whole of 1 colony and then kept in a cool box until bleaching treatment was done at the Exploration Laboratory of Marine Resources, Faculty of Fisheries and the Marine Science University of Brawijaya Malang. For bleaching treatment, the coral colony was then immersed in chlorine solution for up to 1 week and after the soft tissue was completely peeled, the coral was washed with tap water to remove residual chlorine trapped inside the coral framework. Furthermore, the coral skeleton was dried by keeping it in the oven at a temperature of 60oC for 48 hours. The coral skeleton was then morphologically identified by observing skeletal structures, about 1 cm2 samples were taken from coral colony then the morphology of coral skeletal structures was observed used a USB microscope with 100 × magnification. The identification used a method that was described by12.

X-ray Radiography The coral colony was then cut using a mechanical chain-saw so that it was 1 cm thick before photographing used X-ray. The X-ray photograph was performed at Higina Clinic using X-ray Acoma-HF Digital Stationer type HI 500 69 KeV 250 mA for 8 seconds and the result was a negative film in a digital form, then the result of X-ray samples was processed with Photoshop CS6 software and then viewed manually by specifying a dark layer and a bright layer.

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FIGURE 2. The sliced of coral colonies with 1 cm of thickness.

The Sub-Sampling Technique The first step taken was the sample of corals to be analyzed was cleaned using bidest in an ultrasonic bath for 30 minutes. Samples that had been cleaned were then dried using the oven for 7 days at a temperature of 60oC. The dried coral samples were then milled using a hand drill based on a predetermined cycle cycle, then 20 mg was put into a vial bottle put into a vial bottle and then 2 ml of 25% nitric acid was added in the vortex mixer, after which8 mL of boldest was added then a centrifuge was used for 30 minutes at 2,000 rpm. Samples that had completed centrifuge were ready for the ICP-OES test.

Measurement Concentration of Cu The copper in the internal annual band of P. lutea was analyzed using ICP-OES iCAP 7400 Series with ASX 520 autosampler and Qtegra software in Marine Laboratory, Industrial and Environmental Field, Isotope and Radiation Application Center (PAIR), National Nuclear Energy Agency (BATAN). Standard solutions and samples placed on autosampler racks were alternately tested to measure energy intensity using the wavelength of Cu 327.396.

RESULT AND DISCUSSION The P. lutea Extension Rate Figure 3 shows the P. lutea extension rate that was taken from Pantai Kondang Merak. The dark and light band indicated the density of skeleton P.lutea. A pair of dark and light bands represented one year in coral age. The average extension rate in KM1 was 1.09 cm/yr. and KM2 was 1.74 cm/yr. (Table 1).

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TABLE 1. Annual Growth of Coral P. lutea

Year

Annual Growth of P. lutea (cm/year) KM1 KM2

2009 2010 2011 2012 2013 2014 2015 2016

1.46 0.91 1.31 1.1 0.92 0.84 -

1.87 1.39 1.22 2.17 1.86 1.6 2.09 -

Rata-rata

1.09

1.74

FIGURE 3. X-ray image of skeletal density banding of P. lutea that was taken from Pantai Kondang Merak. A=KM1; B=KM2.

The red line in Fig. 3 shows the place of the sub-sampling from annual banding of P. lutea. This line was a vertical growth band of coral; the sub-sample was taken each year from the bottom of to the top of the coral band. KM1 has the highest diameter of about 16 cm and KM2 was 36 cm (Fig. 3). To determine the age of coral, axes were chosen that had the same corallite wall. Each year coral growth has differences in high and low density, which means each year coral has a different response to the environment condition. The X-ray result showed that the coral of KM2 is older (7 yrs.) than KM1. The average extension of both these corals is also different with KM2 faster (1.74 cm/yr.) than KM1 (1.09 cm/yr.). The annual average in P. lutea was different, although they were in the same waters, the physical condition of seawater includes sea surface temperature, nutrient, current, and salinity may have no significant difference in both these locations. The average of the coral extension was inverse with the skeletal density in coral. The higher extension rate means low the carbonate deposited in the coral skeleton.13 The growth of coral cannot be seen only from increasing of annual extension rate of coral but should be seen from another way such as increasing of annual calcification rate, it was the progression of skeleton P. lutea in a gram/cm square in a year. The calcification process in coral where placed in a calcifying fluid among calicodermis and skeleton. It was suggested that transfer of photon will increase of pH and saturation of fluid resulted CaCO3 will crystallize into the coral skeleton to be aragonite Energy is needed to keep the crystallization process continuing and it is estimated that corals allocate about 30% of its energy for this process.14 Corals was divied into 4 layers15-16: they were epidermis, gastrodermis, calicodermis and skeleton. Bicarbonate (HCO3-) from seawater will direct to calicodermis and will lead to calcium (Ca2+) and will be calcium-carbonate (CaCO3), then will be crystallized at the coral skeleton. The ion H+ that resulted from calcification process will be neutralized by hydroxide (OH -) that resulted from a photosynthetic process of zooxanthellae in gastrodermis.

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The global climate change has an expected effect on growth and calcification rate of coral P. lutea, 17stated that the growth rate of coral P. lutea from 8 locations in the Andaman Sea was decreased up to 19.4–23.4% and the calcification rate also decreases up to 23.5%. The increase of sea surface temperature of 0.161oC in the last 46 years may become the main cause of this occurrence.

Accumulation of Cu in P. lutea’s annual band The result of ICP-OES analysis showed that the P. lutea had different absorption of copper metal. In the KM1, Cu concentration was observed between 8.7 to 10.67 ppm, while the KM2 concentration of Cu metals ranged from 12.5 to 30.29 ppm (Figure 4). The lowest absorption of Cu at KM1 occurred in 2014 (8.7 ppm) while KM2 in 2010 was 12.5 ppm. The peak of Cu metal absorption at KM1 occurs in 2013 (10.67 ppm) and KM2 in 2015 is 30.29 ppm.

FIGURE 4. The concentration of Cu recorded in coral annual banding from 2009-2015 at Pantai Kondang Merak, Malang.

The different conditions during the growth period of P. lutea coral will be able to provide a complete profile of heavy metals in the waters just that time, the skeleton of coral only records the number of heavy metal in a limited period of time not similar to mussels, which can accumulate heavy metals in its tissue. Heavy metals bind into the coral skeleton through the feeding process or are absorbed by soft coral tissue.11 The highest concentrations of heavy metals were found in coral zooxanthellae compared with soft tissue or coral skeleton. So it is suspected zooxanthellae have a very big role in taking heavy metals to the coral body.18 Some studies have shown that heavy metals in Porites spp have less heavy metal concentrations than other corals. The location can also determine the absorption of heavy metals in the coral, where corals that live in the ocean far from the coast will have less heavy metal concentrations than coral reefs living in coastal areas and fringing reef.

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Figure 4 showed that Cu’s concentration of KM1 was relatively stable year by year, which means the ability absorption of massive P. lutea against the Cu was same all that time. The coral KM2 showed a different result, from 2010 to 2015 the Cu concentration each year increased up to 2.42 times. The differences in absorption capacity between P. lutea corals from KM1 and KM2 are possible for two reasons: first, differences in coral colony size, although the P. lutea age of KM1 and KM2 are similarly, the coral surface of P. lutea of KM2 is wider so allowing to absorb more Cu from the environment. Second, possibly that P. lutea from KM1 excretes more mucus during the water which results in Cu being trapped in the mucus and cannot enter the coral skeleton network.19 Third, the difference in substrate base in KM1 and KM2 also the depth where the coral lived. In other research on the western coast of Marinduque Island (Philippine), the concentration of Cu metal in coral Porites was very influenced by the rainy season, type of substrate and depth. Rain will be main cause stirred of sediment and brought the settle Cu in sediment can be resuspension and get into the coral network.20 As shown in Figure 4, P. lutea on KM2 has a different absorption in each year. This means the source of Cu in seawater was different each year, in 2012 to 2015 the source of Cu may more abundant combine with not much production of mucus that resulted in high concentration Cu in coral skeleton in those years. The presence of Cu metal in the coral skeleton network suggested replaces the calcium or CaCO 3 atoms that bind on a lattice of coral skeleton matrix. In another way, Cu elements can directly enter the coral skeleton then bounded by aragonite in the calcification process.21 Based on22 research in Nha Trang Bay in the south-west of South China, the high of concentration Cu in P. lutea was caused by the dredging of the harbor and the dredging of the river around the waters. This is reasonable because it was suggested that Cu has been deposited in the sediment will then be a resuspended column of water and possibly absorbed by corals. Cu concentrations in sediments can be more than 100 ppm.8

The Cu concentration in Pantai Kondang Merak and comparative to other studies Cu is an essential metal that is needed by living things as well as the coral P. lutea. In this study, Cu concentrations in the P. lutea skeleton were in the range of 0.84–2.17 ppm and the average of concentration in KM1 and KM2 were 1.09 and 1.74 ppm. In Vietnam, Cu was in the range of 2.15–44.12 ppm with an average of about 5.53 ppm [22]. At Aqaba Bay, the Cu concentrations were 4.7–5.36 ppm11; In Venezuela, Cu concentrations were in the range 3.3–89.5 ppm.23 In Ihatub reef, Marinduque Island, Philippines, it was 9.6 ppm.20 The difference in absorption of corals of Cu heavy metals is very diverse in every country; many factors can influence this condition.

SUMMARY Coral P. lutea can be used as a proxy to see past environmental conditions. In this study, P. lutea corals gave Cu concentrations data from 2009 to 2015 with varying concentrations in each year (annual band), the concentration of 0.84–1.46 ppm (KM1) and 1.22–2.17 ppm (KM2). The high Cu content of the coral skeleton does not depend on the source's sustainability; there are many influencing factors including the coral colony's biological ability to assimilate the heavy metal in its skeleton. It is not yet known exactly how lethal Cu is to coral and this is a topic for future research.

ACKNOWLEDGMENTS We would to thanks our colleagues to Siddiq Pratomo Al-Idrus, Kahindra Donny Anggara, R.M. Agung M. Rizqon S, Saifur Rijal Fakri and Maulana Abdurrahman for helping take the samples and hectic work in the laboratory. This work has been funded by Directorate of Research and Community Service, Directorate General for Research and Development, Ministry of Research, Technology and Higher Education through DIPA Universitas Brawijaya Number: 063/SP2H/LT/DRPM/IV/2017.

REFERENCES 1. 2. 3.

O. M. Luthfi, P. Z. Alviana, G. Guntur, S. Sunardi, A. Jauhari, Research Journal of Life Science 3, 23-30, (2016). T. Agustiadi and O. M. Luthfi, International Journal of Oceans and Oceanography 11, 21-30, (2017). O. M. Luthfi, P. M. Barbara, A. Jauhari, Prosiding Pertemual Ilmiah Nasional Tahunan (PIT) XII ISOI 2015, Banda Aceh, pp. 262-269, (2015).

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Meltzner, J. Aron, D. Colin, Woodroffe. Handbook of Sea-Level Research (Springer, Netherlands, 2015), pp. 125-145. S. Smithers, Encyclopedia of modern coral reefs (Springer, Netherlands, 2011), pp. 430-446. T. Ourbak, T. Corrège, B. Malaizé, F. Le-Cornec, K. Charlier, J. Peypouquet, J Geochem Geophys, Geosys 7, Q03013 (2006). V. Helen, McGregor, Encyclopedia of Modern Coral Reefs (Springer, Netherlands, 2011), pp. 777-785. N. Blossom, Am Chemet Corp, 1-8 (2007). M. Nyström, I. Nordemar, M. Tedengren, Marine Biology 138, 1225-1231 (2001). C. Broun-Fohrlonderand R. Fronfzel-Boymo, European Epi-Marker 11, 1-12 (2007). S. A. Al-Rousan, R. N. Al-Shloul, F. A. Al-Horani, A. H. Abu-Hilal, Marine Pollution Bulletin 54, 19121922 (2007). J. E. N. VeronAustralian Institute of Marine Science, 1-3 (2000) J. M. Lough and D. J. Barnes, Journal of Experimental Marine Biology and Ecology 245, 225-243 (2000). P. L. Jokiel, C. P. Jury, I. B. Kuffner, Coral Reefs at the Crossroads (Springer, Netherlands, 2016), pp. 745. D. Allemand, C. Ferrier-Pagès, P. Furla, F. Houlbrèque, S. Puverel, S. Reynaud, E. Tambutté, S. Tambutté, D. Zoccola, Comptes Rendus Palevol 3, 453-467 (2004). D. Allemand, E. Tambutté, D. Zoccola, S. Tambutté, Coral reefs: an ecosystem in transition (Springer Netherlands, 2011) pp. 119-150. J. T. I. Tanzil, B. E. Brown, A. W. Tudhope, R. P. Dunne, Coral reefs 28, 519-528. (2009) S. Shah E. Lovell, Encyclopedia of Modern Coral Reefs (Springer, Netherlands, 2011), pp. 553-554. C. L. Mitchelmore, E. A. Verde, V. M. Weis, Aquatic Toxicology 85, 48-56, (2007). C. P. David, Marine Pollution Bulletin 46, 187-196. (2003). M. A. Dar, Sedimentology of Egypt 12, 119-129 (2004). A. D. Nguyen, J. X. Zhao, Y. X. Feng, W. P. Hu, K. F. Yu, M. Gasparon, T. B. Pham, T. R. Clark, Coral Reefs 32, 181-193 (2013). C. Bastidas and E. Garcıá , Marine Pollution Bulletin 38, 899-907 (1999).

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The Effectiveness of Leachate Remediation in The Implementation of Unvegetated Constructed Wetland Sophia Laily1, a), Catur Retnaningdyah2), and Bagyo Yanuwiadi2)

1

Environmental Management and Development, Graduate School, University of Brawijay, Malang, East Java, Indonesia 65145 2 Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 a)

Corresponding author: sophiaovilaily@gmail.com

Abstract. The objective of this research was to examine the effectiveness of leachate remediation that is performed through the implementation of a free water surface (FWS) unvegetated constructed wetland system (UCW). The abovementioned remediation was conducted in a glass house with complete randomized design and using a small-scale UCW referred to as UCW reactor. The reactor was designed to replicate a large-scale FWS UCW and was filled with sand and gravel in a 3:5 ratio. The measurements of the leachate quality throughout the remediation experiment were based on hydraulic retention time (HRT) calculation and carried out on the 1st, 5th, 10th, 15th, 21th and 30th days. Subsequently, the resulted homogenous measurements were analyzed using One-way ANOVA while the nonhomogenous ones were analyzed using the Brown-Forsythe test. For further analyses on the resulted statistical data, Turkey-HSD or Games Howell test and Euclidean-distance clustering and biplot were applied. The data representing value decreases in the physicochemical leachate parameters suggest the improvement of the leachate quality throughout the treatment. It was proven that FWS UCW is effective in reducing conductivity, total dissolved solids (TDS), nitrate and orthophosphate contents by 51.31%, 32.94%, 52.25% and 36.24%, respectively on the 5th day. On the 30th day, the leachate quality was further improved as the decreases of the four substances reached 79.64%, 56.28%, 80.58% and 90.39%, respectively.

INTRODUCTION In Indonesia, the sanitary landfill system is a compulsory waste management procedure in final dumpsite as ruled in Law Number 18/ 2008 concerning waste management. The system has so far been considered the best approach to such task on account of its capacity in lending itself to the effective control of pollution caused by leachate and methane, which are the by-products of the solid waste landfill. Leachate is a liquid that seeps or leaches out of a mass of garbage produced by the water content in it which percolates down the waste deposit and in its process it drains and dissolves the soluble materials along with the substances brought about by the decomposing waste matters. Leachate from landfill waste is normally made percolating downward by the gravity and accumulates


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on the impermeable liners of the landfill cell. It is then channeled into leachate collection pipes installed to the floor of the cell. Afterwards, the pipes transport the leachate into a Leachate Processing Plant as its final destination in the leachate collection system before going further for treatment. Leachate Processing Plant, in which the leachate is treated through physic-chemical processes, is a technological installation for a conventional leachate treatment that is still widely used in landfill sites in Indonesia. In this conventional system, the organic matters in the leachate effluent, which is processed through three stabilization tanks (sedimentation, coagulation-flocculation, maturation), could only be reduced to less than 40%. In addition, leachate recirculation is also included in the treatment system for the purpose of keeping the waste moist in the dry season, injecting starter (bacteria) responsible for decomposition, and reusing the leachate effluents which have not met the required quality standard for disposal. Like most final dump sites in Indonesia which undertake a transition into a sanitary landfill system, waste management in TPA Wisata Edukasi Talangagung is carried out through the implementation of sanitary landfill system and leachate treatment in Leachate Processing Plant with less than 40% effectiveness in improving leachate quality (based on the preliminary study). Recirculation has been taken as the best method to reuse leachate effluent. Nevertheless, the plant is getting overburdened and overtasked by the ever-increasing leachate quantity generated from the growing amount of waste mass over time and the continuously reused effluents. Recirculation is liable to cause a number of impacts. Among others are the increasing production of methane, the overflowing leachate, and also the accumulating quantity of volatile organic acid compounds and hazardous non-degradable heavy metal components. The constructed wetland system has been frequently studied and implemented as a method of treating various kinds of wastewater. Constructed wetland (CW) is defined as a form of wetland ecosystem engineered by humans for wastewater management purpose. Common components of the constructed wetland include substrates, vegetation and a hydraulic rotation of wastewater to be managed in its treatment process (involving wastewater inflow and outflow in CW and hydraulic retention time). The reduction of organic substances in wastewater in a constructed wetland is achieved through a number of processes that include physical, chemical and biological processes. In the physical process, there occurs sedimentation and volatilization. Chemical process involves precipitation, adsorption, ion exchange, and ultraviolet radiation. During the biological process in the plant, there is the decomposition of organic matters by microbes, microbial uptake, and plant uptake. The extent of organic matters reduction by plant uptake mechanism only amounts to 5 to 10% of the total complex processes of organic matters reduction within the treatment system performed in Leachate Processing Plant (LPP). It indicates the much greater role of physical and chemical processes in LPP’s operation when hydraulic retention time (HRT) is incorporated in wastewater treatment. HRT allows a certain amount of time for wastewater to make longer contact with the substrates and vegetation. The longer it is exposed to these two components, the better its quality gets. Free water surface (FWS) is one of the types of LPP that creates an aerobic condition for wastewater. The aerobic condition allows the faster process of organic matter decomposition than anaerobic condition does. A field research conducted by A. B. Gupta et al. (2013) 1 in Jaipur, India, has revealed that unvegetated CW of type FWS is more efficient compared with other types in removing various kinds of pollutants except for nitrogen. It is due to the fact that most pollutants with big molecules are likely to settle down at the bottom and create sediment on LPP’s substrates. On the contrary, vegetated CW is better at reducing nitrogen content because plants need to absorb nitrogen to survive. According to Wallace, the degree of total suspended solids (TSS) and turbidity can also be well reduced in an unvegetated FWS CW system but it can easily be back to its previous quantity when resuspension takes place as a result of any turbulence and movement of small animals in the water. This research is aimed at analyzing the effectiveness of leachate remediation that is performed through the implementation of a free water surface (FWS) unvegetated constructed wetland system (UCW).

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EXPERIMENTAL DETAILS Time and Location This research was an ex-situ experiment carried out from January to February 2017 in a glass house of Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya. The leachate sample was taken from the first tank in the Leachate Processing Plant in the Final Waste Disposal Site (TPA) of Talangagung, Kepanjen city, Malang Regency.

Material and Method A small-scale UCW or UCW reactor was designed to resemble a large-scale FWS CW. The reactor was filled with sand and gravel substrates in 3:5 ratio. 9 liters of leachate was poured into the reactor until its surface went up 5 cm above the surface of the substrates (see Figure 1). The reactor was created in a completely randomized design with a hydraulic retention time (HRT) as the independent variable in the experiment. Four replications of UCW reactor were built; each of which was made from the plastic tank or tub 40 cm in diameter and 24 cm in height. HRT is the length of time that a compound (e.g. water) remains in a storage unit. was made from the plastic tank or tub 40 cm in diameter and 24 cm in height. HRT is the length of time that a compound (e.g. water) remains in a storage unit.

FIGURE 1. The cross-section of unvegetated constructed wetland reactor

Data Collection The measurements of physicochemical parameters of leachate were performed at 6 HRTs (Hydraulic Retention Time) which were plotted on the 0, 5th, 10th, 15th, 21st, and 30th days in the Microbiology Laboratory and the Ecology-Animal Diversity Laboratory at the Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya. The leachate quality was observed for 4 parameters namely conductivity, Total Dissolved Solids, Nitrate, and Orthophosphate. The physical conditions of the leachate in the reactors were observed and recorded in photos.

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Data Analysis The data resulted from the measurements were tabulated using a statistics software program, that is SPSS (Software Package for Statistics and Simulation). The data that were identified as normal and homogenous based on Levene test were analyzed using one-way ANOVA test whereas those identified as normal but not homogenous were analyzed using the Brown-Forsythe test. The results of one-way ANOVA and Brown Forsythe analyses that showed significant differences were analyzed further using the Tukey-HSD test (as the follow-up of ANOVA) and Games-Howell test (as the follow-up of Brown-Forsythe) to observe the specific values of the differences. Afterwards, biplot graph statistical processing was applied to group and to describe the overall results of measurements and analyses for all of the observed physicochemical parameters. The percentages of decreases in leachate quality parameters were also tabulated using the relevant Ms. Excel program.

RESULT AND DISCUSSION The overall differences in leachate quality on the 0th, 5th, 10th, 15th, 21st, and 30th days in the UCW reactors are illustrated in the biplot graph as presented in Fig. 2 with the four parameters marked out on it. The leachate quality on the 0th day is characterized by the whole physicochemical parameters showing high values as illustrated in the figure by the group of data in a marine blue polygon. The group of data in the black polygon represents the leachate quality on the 5th day, which indicates significant decreases in Conductivity, TDS, Nitrate, and Orthophosphate. The greater decreases in the four parameters were shown by the data taken from the 10th and the 15th days of observation. In Figure 2, they are represented by the groups of data in the red and purple polygons. It is apparent that the 10th-day group and the 15th-day group are close to each other, which means the decrease percentages in both groups generally show little differences. The 21st-day group and the 30th-day group of data, which are represented by the blue and orange polygons respectively, also show a little gap from each other. As in the case of the 10th-day group and the 15th-day group, the data of leachate quality in the 21st-day group show insignificant difference from those in the 30th-day group. The amounts of decrease of Conductivity, TDS, Nitrate, and Orthophosphate on the 30th day are 79.64%, 56.28%, 80.58%, and 90.39% respectively. The percentages of the decreases for the four physicochemical parameters of leachate quality can be seen in Table 2. Table 1 presents the mean values of each parameter that were obtained from the observations on the 0th, 5th, 10th, 15th, 21st, and 30th days. Figure 3 illustrates the diagrams that represent the correlations of those values of the observed decreases shown in Table 1 for all the parameters of leachate quality. Conductivity that theoretically correlates positively with TDS is illustrated on combo chart (a) in Figure 3. TDS has positive correlation with conductivity because TDS contains soluble inorganic compounds such as Sodium (Na), Calcium (Ca), Magnesium (Mg), Bicarbonate (HCO3), Sulphate (SO4), Chloride (Cl), Iron (Fe), Strontium (Sr), Kalium (K), Carbonate (CO 3), Nitrate (NO3), Fluoride (F), Boron (B), and Silica (SiO2). The positive correlation enables the estimation of TDS values using a constant (0.55–0.75) depending on the kind of water involved in it (Effendi, 2003) 2. The highest value of conductivity was observed on the 0th day, which was 18.30 μSiemens/cm. It was steadily declining throughout the experiment and reached 3.72 μSiemens/cm on the 30th day. This decrease indicates the lessening number of inorganic ions through the processes of sedimentation, diffusion, and volatilization. HRT treatment provided the necessary amount of time for the unstable compounds to bond and coagulate. According to Wallace, et al. (2006)3, removal mechanism in FWS wetlands can be classified into the physical, chemical, or biological process. A specific contaminant may be affected by two or more mechanisms acting simultaneously or sequentially, depending upon the contaminant and its location within the wetland. Precipitation reaction has an important role in reducing the number of metal ions such as iron, copper, and nickel. When these physical and chemical processes are taking place, the biological process by microbes is also in progress in the UCW reactor involving microbial degradation and uptake. As in the case of the inorganic ions aforementioned, orthophosphate decreased significantly not only by precipitation and adsorption but also by sedimentation and microbial uptake. Nitrogen, which includes Nitrate (NO 3-

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), Nitride (NO2-), Ammonia (NH3), Ammonium (NH4+), was significantly decreasing by microbial uptake and transformation processes. It also underwent sedimentation, adsorption and volatilization processes. The mean values of nitrate and orthophosphate decreases are shown on combo chart (b) in Fig. 3.

FIGURE 2. Biplot Graph of leachate quality parameters in Unvegetated CW reactor in 6 hydraulic retention times

b 10000

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FIGURE 3. Mean values of physicochemical parameters measured throughout a series of HRT and illustrated in combo chart: (a) conductivity and TDS, and (b) nitrate and orthophosphate

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TABLE 1. Mean Values of leachate quality for the four physicochemical parameters HRT 0 5 10 15 21

30

Conductivity 18.30 8.91 9.45 6.17 3.72

TDS 9644.38 6471.88 6287.40 4606.77

Nitrate

Orthophosphate

6.67 3.18 3.11 2.64 1.78

1196.528

0.554

5.34 3.41 2.23 2.69 1.11 0.53

TABLE 2. The decrease percentages of leachate quality in the four physicchemical parameters in 5 HRT HRT Conductivity TDS 5 51.32 32.89 10 *) *) 15 48.35 34.81 21 66.28 52.23 30 79.67 56.46 Note: *) no measurement is performed

Nitrate

Orthophosphate

52.27 53.38 60.40 73.37 80.55

36.16 58.19 49.67 79.18 90.15

SUMMARY The effectiveness of leachate remediation using unvegetated constructed wetland system in this research has been proven. The improvement in leachate quality was indicated by the decreases observed in four physicochemical leachate parameters. The decreases of conductivity, total dissolved solids (TDS), nitrate and orthophosphate contents had reached 51.31%, 32.94%, 52.25% and 36.24% respectively on the 5th day. By the 30th day, the decreases of the four substances were up to 79.64%, 56.28%, 80.58% and 90.39% respectively.

ACKNOWLEDGMENTS I am indebted to the manager of Final Waste Disposal Site of Talangagung, Kepanjen city, Malang Regency, for allowing me to obtain some material from the site for my research. I also greatly owe the district government of Malang Regency for generously providing me the opportunity to pursue my master degree in the Postgraduate Program of Environmental Management and Development at the University of Brawijaya.

REFERENCES 1. 2. 3.

H. Effendi, Telaah Kualitas Air Bagi Pengelolaan Sumber Daya dan Lingkungan Perairan (Kanissius, Yogyakarta, 2003) A. B. C. Gupta, R. Tushali, Journal of Environmental Research And Development 7, 4A (2013). S. D. Wallace, R. L. Knight, Small-scale constructed wetland treatment systems : feasibility, design criteris, and O&M requirements (Water Environment Research Foundation, USA, 2006).

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Geo Techno Park Potential at Arjuno-Welirang Volcano Hosted Geothermal Area, Batu, East Java, Indonesia (Multi Geophysical Approach) Sukir Maryanto1, 2, a) 1

Department of Physics, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145. 2 Center of Energy and Natural Resources, Institute of Research and Community Services, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 a)

Corresponding author: sukir@ub.ac.id

Abstract. Arjuno Welirang Volcano Geothermal (AWVG) is located around Arjuno-Welirang Volcano in Malang, East Java, about 100 km southwest of Surabaya, the capital city of East Java province, and is still an undeveloped area of the geothermal field. The occurrence of solfatara and fumaroles with magmatic gasses indicated the existence of a volcanic geothermal system in the subsurface. A few hot springs are found in the Arjuno-Welirang volcanic complex, such as Padusan hot spring, Songgoriti hot spring, Kasinan hot spring, and Cangar hot spring. Multi geophysical observations in AWVG complex was carried out in order to explore the subsurface structure in supporting the plan of Geo Techno Park at the location. Gravity, Magnetic, Microearthquake, and Electrical Resistivity Tomography (ERT) methods were used to investigate the major and minor active faulting zones whether hot springs circulation occurs in these zones. The gravity methods allowed us to locate the subsurface structure and to evaluate their geometrical relationship base on density anomaly. Magnetic methods allow us to discriminate conductive areas which could correspond to an increase in thermal fluid circulation in the investigated sites. Micro-earthquakes using particle motion analysis to locate the focal depth related with hydrothermal activity and electrical resistivity tomography survey offers methods to locate more detail subsurface structure and geothermal fluids near the surface by identifying areas affected by the geothermal fluid. The magnetic and gravity anomaly indicates the subsurface structure of AWVG is composed of basalt rock, sulfide minerals, sandstone, and volcanic rock with high minor active fault structure as a medium for fluid circulation. While using microearthquake data in AWVG shown shallow focal depth range approximate 60 meters which indicates shallow hydrothermal circulation in AWVG. The geothermal fluid circulation zones along the fault structure resulted in some hot springs in a central and north-western part of AWVG detected by the Electrical Resistivity Tomography, appear to be well correlated with corresponding features derived from the gravity, magnetic, and micro-earthquake survey. We just ongoing process to develop Arjuno Welirang Volcano & Geothermal Research Center (AWVGRC) located at Universitas Brawijaya Agro Techno Park, Cangar in the flank of Arjuno Welirang volcano complex. Due to our initial observations, AWVG has a great potential for a pilot project of an educational geo technopark development area.

INTRODUCTION Geothermal energy potential in Indonesia was estimated to reach 28,910 MW or approximately 40% of the world’s potential and spread in 256 locations. From this potential, about 203 locations (±80%) are spread in volcanic areas which are called volcano-geothermal, while about 53 locations (±20%) are spread in non-volcanic areas The number of each geothermal prospect area is 84 regions in Sumatera, 76 in Java, 51 in Sulawesi, 21 in Nusa Tenggara, 3 in Papua, 15 in Maluku and 5 prospects in Kalimantan. The geothermal system in Indonesia is mostly volcanic geothermal system associated with Quaternary volcanoes which generally located in the Quaternary volcanic arc extending from Sumatera, Java, Bali and Nusa Tenggara, also parts of Maluku and North Sulawesi. This system is a hydrothermal system with a temperature above 2250 °C


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and some of them have medium temperatures (1500–2500 °C). There are 159 locations (62.1%) of the geothermal area at the preliminary investigation and 82 locations (32.03%) have been surveyed in detail through surface exploration without temperature drainage drilling. Indonesia currently produces 1,189 MW of geothermal (4.3%) of its total geothermal energy and it is utilized for power generation. The largest power generation is in West Java of 1057 MW (20% of reserves), followed by Central Java 60 MW, North Sulawesi 60 MW, and North Sumatera 12 MW. Geothermal systems in Indonesia are usually composed of medium volcanic rock (andesite-basaltic) to acid volcanic rock and generally have reservoir characteristics >1.5 km with a high-temperature reservoir (~250°–370 °C). Active volcanic areas have young volcanic rocks with high-temperature conditions and large magmatic gas content. Space between rocks is relatively small, due to tectonic activity that has not been too dominant in forming intensive gaps/cracks as reservoir rocks. The inactive volcanic areas generally have relatively older volcanic rocks and experience strong tectonic activity to form rocks permeability through intensive cracks and fissures. In these conditions, it is usually formed medium to high temperatures with fewer magmatic gas concentrations. Generally, the volcanic system in Indonesia can be divided into Strato-volcano system, volcanic complex system, and caldera system. The volcano-tectonic geothermal system is associated with graben and volcanic cones, for example in Sumatera fault system (Semangko fault).

(a)

(b)

FIGURE 1. Distribution of active volcanoes and geothermal potentials in Indonesia (a) Map of type A, B, and C volcano distribution as well as tectonics in Indonesia; (b) Distribution maps of volcano geothermal potentials (80%) and non-volcano geothermal (20%) in Indonesia

A non-volcanic system is a geothermal system that is not directly related to volcanism and not included in the quaternary volcanic route. The non-volcanic environments in western Indonesia generally spread in the eastern part of the Sundaland (Sunda exposure) because rocks that constitute the Asian continental crust such as metamorphic rocks and sediments dominate it. In eastern Indonesia, non-volcanic environments are located in the arm and leg of Sulawesi, Maluku islands to Irian and are dominated by granitic, metamorphic and marine sediments rocks. East Java has considerable volcano-geothermal energy potential, due to the presence of Mount (Mt.) Arjuno Welirang, Mt. Wilis, Mt. Kelud, Mt. Bromo, Mt. Semeru, Mt. Lamongan, Mt. Ijen, Mt. Iyang Argopuro, etc. Around 11 locations of geothermal potential in East Java are estimated to generate the energy of 1206 MWe or nearly 5% of total geothermal potential in Indonesia. Three of the 11 locations (Arjuno-Welirang, Wilis Argopuro, and BlawanIjen) are estimated to have 274 MWe of possible reserves and resources of 240 MWe. If exploration efforts for other sites have done, it is certain that the total number of resources (515 MWe) will increase. The potential still not utilized except for tourist destinations. The Arjuno Welirang Volcano Geothermal (AWVG) located around of Arjuno-Welirang Volcano in Malang, East Java about 100 km southwest of Surabaya, the capital city of East Java and is still an undeveloped geothermal prospect. The occurrence of solfatara and fumaroles with magmatic gasses indicated the existence of a volcanic geothermal system in the subsurface. A few hot springs are found in the Arjuno-Welirang volcanic complex, such as Padusan hot spring, Songgoriti hot spring, Kasinan hot spring, and Cangar hot spring. The high-temperature geothermal system characterized by the presence of thermal features that emitted solfatara and fumaroles with the high content of sulfur deposit. The heat source and up-flow zone are under Mount Welirang’s summit that associated with andesitic1.

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The geothermal system in Java can be classified into volcano-hosted and fault-hosted. Based on these classifications, AWVG were considered as volcano-hosted. The Na/K and Na-K-Ca geothermometer were applied to estimate the highest and the lowest temperature in the geothermal system. These two methods were applied to calculated geothermal temperature Arjuno-Welirang and yield range from 217 °C to 305 °C2. In geothermal exploration, understanding of the structural relationship between faults and regions of hydrothermal upwelling is very important. The magnetic3, gravity survey methods are often classified together as structural methods. In this sense, the structural methods are primarily an extension of geological mapping. There is also considerable evidence showing that some of the anomalies mapped by the structural methods in geothermal areas may be directly caused by the effect of the hydrothermal system on the host rock. On the other hand, the electrical methods may also be called structural, to the extent variations in porosity cause the resistivity variations, shows that there is no sharp distinction possible between methods which map the thermal and the structural parameter of a hydrothermal system. To understand geothermal prospect in CGF was conducted using the magnetic method, gravity method, and the Electrical Resistivity Tomography (ERT) method.

Geological Setting Arjuno-Welirang volcano is located in Sololane quaternary, flanked by Kendeng Lane in the north and southern mountain in the south. The geological history of this area is dominated by late Quaternary volcanic rocks, both lava, and pyroclastic. Geological structures such as faults, the caldera rim structures, and other circular features are indicated by remote sensing data as shown in Figure 1. The circular feature correlated with the collapse zone that formed as a result of Pre Arjuno-Welirang volcanic eruption. Multiple extensional events and emplacement of the CGF have resulted in the anomalously thin crust that provides a shallow regional heat source and high geothermal gradients. The Cangar fault caused by converging of old Anjasmara Mountain and Welirang Mountain appears to be a controlling factor governing the location of Cangar hot spring or further called Cangar Geothermal Field (CGF), whereas the Padusan fault controls the Padusan hot spring. The temperature reservoir of the Arjuno-Welirang geothermal system could be categorized as a high-temperature geothermal system. The reservoir is possibly composed of quaternary volcanic rock as a result of stratigraphic correlation based on surface lithology. The high content of sulfur deposits indicates the reservoir fluid is acid, which is obviously influenced by active magmatic.

3 FIGURE 2. Geological Map of Cangar Geothermal Field, East Java, Indonesia4

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EXPERIMENTAL DETAILS Magnetic and Gravity Survey The objective of the magnetic method is to examine subsurface geology based on the anomaly of the Earth’s magnetic field resulting from the magnetic properties of the underlying rocks. In general, the magnetic content of rocks or called susceptibility is extremely variable depending on the type of rock and the environment it is in such as dykes, faults, and lava flows. In a geothermal environment, due to high temperatures, the susceptibility decreases1. Rocks in the geothermal system also have lower magnetization than the surrounding rock due to demagnetization by hydrothermal alteration process, which converts existing minerals to become paramagnetic or diamagnetic. The low magnetic values can be interpreted zones and reservoir potential as a heat source11. Temporal changes in the Earth’s field during the period of the survey must be considered to give more accuracy to the magnetic anomaly maps. Deviations in the intensity of Earth’s magnetic field are caused by the time difference measurements and the effect of sunlight in a day. Total magnetic field data processing was done through corrections: daily (drift correction), and the IGRF (International Geomagnetic Reference Field). Filtering using upward continuation was done to the total magnetic anomaly for obtained regional and residual magnetic anomalies; it was done by reducing the total magnetic anomaly to the even surface at the highest topographic. To get the exact position of the cross-section which is also used pseudo gravity transformation considerations, and horizontal gradient of total magnetic anomaly5. The gravity method is the measurement of the Earth’s gravity to find the local density information surrounding the formation and learn something about the irregularities of the earth6. The result of gravity measurement at the surface varies, besides due to irregularities of the Earth, is also affected by the altitude, the location of the buried bodies around the Earth and rock density conditions beneath the Earth’s surface. Therefore, it is necessary to have some correction, latitude correction, and free air correction. Terrain correction is also used to take account of the gravitation changing due to the terrain surrounding the measurement point. Bouguer correction was applied to the dataset to take account of rock thickness between the current and base station elevation. After applying all corrections, the value of the gravity anomaly is known as the Bouguer anomaly.

Electrical Resistivity Tomography ERT (Electrical Resistivity Tomography) is a form of high-resolution 2D or 3D geology of the subsurface resistivity value. ERT is often used to solve geological problem, including determine position of active fault and dislocating of formation before paleoseismology study7, 8, 9,10,11,12,13, understanding characteristic of fault zone and discover fluid and estimates wide of area investigate14,13 depiction of the geological structure of basin sedimentation15 and volcanic area16,17. The ERT method gives more detailed information on horizontal and vertical layers, but the process of data acquisition requires a longer time. ERT measurements were performed by varying the electrode spacing and move the overall position of the electrode configuration on a trajectory to obtain information on the resistivity distribution both vertically and laterally, or within a stretch of the electrode determines the depth of the soil layer of the survey area. The configuration used is the Wenner configuration. Data acquisition of geoelectrical resistivity, mapping the hot springs area Cangar consist in four line. Line length ranges from 90–120 meters with the position of each line track surrounding the hot springs.

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FIGURE 3. Regional anomaly contour map using interval 100 nT, which red triangle indicates data point and pink cross indicate geothermal manifestationThe residual anomaly contour map shows magnetic values in the range between –1000 nT and 680 nT. Suspected areas of geothermal fluid alteration have a small magnetic intensity in the anomaly contour map (Figure 3). A crosssection was used to understand geology structure in the subsurface. Qualitative interpretation of the AB cross section from West to East shows an area dominated by igneous rock such as basalt and granite, and also a sedimentary rock, sandstone. Calcite and sulfide were found in 0–1500 meters as a result of intrusion from fault zone and activity of Arjuno-Welirang (Figure 4). The subsurface structure of the CD cross section was similar to the AB cross-section, where the rock structure was dominated by igneous rock such as basalt, porphyry, and diabase. Also, there are mineral rocks such as calcite and sandstone. In the top layer at a depth of 0–1253 meters are calcite rocks, porphyry, sandstone, and rock diabase. The presence of these minerals was the result of intrusion across basalt rock through the fracture. Whereas at a depth of 1253–2000 meters igneous rock that is basalt and diabase still dominated (Figure 5).

030012-5

FIGURE 4. Residual anomaly contour map using interval 40 nT, which red triangle indicates data point and pink cross indicate geothermal manifestation

FIGURE 5. Cross section AB profile

030012-6

FIGURE 6. Cross section CD profile

In contrast with magnetic anomalies, we perform the Bouguer anomaly in Figure 6, showing range in the study area between 15 and 120 mGal. The low anomaly was located in the north and southwest of the hot spring, while a high anomaly was located to the east of the hot spring. The high-density contrast shows that there are cracks in the area. The Bouguer anomaly shows a longitudinal fracture in the north-south direction and in lines with this fault, there is a manifestation of hot spring in the surface. These phenomena can occur in this area because groundwater that has been heated rise to the surface through the cracks of rocks. The residual anomaly contour map in Figure 7 shows an anomaly in the range between –8 and 14 mGal, which is much smaller than the value of Bouguer anomaly. In the residual anomaly, the low and high anomaly is located adjacent and within the same area as the Bouguer anomaly.

FIGURE 7. Bouguer anomaly map with contour interval is 5 mGal, black star dot showing data collecting point and red dot indicates hot spring manifestation

030012-7

FIGURE 8. Residual anomaly contour using interval 1 mGal, black star dot showing data collecting point and red dot indicate hot spring manifestation

An estimation of subsurface conditions done by modeling the geology in the area alleged that AA’ and BB’ like Figure 8. The AA’ line across the fracture on residual anomalies, whereas the BB’ line across the area indicates the manifestation of hot spring. Rock types in AA’ and BB’ line have the same type of variations in thickness viewing in Figures 9 and 10. On the top layer along the path AA’ are sandstone (1.7 to 2.3 g/cm3), igneous lava (2.8 to3.0 g/cm3), rock basalt (2.7 to 3.3 g/cm3), and sulfide mineral (2.8 to 3.0 g/cm3). The sulfide mineral basalt rocks broke through fractures due to Arjuno-Welirang activity. While the hot spring located CGF comes from groundwater by heating basalt rocks and lava rocks which are volcanic rocks of Arjuno-Welirang. This result agrees with the result from the magnetic data.

FIGURE 9. Residual anomaly contour map in 1 mGal interval, black line indicates the modeled area and red dot indicate hot spring manifestation

030012-8

FIGURE 10. AA’ cross-section residual anomaly contour with variation color to indicate each rock density composition. The red color from dark red to light red respectively is rock basalt (2.7 to 3.3 g/cm3), igneous lava (2.8 to 3.0 g/cm3), and sandstone (1.7 to 2.3 g/cm3). Blue color indicates the sulfide minerals (2.8 tp 3.0 g/cm3)

FIGURE 11. BB’ cross section from residual anomaly contour (color information same as Fig. 9)

Processing electrical resistivity mapping subsurface was conducted to estimate the crack as a tunnel where hot fluid flows and emerging the surface as hot spring. Line 1 shows the area dominated by fracture/cracks and sediment erosion. A number of cracks in the research area emergence hot springs with the highest temperature at the source of the most southwest (Figure 11). Line 2 (Figure 12) did not indicate any alleged lava rock as a heat source, but the result of erosion sedimentation found in slopes around the hot springs. Result interpretation of Line 3 indicates sandstone, tuff, and igneous rock. Sandstone layer existed as a result of erosion and sedimentation of the stream (Figure 13). The convergence between the tuff and sandstone showed a crack but did not indicate the source of water at the site. Line 4 (Figure 14) shows layers dominated the sandstone as sedimentation area which is the result of river sedimentation and lava rock not found. A small value of resistivity is located in the middle of the line.

030012-9

FIGURE 12. Resistivity profile Cangar Line 1

FIGURE 13. Resistivity profile Cangar Line 2.


030012-10


Vertical Electrical Sounding (VES) was also performed in this area to obtain more detail of the geological condition vertically. Interpretation of the data of this area suggests that the geological condition is dominated by lava rock with resistivity value >1000 Ohm meter in layers near the surface, then there is a layer of tuff with a variation of sand. The tuff layer has inserts of sandstone suspected as a result of erosion from the nearest volcanic area. The result of processing VES shows the flow of hot water with resistivity value below 10 Ohm meter, at a depth of 24.7 meters with a thickness of ±10 meters. This result is reinforced by the presence of lava rock layers alleged head source located below the rock layer has a low resistivity (18 h) for increasing the absorbance by 0.3.

050008-4

TABLE 2. Acid tolerance of Lactobacillus plantarum in MRS broth (pH 2–6.2) measured by the time taken to reach absorbance 0.3 units at 620 nm

Time* (h) Strain

MRS pH 2.0

dt (min)

Time* (h) MRS pH 3.0

dt (min)


dt (min)


dt (min)


dt (min)

Time* (h) control MRS pH 6.2

S1.30

>18

n.a

>18

n.a

>18

n.a

6.1

66

5.2

12

5.2

GD.1

>18

n.a

>18

n.a

>18

n.a

7.6

144

4.8

-18

5.3

SL2.2

>18

n.a

>18

n.a

>18

n.a

16.7

678

5.7

18

5.4

SL0.17

>18

n.a

>18

n.a

>18

n.a

>18

n.a

7.6

78

6.3

SL2.10

>18

n.a

>18

n.a

>18

n.a

>18

n.a

8.0

60

7.0

SL3.4

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.3

>18

n.a

>18

n.a

>18

n.a

>18

n.a

7.4

36

6.8

SL2.7

>18

n.a

8.0

-18

21.2

774

8.8

30

7.9

-24

8.3

SL3.1

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.2

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.4

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.12

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL3.2

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL3.5

>18

n.a

>18

n.a

>18

n.a

4.6

36

4.0

0

4.0

SL3.3

>18

n.a

>18

n.a

>18

n.a

9.4

36

8.3

-30

8.8

SL3.7

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL3.6

>18

n.a

>18

n.a

>18

n.a

5.3

132

3.3

6

3.2

SL3.8

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.5

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

GD.6

>18

n.a

>18

n.a

>18

n.a

10.3

-18

9.8

-42

10.6

*Time (h) required to increase absorbance by 0.3 units at 620 nm in each medium. t Delay of growth between the control culture and low pH culture in minutes. n.a: not available

On the other hand, different results were shown when the 20 strains of L. plantarum were cultivated in MRS broth supplemented with oxgall (0.3%, 0.5%, and 1%). Although supplementation of bile (oxgall) in the medium also inhibited the growth rate of the tested strains, the number of tolerant strains was higher than when they were grown in acidic condition. The higher the concentration of the oxgall, the longer the delay observed. Among the tolerant strains (S1.30, GD.1, SL2.7, SL3.5, and SL3.6), Lactobacillus plantarum S1.30 and L. plantarum GD.1 were the most tolerant strains against 1% of oxgall, which had the delay time of 210 min. When considering the other oxgall concentration (0.3% and 0.5%), L. plantarum S1.30 exhibited a lower delay time (lag phase) compared to other tolerant strains.

050008-5

TABLE 3. Bile tolerance of Lactobacillus plantarum in MRS broth measured by the time taken to reach absorbance of 0.3 units at 620 nm

Strain

Time* (h) MRS bile 0.3

t

d (min)


t

d (min)


t

d (min)

Time* control

(h)

MRS 6.2

pH

S1.30

8.2

180

8.0

168

8.7

210

5.2

GD.1

10

288

11

342

8.7

210

5.3

SL2.2

>18

n.a

3.5

-114

>18

n.a

5.4

SL0.17

>18

n.a

9.5

198

>18

n.a

6.3

SL2.10

>18

n.a

>18

n.a

>18

n.a

7.0

SL3.4

>18

n.a

>18

n.a

>18

n.a

>18

GD.3

>18

n.a

>18

n.a

>18

n.a

6.8

SL2.7

11.6

198

13.7

324

20.8

762

8.3

SL3.1

>18

n.a

>18

n.a

>18

n.a

>18

GD.2

>18

n.a

>18

n.a

>18

n.a

>18

GD.4

>18

n.a

>18

n.a

>18

n.a

>18

GD.12

>18

n.a

>18

n.a

>18

n.a

>18

SL3.2

>18

n.a

>18

n.a

>18

n.a

>18

SL3.5

5.9

114

5.9

114

8.1

246

4.0

SL3.3

>18

n.a

>18

n.a

>18

n.a

8.8

SL3.7

>18

n.a

>18

n.a

>18

n.a

>18

SL3.6

5.1

114

5.1

114

6.9

228

3.2

SL3.8

>18

n.a

>18

n.a

>18

n.a

>18

GD.5

>18

n.a

>18

n.a

>18

n.a

>18

GD.6

>18

n.a

>18

n.a

>18

n.a

10.6

*Time (h) required to increase absorbance by 0.3 units at 620 nm in each medium. t Delay of growth between the control culture and low pH culture in minutes. n.a: not available

To confirm whether these five strains of L. plantarum tolerant to 1% of oxgall can survive in MRS broth at pH 2, the selected strains were grown in MRS broth at pH 2 for 3 h and then the survival rate (%) was calculated by dividing the number of viable cell before and after incubation (Table 4). The highest survival rate was L. plantarum SL2.7 with the survival rate of 94.9%.

050008-6

TABLE 4. Survival rates of five strains of L. plantarum in MRS broth at pH 2 (n=2, x±SEM)

Strains

Tolerance to MRS broth at pH 2 (log CFU/mL) 0h 3h 8.42±0.01 6.38±0.10 8.28±0.00 7.12±0.12 8.28±0.01 7.85±0.10 8.39±0.02 7.78±0.18 8.31±0.01 7.61±0.10

S1.30 GD.1 SL2.7 SL3.5 SL3.6

Survival rate (%) 75.7 85.9 94.9 92.7 91.5

By measuring the delay time of 29 strains of L. lactis subsp. lactis and one strain of E. faecium in the M17 medium at pH range from 2–6.2, a number of strains of L. lactis subsp. lactis (SL3.34, SL1.9, D1.1, SL0.15, DC.6, SL1.18 and SL3.27) showed a reduced delay time when pH increased from 5 to 6.2 (Table 5). These strains were sensitive to low pH (below pH 5). After these selected strains were grown in M17 broth at pH 2 for 3 h, only two strains of L. lactis subsp. lactis (SL3.34 and SL3.27) were able to survive in the low pH, with survival rates of 66.3% and 51.3%, respectively (Table 6). This indicates that L. lactis subsp. lactis SL3.34 demonstrated a better tolerance to low pH than other strains. TABLE 5. Acid tolerance of Lactococcus lactis subsp. lactis and Enterococcus faecium in M17 broth (pH 2–6.2) measured by the time taken to reach absorbance 0.3 units at 620 nm

Strain

Time* (h) pH 2.0

t

d (min)

Time* (h) pH 3.0

t

d (min)

Time* (h) pH 4.0

t

d (min)

Time* (h) pH 5.0

t

d (min)

Time* (h) pH 6.0

t

d (min)

Time* (h) control pH 6.2

SL3.34

>18

n.a

>18

n.a

>18

n.a

7.8

198

3

48

4.5

SL1.9

>18

n.a

>18

n.a

>18

n.a

>18

n.a

7.6

78

6.3

D2.15

>18

n.a

>18

n.a

>18

n.a

>18

n.a

7.5

42

6.8

SL0.3

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

9.2

SL1.9

>18

n.a

>18

n.a

>18

n.a

14

612

6.0

132

3.8

SL3.32

>18

n.a

>18

n.a

>18

n.a

2.8

42

2.2

0

2.2

D1.1

>18

n.a

>18

n.a

>18

n.a

12.2

480

4.5

18

4.2

SL1.16

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL0.11

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

D2.17

>18

n.a

>18

n.a

>18

n.a

>18

n.a

5.7

138

3.3

SL0.15

>18

n.a

>18

n.a

>18

n.a

14.2

570

6.5

108

4.7

D1.9

>18

n.a

>18

n.a

>18

n.a

>18

n.a

3.6

48

2.8

SL2.15

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

13.1

SL3.16

>18

n.a

>18

n.a

>18

n.a

>18

n.a

6.7

108

4.8

DC.6

>18

n.a

>18

n.a

>18

n.a

6.7

282

2.5

24

2.1

050008-7

D3.6

>18

n.a

>18

n.a

>18

n.a

>18

n.a

2.7

36

2.1

SL3.19

>18

n.a

>18

n.a

>18

n.a

>18

n.a

2.7

24

2.3

D1.2

>18

n.a

>18

n.a

>18

n.a

>18

n.a

5.3

138

2.9

D1.7

>18

n.a

>18

n.a

>18

n.a

>18

n.a

7.3

96

5.8

SL3.14

>18

n.a

>18

n.a

>18

n.a

>18

n.a

2.6

24

2.2

SL1.18

>18

n.a

>18

n.a

>18

n.a

>18

n.a

6.7

114

4.8

SL0.10

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL3.24

>18

n.a

>18

n.a

>18

n.a

>18

n.a

2.7

30

2.2

SL3.31

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL3.27

>18

n.a

>18

n.a

>18

n.a

>18

n.a

6.8

108

5.0

SL2.14

>18

n.a

>18

n.a

>18

n.a

>18

n.a

6.3

30

5.8

D2.9

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

SL1.17

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

D1.8

>18

n.a

>18

n.a

>18

n.a

>18

n.a

4.7

78

3.4

E. faecium SL3.26

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

n.a

>18

*Time (h) required to increase absorbance by 0.3 units at 620 nm in each medium. t Delay of growth between the control culture and low pH culture in minutes. n.a: not available TABLE 6. The survival rate of seven strains of Lactococcus lactis subsp. lactis in M17 broth at pH 2 (n=2, x±SEM)

Strains SL3.34 SL1.9 D1.1 SL0.15 DC.6 SL1.18 SL3.27

Tolerance to M17 broth at pH 2 (log CFU/mL) 0h 3h 7.98±0.07 5.72±0.12 8.22±0.05 0 7.29±0.03 0 8.23±0.06 0 8.15±0.04 0 8.23±0.04 0 8.22±0.04 4.89±0.11

Survival rate (%) 66.3 0 0 0 0 0 51.3

On the other hand, the presence of bile tolerance in Lactococcus lactis subsp. lactis was greater than acid tolerance (Table 7). Sixteen strains of Lactococcus lactis subsp. lactis exhibited tolerance to 1%, 0.5% and 0.3% of oxgall. Enterococcus faecium was sensitive to these three concentrations of oxgall. Unfortunately, strains of SL3.34 and SL1.9 were sensitive to these oxgall concentrations. However, L. lactis subsp. lactis strain SL3.34 was still regarded as a potential probiotic candidate due to its greater survival rate at pH 2 pending further probiotic characterization with antibiotic susceptibility and adhesion to Caco-2 cells assessments. Tolerance to low pH and resistance to high concentrations of bile salt was used as the preliminary selection testing of the LAB isolates. Ability to tolerate exposure to these conditions allows the probiotic organism to pass unharmed through the upper gastrointestinal tract. 28 The best strain to survive under low pH was L. plantarum

050008-8

SL2.7, while strain S1.30 showed the greatest growth in media containing high concentrations of bile salt. In case of L. lactis subsp. lactis strains, four strains were tolerant to slightly acidic media (pH 5-6) with strain SL3.34 demonstrating the highest survival rate. Strain SL3.34 had a moderate survival rate (66.3%) for 2 h at pH 2; however, this strain was not resistant to a high concentration of bile salt. Interestingly, the number of L. lactis subsp. lactis isolates able to resist to bile salt was much greater than that of L. plantarum strains. These observations are in agreement with a previous study.14 In fact, only a small proportion of LAB isolated from fermented products has a good tolerance to low pH and bile salts (in vitro), compared with LAB strains obtained from the gastrointestinal tract of animals or humans.29

Molecular Detection of msa and bsh Genes All strains of L. plantarum tested (20 strains) as well as the L. plantarum type strain ATCC 14917T showed the expected band size for both msa (1,740 bp) and bsh (919 bp) genes (Figure 1). Thus, all strains met the requirement to be considered as probiotic cultures due to the presence of this probiotic marker. TABLE 7. Bile tolerance of Lactococcus lactis subsp. lactis and Enterococcus faecium in M17 broth measured by the time taken to reach an absorbance of 0.3 units at 620 nm

Time* (h) Strain

M17 0.3

bile

dt (min)

Time* (h) M17 bile 0.5

dt (min)

Time* (h) M17 bile 1.0

dt (min)

Time* (h) control M17

SL3.34

>18

n.a

>18

n.a

>18

n.a

4.5

SL1.9

>18

n.a

8.8

162

>18

n.a

6.3

D2.15

>18

n.a

>18

n.a

>18

n.a

6.8

SL0.3

>18

n.a

>18

n.a

>18

n.a

9.2

SL1.9

3.5

-18

3.2

-42

3.3

-30

3.8

SL3.32

2.2

0.0

2.0

-12

>18

n.a

2.2

D1.1

7.8

222

6.2

120

7.8

222

4.2

SL1.16

>18

n.a

>18

n.a

>18

n.a

>18

SL0.11

>18

n.a

>18

n.a

>18

n.a

>18

D2.17

3.0

-24

2.8

-30

2.8

-30

3.3

SL0.15

4.8

12

3.5

72

4.3

18

4.7

D1.9

2.8

6.0

2.8

0.0

3.1

18

2.8

SL2.15

>18

n.a

>18

n.a

>18

n.a

13.1

SL3.16

6.4

96

5.3

30

9.3

270

4.8

DC.6

2.4

18

2.1

0.0

2.2

6.0

2.1

D3.6

1.8

-18

1.8

-18

2.0

-6.0

2.1

SL3.19

2.0

-18

1.8

-24

1.9

-18

2.3

D1.2

2.7

-18

2.8

-12

2.9

0.0

2.9

050008-9

pH

D1.7

6.3

-30

5.3

-30

9.4

222

5.8

SL3.14

2.0

-12

1.8

-18

1.9

-18

2.2

SL1.18

4.8

0.0

4.2

-36

5.2

24

4.8

SL0.10

>18

n.a

>18

n.a

>18

n.a

>18

SL3.24

2.2

0.0

2.0

-12

2.2

0.0

2.2

SL3.31

>18

n.a

>18

n.a

>18

n.a

>18

SL3.27

4.8

-12

4.3

-42

8.6

216

5.0

SL2.14

>18

n.a

>18

n.a

>18

n.a

5.8

D2.9

>18

n.a

>18

n.a

>18

n.a

>18

SL1.17

>18

n.a

>18

n.a

>18

n.a

>18

D1.8

3.8

18

2.8

-36

2.9

-30

3.4

E. faecium SL3.26

>18

n.a

>18

n.a

>18

n.a

>18

*Time (h) required to increase absorbance by 0.3 units at 620 nm in each medium. t Delay of growth between the control culture and low pH culture in minutes. n.a: not available Lane M: 100 bp DNA marker; lane 0: sterile milliQ H2O; lane 1: L. plantarum S1.30; lane 2: L. plantarum S2.7; lane 3: L. plantarum S2.5

M 1

2

3

4 M 0 1

2

1,000 bp 1,000 bp 500 bp 500 bp

(A)

(B)

FIGURE 1. Representation of amplification products of msa gene – 1,740 bp (A) and bsh gene – 919 bp (B) Lane M: 100 bp DNA marker; lane 0: sterile milliQ H2O; lane 1: L. plantarum S1.30; lane 2: L. plantarum S2.7; lane 3: L. plantarum S2.5

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The presence of the msa and bsh genes, encoding for mannose-specific adhesion and bile salts hydrolase, in L. plantarum strains indicates mucosal adhesion properties, and resistance to toxic bile salts in the gastrointestinal tract and is associated with the ability to reduce blood cholesterol concentration, respectively. 30,31 All L. plantarum strains demonstrated these probiotic markers, indicating that the strains could be expected to be resistant to bile salts and able to colonize intestinal epithelial cells. Although all examined strains carried the bsh gene, only five strains were resistant to a high concentration of bile salt. This could be due to sequence variation in the bsh gene in L. plantarum.32

Antimicrobial Activity and Bacteriocin Detection All strains of L. plantarum isolated from dadih and dangke were evaluated for their antimicrobial activity against a range of pathogenic bacteria either Gram-negative or Gram-positive (data not shown). The indicator bacteria used in this study are listed in Table 1. The tested bacteria were not able to inhibit two indicator bacteria used, namely B. cereus and S. aureus ATCC 25923. In general, Gram-positive bacteria were more sensitive than Gram-negative bacteria as shown by the sizes of the inhibition zones. The Gram-positive bacteria targeted were Enterococcus faecalis ATCC 29212, Listeria monocytogenes and Streptococcus pyogenes ATCC 10389. By assessment of these indicator bacteria, the largest inhibition zones were exhibited by L. plantarum S1.30 and L. plantarum ATCC 14941T. However, these antimicrobial producers also demonstrated an antagonistic activity against E. coli strain 99364-1 and 99386-3. LAB can produce some antimicrobial substances for their defense and survival strategy against other microbes in their ecological niche. One of the antimicrobial substances that have recently emerged as important in the food industry is bacteriocins. In this study, the presence of bacteriocin from all strains of L. plantarum was assessed through an agar-based method and a PCR-based method. Detection of bacteriocin performed by agar well-diffusion assay failed to reveal the activity of any bacteriocin (data not shown). Thus, a PCR-based technique was applied to detect the presence of a specific gene for regulating bacteriocin activity especially for L. plantarum, that is plantaricin A (plnA). The PCR amplification resulted in 450 bp of DNA fragment from all strains of L. plantarum (Figure 2).

FIGURE 2. Amplification products of plantaricin A (450 bp) from representative’s strains of Lactobacillus plantarum Lane M: 100 bp DNA marker; lane 1: L. plantarum S1.30; lane 2: L. plantarum ATCC 14941T, lane 3: L. plantarum SL3.5; lane 4: L. plantarum SL3.6; lane 5: L. plantarum SL3.7; lane 6: L. plantarum SL3.8; lane 7: L. plantarum SL2.4; lane 8: L. plantarum SL2.6; lane 9: L. plantarum SL2.9; lane 10: L. plantarum SL0.17; lane 11: L. plantarum GD.1; lane 12: L. plantarum GD.2

The strain with the highest potential to produce antimicrobial substances was L. plantarum S1.30. Antibacterial activity of this strain was previously elucidated, 18 and it was included in this current study due to its potential antimicrobial activity. Since bacteriocin detection using agar-based method failed, the presence of the plantaricin A (plnA), the gene responsible for bacteriocin production, was used and the gene was detected in all L. plantarum strains through a PCR-based method. This result was in agreement with previous studies that organic acids,

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particularly lactic acid were the main key player in the antagonistic activity. 33,34 Lactobacillus plantarum S1.30 has a bacteriostatic effect against L. sakei ATCC 15521 and L. monocytogenes in vitro as a result of increasing the acidity level.18 The failure of bacteriocin detection using conventional techniques probably relates to unsuitable culture medium conditions (pH, temperature, and ingredients) and the regulatory mechanism of bacteriocin biosynthesis which is associated with the quorum sensing mechanism in these bacteria. 35,36

Antibiotic Resistance and Adhesion to Caco-2 Cells The results of antibiotic susceptibility testing of six strains of L. plantarum, two type strains (L. plantarum ATCC 14917T and L. rhamnosus GG), and one strain of Lactococcus lactis subsp. lactis using disk diffusion agar are listed in Table 8. In general, all the L. plantarum strains and the reference strains were sensitive to tested antibiotics, except to vancomycin. However, Lactococcus lactis subsp. lactis strain SL3.34 was sensitive to all antibiotics used. Lactobacilli are characterized as naturally resistant to vancomycin. 37 The vancomycin resistance is chromosomal encoded and not transferable to other microbial species. 34 Therefore, all strains showing a good survival rate against low pH and bile salt (Table 8) were appropriate for further probiotic characterization. In contrast, strains of L. plantarum DH1 and Pediococcus acidilactici DH7 isolated from dadih showed low levels of resistance to chloramphenicol, up to 5 μg/mL.38 The strains that showed a tolerance to low pH, bile salts, sensitivity to almost all of tested antibiotics and additional probiotic features (the presence of msa and bsh genes) were further analyzed for their ability to adhere to Caco-2 cells. The adhesion ability may be linked to competitive exclusion of pathogens and the immunomodulatory system.39 Lactobacillus plantarum strains S1.30, SL2.7, and SL3.5 were selected for the adhesion test. Strains of SL2.7 and SL3.5 were selected because they have a greater survival rate at pH 2. While, strain S1.30 was also chosen because this strain exhibited a potent antimicrobial activity compared with other strains, although it has a moderate survival rate at low pH. The highest adhesion ability has been shown by L. plantarum strain S1.30 (82.24±0.14%), followed by strain SL2.7 (80.26±0.39%), Lactobacillus rhamnosus GG (78.89±0.64%) and strain SL3.5 (78.34±0.16%). Meanwhile, Lactococcus lactis subsp. lactis strain SL3.34 and SL2.1 exhibited adhesion to Caco-2 cells as much as 73.94±1.26% and 73.44±1.51%, respectively. Therefore, L. plantarum strain S1.30 and Lactococcus lactis subsp. lactis SL3.34 were the best probiotic candidates representing each genus. The adhesion ability of L. plantarum S1.30 was strengthened with the presence of msa gene in its genome. The adhesion capability of Lactobacillus is dependent on the isolation source; the adhesion will be higher if the strains are derived from human feces or from buffalo milk, rather than from cheese. 40 Kaushik et al.41 have previously characterized L. plantarum Lp 9 isolated from raw buffalo milk. In addition to probiotic parameters utilized in this current study, L. plantarum Lp9 also demonstrated a good hydrophobicity, cellular autoaggregation, the presence of the gene for mucus-binding and fibronectin-binding protein and antioxidative activity.41 Therefore, L. plantarum S1.30 is a potential candidate to be used for further in vivo tests. Among members of genus Lactobacillus, L. plantarum is the most versatile microorganism and is widely distributed in diverse ecological niches such as plants/ vegetables, meat, fish, dairy products and mammalian gastrointestinal tract.42,43 Consequently, L. plantarum exhibiting probiotic properties have been detected and characterized in many NFM products.8,16,39,44,45 Meanwhile, L. lactis subsp. lactis derived from NFM products has also been previously shown to demonstrate probiotic characteristics. 14,46–48 However, the prevalence of L. lactis subsp. lactis with probiotic properties in NFM products is less frequent than L. plantarum. Considerable variation among presumptive probiotic candidates was normally observed when the characterization of probiotic properties was performed. This variability may be associated with variation in their genome sequences and ecological niches. Therefore, the probiotic selection is urgently required to obtain the best candidate.

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TABLE 8. Antibiotic resistance and adhesion percentage of Caco-2 cells of selected strains of Lactobacillus plantarum and Lactococcus lactis subsp. lactis

Inhibition zone diameter (mm) Strains

% adhesion on Caco-2

AMP (10 μg)

CMN (2 μg)

HLS (300 μg)

ERY (15 μg)

VAN (30 μg)

CHL (30 μg)

TET (30 μg)

L. plantarum S1.30

45

30

23

37

-

40

30

82.24±0.14b

L. plantarum SL2.7

45

30

25

38

-

40

30

80.26±0.39b

L. plantarum SL3.5 L. plantarum GD1 L. plantarum SL2.2 L. plantarum SL3.6

45

30

25

38

-

40

30

78.34±0.16b

45

30

25

37

-

40

30

n.d

43

30

24

37

-

40

30

n.d

45

30

25

38

-

40

30

n.d

L. plantarum ATCC 14917

46

24

30

34

-

40

30

n.d

L. rhamnosus GG

41

36

35

43

-

44

44

78.89±0.64b

L. lactis SL3.34

40

30

48

62

60

56

34

73.94±1.26a

L. lactis SL2.1

n.d

n.d

n.d

n.d

n.d

n.d

n.d

73.44±1.51a

*Ampicillin- AMP (≤19 R, >23 S), Clindamycin-CMN (≤14 R, >20 S), Streptomycin-HLS (≤8 R, >15 S), Erythromycin-ERY (≤13 R, >19 S), Vancomycin-VAN (≤14 R, >18 S), Chloramphenicol-CHL (≤12 R, >18 S), Tetracycline-TET (≤14 R, >18 S) [27] % adhesion on Caco-2 cells was expressed as percentage of the viable bacteria (log cfu/ml) compared to their initial population. Data of adhesion test are expressed as mean (%) ± SEM. Different letters are significantly different (P < 0.05) n.d: not determined

SUMMARY The results presented in this study indicate that L. plantarum S1.30 and L. lactis subsp. lactis SL3.34 isolated from dadih possess probiotic traits (in vitro) and therefore could be considered appropriate probiotic candidates for further in vivo examination. Lactobacillus plantarum S1.30 representing genus Lactobacillus demonstrated some probiotic properties such as tolerance to low pH and bile salts, antimicrobial activity and the presence of a bacteriocin regulating gene (plantaricin A) and msa and bsh genes, susceptibility to antibiotics and ability to adhere to Caco-2 cells. From these probiotic features, only antimicrobial activity and the presence of msa and bsh gene were not demonstrated by L. lactis subsp. lactis SL3.34. However, this strain was selected as representative of the dominant genus/ species in dadih. However, further studies are needed on the evaluation of additional important probiotic traits such as assimilation of cholesterol, antioxidant activity, anti-inflammatory properties as well as lysozyme resistance.

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AKNOWLEDGEMENT The authors acknowledge the scholarship of Adelaide Scholarship International (ASI) in supporting this research.

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M. E. Fadda, V. Mossa, M. Deplano, M. B. Pisano and S. Cosentino, LWT - Food Sci. Technol 75, 100-106 (2017). S. Liu and Y. Han, “Microbial diversity in fermented foods with an emphasis on bacterial fermentation and health benefits, in Microorganism and fermentation of traditional foods,” Edited by R.C. Ray and D. Montet (CRC Press, New York, 2015), p. 49. B. V. Kumar, S. V. N. Vijayendra and O. V. S. Reddy, J. Food Sci. Tech. 52, 6112-6124 (2015) A. B. Shori, Food Biosci 13. 1-8 (2016) W. R. Sybesma, Kort, and Y.K. Lee, Trends Biotechnol 33, 197-200 (2015). A. Lourens-Hattingh, and B.C. Viljoen, Int. Dairy J. 11, 1-17 (2001). P. Kandylis, K. Pissaridi, A. Bekatorou, M. Kanellaki and A.A. Koutinas, Curr. Opin. Food Sci. 7, 58-63 (2016). K. Angmo, A. Kumari, Savitri and T. C. Bhalla, LWT - Food Sci. Technol. 66, 428-435 (2016). W. Zhang, D. Yu, Z. Sun, R. Wu, X. Chen, W. Chen, H. Meng, S. Hu and H. Zhang, J. Bacteriol 192, 52685269 (2010). B. Guo, Z. Wu and J. Ye, European patent EP (2009). U. K. Shandilya, A. Sharma, R. Kapila and V. K. Kansal, J. Sci. Food Agric. 96, 3180-3187 (2016). J. M. Mathara, U. Schillinger, P. M. Kutima, S. K. Mbugua, C. Guigas, C. Franz and W. H. Holzapfel, Curr. Microbiol 56. 315-321 (2008). I. S. Surono, F. P. Koestomo, N. Novitasari, F. R. Zakaria, Yulianasari and Koesnandar, Anaerobe 17, 496-500 (2011). I. S. Surono. Asian-Australas. J. Anim. Sci. 16, 726-731 (2003). I. S. Surono, U. Pato, Koesnandar and A. Hosono, Asian-Australas. J. Anim. Sci. 22, 119-123 (2009). M. C. Collado, I. S. Surono, J. Meriluoto and S. Salminen, J. Food Prot. 70, 700-705 (2007). F. Nur, Hafsan, and A. Wahdiniar, Biogenesis 3, 60-65 (2015). Y. D. Jatmiko, M.D. Barton, and M. de Barros Lopes, “Isolation and antimicrobial potency of indigenous lactic acid bacteria isolated from dadih, a traditional fermented buffalo milk from Indonesia,” 2010, Master thesis, University of South Australia, 2010. M. du Toit, C. M. A. P. Franz, L. M. T, Dicks, U. Schillinger, P. Haberer, B. Warlies, F. Ahrens and W. H. Holzapfel, Int. J. Food Microbiol 40, 93-104 (1998). K. Ramasamy, N. Z. A. Rahman, S. C. Chin, N. J. Alitheen, N. Abdullah and H. Y. Wan, Int. J. Food Sci. Technol 47, 2175-2183 (2012). U. Schillinger and F.K. Lucke, Appl. Environ. Microbiol 55, 1901-1906 (1989). F. Zhou, H. Zhao, F. Bai, P. Dziugan, Y. Liu and B. Zhang, Czech J. Food Sci. 32. 430-436 (2014). A. Remiger, M. A. Ehrmann and R. F, Syst. Appl. Microbiol 19, 28-34 (1996). I. Klare, C. Konstabel, S. Műller-Bertling, R. Reissbrodt, G. Huys, M. Vancanneyt, J. Swings, H. Goossens and W. Witte, Appl. Environ. Microbiol 71, 8982-8986 (2005). W. P. Charteris, P. M. Kelly, L. Morelli and J. K. Collins, J. Food Prot. 61, 1636-1643 (1998). E. B. Minelli, A. Benini, M. Marzotto, A. Sbarbati, O. Ruzzenente, R. Ferrario, H. Hendriks and F. Dellaglio, Int. Dairy J. 14, 723-736 (2004). J. D. Babot, E. Arganaraz-Martinez, L. Saavedra, M. C. Apella and A. Perez Chaia, Res Vet Sci 97, 8-17 (2014). M. Bull, S. Plummer, J. Marchesi and E. Mahenthiralingam, FEMS Microbiol. Lett. 349, 77-87 (2013). M. Kizerwetter-Świda and M. Binek, Pol. J. Vet. Sci. 19, 15-20 (2016). A. K. Patel, R. R. Singhania, A. Pandey and S. B. Chincholkar, Appl. Biochem. Biotechnol 162. 166-180 (2010).

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Species Composition of Mosquito and Public Perception about Dengue Vector of Hemorrhagic Fever in Bareng Tenes Malang Zulfaidah Penata Gama1, a) and Jenvia Rista Pratiwi1, b) 1


Corresponding author: zulfaidahpenatagama@gmail.com b) Jenviapratiwi24@gmail.com

Abstract. Dengue Hemorrhagic Fever (DHF) is a mosquito-borne tropical disease caused by the dengue virus. Aedes aegypti and Aedes albopictus are the mosquito vectors of DHF. Malang city was an endemic region of dengue disease in East Java. One of the villages that had a high number of DHF cases was Bareng Tenes. The Case Fatality Rate (CFR) in Malang city totaled 5 patients out of 879 cases (Health Department of Malang city, 2010). Bareng Tenes RW 02 was one of the densely populated regions of Malang city. The objectives of this research were to identify mosquito composition and to analyze the public perception about the DHF vectors in Bareng Tenes RW 02 Malang. This research used two kinds of survey methods of mosquitoes. The first method for collecting larvae was used by direct capture using pipettes from artificial containers and the second method was collecting egg of mosquitoes by using an ovitrap. Public perception was calculated using the questionnaire technique. The accidental sampling technique in this research was Likert scale. The composition of mosquitoes found in Bareng Tenes RW 02 was Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. The mosquito survey showed that Aedes aegypti was the dominant species and the IVI value for the ovitrap survey was 118.06% while the value of IVI for the larval survey was 103.51%. Based on the public perception data, it showed that the community has a very good understanding of DHF knowledge, DHF vectors and ways of DHF prevention, but the undertaken activities by the community have not yet appeared to control the mosquito population especially for their larvae.

INTRODUCTION Dengue Hemorrhagic Fever (DHF) is a mosquito-borne tropical disease caused by the dengue virus. DHF is one of the most common diseases, especially in tropical and subtropical climates such as Indonesia. DHF is caused by four interconnected viruses (Family Flaviviridae Genus Flavivirus) DEN-1, DEN-2, DEN-3, and DEN-4. This virus consists of a single strand of RNA in the same genus as Yellow Fever and West Nile viruses1. Aedes aegypti and Aedes albopictus are the mosquito vectors of DHF. The life cycle of involves both mosquitoes in water and air. The eggs hatch, develop into pupae in the water, then become imago in the air. Larvae of Aedes sp. are found in good water. They are usually found in puddles contained in artificial water reservoirs such as drums, bathtubs, barrels, buckets, and others. They are also found in natural water reservoirs such as tree holes, banana leaves, taro leaves, stone holes, as well as artificial water reservoirs such as flower vases, used tires, bottles, birds drinking, etc2. Mosquitoes are holometabola animals consisting of egg phase, larval phase, pupa phase and adult mosquito (imago) phase. Mosquitoes need nine to ten days to metamorphose. Mosquito eggs hatch into larvae, which takes one to two days with air temperature 20–40 ºC. The development of larvae into pupa takes four to nine days depending on the air temperature, place, the state of water and the availability of suitable food. The stage from the pupa to becoming a mosquito needs two to three days. The pupa phase is an inactive phase, so it does not require food to become an adult mosquito. The whole mosquito takes seven to 14 days for one life cycle2. 8th International Conference on Global Resource Conservation (ICGRC 2017) AIP Conf. Proc. 1908, 050009-1–050009-9; https://doi.org/10.1063/1.5012733 Published by AIP Publishing. 978-0-7354-1600-0/$30.00

050009-1

Female mosquitoes need blood for protein sources during the egg-maturation process. It causes mosquitoes to have the ability as vectors of some diseases3. Male mosquitoes will suck nectar as a source of glucose in the process of energy formation4. Mosquitoes are commonly found in dark places sheltered from the sun, and in calm clear water. Mosquitoes have a place of indulgence inside the house as well as outdoors. Some of the mosquitoes that become vectors of dengue disease are Ae. aegypti and Ae. albopictus. Filariasis is transmitted by Aedes sp., Anopheles sp., Culex sp., and Mansonia sp. Chikungunya disease is transmitted by mosquitoes Ae. aegypti, Ae. albopictus, Cx. fatigans and Mansonia sp. Ae. aegypti is found in many houses and in buildings5. Approximately 500,000 to 1,000,000 people worldwide experience dengue per year, so this disease was becoming the most common disease in the world. The disease was common in urban areas. Female mosquitoes suck human blood to meet the nutritional needs of the egg maturation process. These habits cause mosquitoes to have the potential as a disease vector. DHF is also commonly found in tropical and subtropical regions, especially Southeast Asia, Central America, America and the Caribbean6. Malang city was in endemic regions of dengue disease in East Java. Malang city is one of the endemic areas of dengue disease. In 2010, the number of Case Fatality Rate (CFR) in Malang city totaled 5 patients out of 879 cases. DHF cases fluctuated during 2011 to 2015. In August 2016, CFR reached 2 patients out of 442 patients. One of the villages that has a high number of DHF cases was Bareng Tenes. In 2010, there were 83 dengue fever patients recorded in local government clinic of Bareng Tenes Village. The number of DHF patients in Bareng Tenes Village fluctuates every year. In 2011 there were 10 patients. In 2012 this increased to 18 patients and increased again in 2013 to 34 patients. In 2014 dengue fever cases in the local government clinic, Kelurahan Bareng decreased to 10 patients. In 2015 there was an increase in the number of cases of dengue fever with a total of 23 patients. In August 2016, there were 35 patients recorded by Malang City Health Office. This figure is quite high compared with the number of patients in other villages7. Bareng Tenes RW 02 was one of the densely populated regions of Malang city. Therefore it is necessary to do research about the type of mosquitoes that exist around the area of Bareng Tenes Village RW 02 Malang to ensure the presence of vector DHF disease. The objectives of this research were to identify mosquitoes composition and to analyze public perception about the DHF vectors in Bareng Tenes RW 02 Malang.

MATERIAL AND METHODS Study Area Bareng Tenes located in Bareng Tenes Village, Klojen, East Java. Bareng Tenes has an area of about 10,650 km2 with a plain height of 4.44 mdpl. The temperature in Bareng Tenes approximately between 23.2 and 24.5ºC, with a maximum air temperature of 29.2ºC and minimum of 19.8ºC. Rainfall in the rainy season is between 30 and 526 mm with an average of 44 mm / year. The air humidity in Bareng Tenes is approximately 78–86%8. The location of the sampling is determined by the house and the location of the stream (Figure 1). Based on research that has been done by Lestari, et al. (2009), the sampling of mosquito larvae was conducted in ten locations, where the location was a house of residents based on the area of the house near the river.

FIGURE 1. Sampling site in Bareng Tenes Village RW 02, Klojen, Malang, East Java

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Larvae survey The first method was the larvae survey. This method for collecting larvae involved direct capture using pipettes from artificial containers inside and outside. Indoor container included bathtubs, buckets, jars, drinking water/dispensers, and aquariums. Outdoor water reservoirs included used tires, fish ponds, fountain ponds, used tins, used drums, used bottles, used glasses and used flower pots. The larva was obtained, then placed in a bottle and labeled and taken to the laboratory and carried out the maintenance of the mosquito larvae before the species is identified9. Rearing larvae until imago and identified based on Direktorat Jendral Pengendalian Penyakit dan Penyehatan Lingkungan10.

Ovitrap survey The second method for collecting egg of mosquitoes was carried out by an ovitrap. It was based on previous research [16] [9] that use ovitrap as a control of mosquito populations. Surveys with ovitrap were performed using a plastic cup 250 mL and black. The plastic cup has a base diameter of 4.5 cm and a diameter of 8.5 cm at the top. Each plastic cup was filled with water with as high as 4 to 4.5 cm. Next, a 5 × 10 cm filter paper was used that surrounded the wall as the mosquito lays the egg [modified 12]. The ovitraps were placed inside and outside, then collected after 4 days. The eggs were reared until they reached the imago phase and identified based on Direktorat Jendral Pengendalian Penyakit dan Penyehatan10.

Public perception Public perception was ascertained using the questionnaire technique. The accidental sampling technique in this research was the Likert scale13. The number of respondents taken can use calculations with the Slovin formula14. Analyzed with equation 1 to obtain an interpretive value from the Likert scale. ‫ ݅ܣ‬ൌ

௔Ǥସା௕Ǥଷା௖ǤଶାௗǤଵ ௔ା௕ା௖ାௗ

(1)

Note: Ai = perception for question i a = the number of respondents choose answer 4 b = the number of respondents choose answer 3 c = the number of respondents choose answer 2 d = the number of respondents choose answer 1

Density Figure The density figure can be an indicator of an area against the risk of DHF transmission. The density figure is obtained from several indicators, such as the free number of larvae (FNL), Container Index (CI), House Index (HI), and Breteu Index (BI). FNL was obtained from the division between the number of houses not found larva and the total number of houses examined. HI was obtained from the division between the number of houses found larvae and the total number of houses examined. CI was obtained from the division between the number of containers found larvae and the total number of containers examined. BI was obtained from the division between the number of positive larvae containers and the total number of houses examined15. The interpretation of each indicator is presented in Table 1, while larva density categorization is presented in Table 2. TABLE 1. Category of entomological parameters against the risk of DBD transmission

Entomological parameters House Index (HI) ≥ 5 % House Index (HI) ˂ 5 % Countainer Index (CI) ≥ 10 % Countainer Index (CI) ˂ 10 % Breteau Index (BI) ≥ 50 Breteau Index (BI) ˂ 50

Interpretation of Risk of Transmission High Risk Low Risk High Risk Low Risk High Risk Low Risk

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TABLE 2. Density figure category

High

Mediu m

Low

Density figure (DF) 1 2 3 4 5 6 7 8 9

House Index (HI) 1-3 4-7 8-17 18-28 29-37 38-49 50-59 60-76 ˃77

Countainer Index (CI) 1-2 3-5 6-9 10-14 15-20 21-27 28-31 32-40 ˃41

Breteau Index (BI) 1-4 5-9 10-19 20-34 35-49 50-74 75-99 100-199 ˃200

Maya Index The Maya index is used to estimate high-risk areas as a breeding ground for mosquito larvae, with HRI (Hygiene Risk Index) and BRI (Breeding Risk Index) as an indicator. Both indicators have three criteria of height, medium and low determined by the highest distribution calculation. The MI category is determined by a 3×3 matrix. The matrix of the 3×3 component of HRI and BRI is presented in Table 3.

HRI

TABLE 3. Matrix of 3x3 component of Breeding Risk Index (BRI) and Hygiene Risk Index (HRI) in Maya Index (MI)

1 (low) 2 (medium) 3 (high)

BRI 1 2 (low) (medium) BRI1/ HRI1 BRI2/HRI2 (low) (low) BRI1/ HRI2 BRI2/ HRI2 (low) (medium) BRI1/ HRI3 BRI2/ HRI3 (medium) (high)

3 (high) BRI3/HRI1 (low) BRI2/ HRI3 (high) BRI3/ HRI3 (high)

RESULT AND DISCUSSION Composition of mosquitoes in Bareng Tenes Village RW 02 The composition of mosquitoes found from the survey results for three weeks in Bareng Tenes Village RW 02 consists of three species. The three species are Aedes aegypti, Aedes albopictus and Culex quinqeufasciatus. The difference between three species of mosquito can be seen in the thorax. Ae. aegypti has black scutum with two white line in dorsal scutum between two curve line. Ae. albopictus has black scutum with one white line in the dorsal scutum. Cx. quinquefasciatus has black proboscis and leg. Three species found in Bareng Tenes are presented in Figure 2.

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(a)

(b)

(c)

FIGURE 2. Mosquitoes found in Kelurahan Bareng Tenes. Note: a. Ae. aegypti, b. Ae. albopictus, and c. Cx. quinquefasciatus

The abundance of mosquitoes The abundance of mosquitoes found in Bareng Tenes Village RW 02 is presented in Tables 4 and 5. Sampling was conducted on three species for three weeks. The three species are Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus. The dominant mosquito species in the ovitrap survey is Ae. aegypti with 10 of 21 individuals. This is supported by the abundance in the larvae survey, which was 40 of 59 individuals. TABLE 4. The results of mosquitoes abundance based on ovitrap survey

Species

Mosquito abundance (indv) 10 9 2 21

Aedes aegypti Aedes albopictus Culex quinquefasciatus Total

TABLE 5. The abundance of mosquitoes with larvae survey

Species

Mosquito abundance(indv) 40 16 3 59

Aedes aegypti Aedes albopictus Culex quinquefasciatus Total

Important Value Index (IVI) The survey of mosquito larvae in Bareng Tenes Village RW 02 found three species in ovitrap and larvae survey method, that is Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus (Figure 3). For larvae survey, Ae. aegypti is the dominant species with an IVI of 103.51% followed by Ae. albopictus of 69.01% and Cx. quinquefasciatus of 27.49%. The same result was reached in the ovitrap survey with the dominant species being Ae. aegypti with IVI of 118.06% followed by Ae. albopictus 58.33% and Cx. quinquefasciatus 23.61%.

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(a) (b) FIGURE 3. IVI (%) mosquitoes in Bareng Tenes Village RW 02. Note a. results of the ovitrap survey and b. results of larvae survey

Public Perception about Mosquitoes in Bareng Tenes Village RW 02 Public perception in Bareng Tenes Village RW 02 was conducted by interview method using interview guidelines for the community. The number of respondents based on the slovin formula calculation is 97 respondents. Respondents selected were respondents who were often at home with an age range of about 20–70 years. The community knowledge about dengue disease resulted in a Likert scale of 3.08. Public knowledge about mosquitoes as a vector of DHF disease resulted in a Likert scale of 2.91. Knowledge of how dengue prevention is produced value a Likert scale of 2.90. Community action on how to prevent dengue fever resulted in a Likert scale of 2.23. Patients with DHF in Bareng Tenes Village RW 02 were obtained with Likert scale 1.80. This result is presented in Figure 4. 3.50

Likert scale

3.00 2.30

2.50 2.00 1.50 1.00 0.50 0.00

Series1

Knowledge about DHF

Knowladge about mosquitoes as DHF vectors

Knowledge about DHF preventing

DHF preventing has been taken

Experience about DHF around

3.08

2.91

2.90

2.23

1.80

Question FIGURE 4. Likert scale in each question of Public perception in Bareng Tenes Village RW 02

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Density Figure The density figure is a density of mosquito larvae with a free number of larvae (FNL), Container Index (CI), House Index (HI), and Breteu Index (BI) as indicators. The density figure value shows the area’s potential for breeding mosquitoes and related DHF diseases. The value FNL is 30%; the value of HI is 70%. The value of CI obtained 28.05%. The value of BI obtained 230. It is presented in Table 5. TABLE 5. The results of the calculation of Free Number of Larvae (FNL), House Index (HI), Container Index (CI) and Bretau Index (BI)

FNL %

HI %

CI %

BI

30

70

28,05

230

Maya Index The Category Maya Index with HRI and BRI is presented in Table 6. Two areas were in the high category, one area in the medium category and the rest included in the low category of ten areas. The low category is found on R1, R2, R3, R4, R8, and R10. The high category is found in R6 and R7 areas. The medium category is found in R5, then the low category is found in the R1, R2, R3, R4, R8, R9 and R10 areas. The category about the potential risk of DHF according to hygiene (HRI) and potential breeding of mosquitoes (BRI) around areas. TABLE 6. Category Maya Index with HRI (Hygiene Risk Index) and BRI (Breeding Risk Index)

Category HRI

Category BRI

Category MI

R1

low

low

low

R2

low

low

low

R3

low

low

low

R4

low

low

low

R5

low

high

medium

R6

medium

high

high

R7

high

medium

high

R8

low

low

low

R9

low

low

low

R10

medium

low

low

The bodies of Ae. aegypti are smaller than Ae.albopictus. The body colors are browner than Ae. alopictus. The legs and abdomen are black with white stripes. Both have a different color of scutum. Ae. aegypti has black scutum with two white line in dorsal scutum between two curve line. Ae. albopictus has black scutum with one white line in dorsal scutum. Cx. quinquefasciatus has black proboscis and leg16,10. The dominant species in Bareng Tenes Village RW 02 are Ae. aegypti. It causes Bareng Tenes Village RW 02 is a densely populated area13. Mosquitoes need human blood for the process of egg maturation. It causes mosquitoes to be vectors of the disease. Human blood is preferred by Aedes sp. because it has amino acids and ions that are needed in the process of oogenesis, egg cell maturation, the formation of the fat body through the previtelogenic process, and vitellogenin17. Serotonin and adrenaline in human blood are needed to stimulate the gonadotropic hormones necessary for18.

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Actually, people have known DHF is a contagious disease that causes death. This is supported by Fauzy et al.’s research19, which states that the public has known that DHF is a very dangerous disease. The symptoms appear difficult to detect because they are almost the same as other diseases. Most people know that mosquitoes are a vector of dengue disease characterized by white stripes on their legs and body. Mosquitoes are active in the morning until late afternoon, so some people conclude that sleeping in the day is very dangerous because it can provide opportunities to be bitten by dengue mosquitoes. DHF is a viral disease and no treatment can stop the development of this. Therefore, the medical profession just eliminates the symptoms that arise from the patient. Efforts that can be made are to avoid the sucking of mosquitoes with the actions of PSN, fogging, larvacide and 3M19. Moreover, it reduces the potential place for mosquito breeding. Most people control the inside more than the outside of the house. This can be seen from the number of containers such as flower pots, used tins, used glass and others. DHF prevention measures with 3M are not always performed by people. According to the density figure and MI, Bareng Tenes Village RW 02 has a high population density of larvae than South Tangerang City. R6 and R7 areas are the high potentials of mosquitoes. The high larvae density and MI can cause the potential of DHF to also be high. The R6 and R7 areas have a good temperature for mosquito oviposition. This is shown by the mosquitoes found in containers. Both areas have many plants in front of the house. The mosquito population can be controlled by cleaning or disposing of unused containers and potentially as a breeding ground for mosquitoes. The containers include used cups, used plates and other dark colors. 3M plus action is also mandatory for all citizens. It is important that these actions are taken, to reduce the potential of oviposition mosquitoes, whereas for locations with low MI category it is advisable for local residents to maintain the condition.

SUMMARY The composition of mosquitoes is Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Aedes aegypti is the dominant species and the value of IVI is 118.06 % in ovitrap surveys and 103.51 % in larvae surveys. Public knowledge of DHF is good, but prevention has not been done optimally. That perception was supported by 30% FNL with a high-density figure and Maya Index; there are 2 areas in RT 3 that are a high potential area of DHF.

ACKNOWLEDGMENT The authors thank for chief and inhabitant of Bareng Tenes, Malang for research permission. Special thank to Biology Department of Universitas Brawijaya that give permission for finishing this research.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

C. R. Howard, Journal Elsevier Perspective Medical Virology 11. 205-210 (2005). S. Soegijanto, Demam Berdarah Dengue (Airlangga University Press, Surabaya, 2004). I. W. Supartha, Pengendalian terpadu vektor virus Demam Berdarah Dengue Aedes aegypti (Linn) dan Aedes albopictus (Skuse) (Diptera: Culicidae), http://dies.undud.ac.id, date accessed December 10, 2016. D. Natadisastra and R. Agoes, Parasitologi kedokteran ditinjau dari organ tubuh yang diserang, (EGC, Jakarta, 2009). W. H. Cahyati dan Suharyo, Jurnal Kesehatan Masyarakat 2, 11-15 (2006). B. R. Guerdan, American Journal of Clinica Medicine Spring 7, 51-53 (2010). Health Department of Malang city (private communication). Akaibara. Profil Kelurahan Bareng, Kecamatan Klojen, Kota Malang, http://ngalam.co/2016/02/15/profilkelurahan-bareng-kecamatan-klojen-kota-malang/, date accessed November 01, 2016. M. Islamiyah, A. S. Leksono dan Z. P. Gama, Jurnal Biotropika 1, 80-85 (2013). Direktorat Jendral Pengendalian Penyakit dan Penyehatan Lingkungan, Kunci identifikasi nyamuk Aedes. (Depatemen Kesehatan Republik Indonesia, Jakarta, 2008). S. A. Ritchie, S. Long, A. Hart, C. E. Webb and R. C. Russell, Journal of the American Mosquito Control Association 19, 235-242 (2003). O. Wan-Norafikah, W. A. Nazni, S. Noramiza, S. Shafa`Ar-Ko`Ohar, S. K. Heah, A. H. Nor-Azlina, M. Khairul-Asuad and H. L. Lee, Jurnal Sains Malaysiana 41, 1309-1313 (2012). H. Nawawi, Metode penelitian bidang social (Gajah Mada University Press, Yogyakarta 2001).

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14. R. A. Putra, Suprayogi and S. Kahar, Jurnal Geodesi Undip 2, 1-12 (2013). 15. H. Mutiara, “Analisis spasial kepadatan larva, Maya Index dan kejadian Demam Berdarah Dengue,” Minithesis. Universitas Negeri Semarang, 2016. 16. Direktorat Jendral Pengendalian Penyakit dan Penyehatan Lingkungan, Kunci Identifikasi Culex jentik dan dewasa di Jawa. (Depatemen Kesehatan Republik Indonesia, Jakarta, 1989). 17. S. Mengko dan J. S. B.. Jurnal e-Biomedik 4. 1-7 (2016). 18. S. U. Palgunadi dan A. Rahayu. Jurnal e-library Medical Faculty Wijaya Kusuma Surabaya University 2, 1-6 (2011). 19. S. Fauzy, Z. Sugiyanto dan Nurjanah. Persepsi masyarakat terhadap risiko DBD dan cara pencegahannya di Kelurahan Sedangmulyo Kecamatan Tembalang Kota Semarang Tahun 2014. Artikel Ilmiah http://eprints.dinus.ac.id/6616/1/jurnal_13180.pdf, date accessed Mei 27, 2017. 20. B. D. Lestari, Z. P. Gama, dan B. Rahadi. Identifikasi nyamuk di Kelurahan Sawojajar Kota Malang. http://www.academia.edu, date accessed 10 November 10, 2016 .

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Influence of CSN1S2 Protein from Caprine Milk Etawah Breed (EB) on Histology of Microglial Cells in Rat (Rattus norvegicus) Type-2 Diabetes Mellitus (T2DM) Margareth Rika1, 2, b), Fatchiyah1, 2, a) 1

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145 2 Research Group of Smart Molecule and Genetic Resources, University of Brawijaya a)

Correspondence author: fatchiya@ub.ac.id b) margarethrikawgl@gmail.com

Abstract. Type-2 diabetes mellitus (T2DM) is a degenerative disease that causes an imbalance in the metabolism. The aim of this research is to determine the influences of CSN1S2 on the structure of microglial cells in T2DM. Rats (Rattus norvegicus) were divided into eight groups of treatment with looping three times each between treatment groups (CM) Control. The control is given a milk treatment with doses of 375 mg/kg (CM375), 750 mg/kg (CM750), and 1500 mg/kg (CM1500), T2DM (DMK), and T2DM with CSN1S2 375 mg/kg dose (DM375), 750mg/kg (DM750), and 1500 mg/kg (DM1500). The animal model T2DM was induced by a high-fat diet in the form of feed followed by injection of STZ (dose of 25 mg/kg of animal treatment) and treatment of CSN1S2 for 28 days. Brain organs were taken and analysed in histopathology stained by Hematoxylin-eosin (HE) and observed using Olympus BX53. Based on the results, it was concluded that CSN1S2 protein is influential for induction of microglial cell proliferation in animal models of T2DM, as immunity responds to the inflammatory condition in T2DM.

INTRODUCTION Indonesia is a Pacific Asian country with the seventh largest rate of diabetes mellitus in the world.1 Type II diabetes mellitus (T2DM) is marked by a hyperglycemic condition and insulin resistance that induces some abnormal metabolic activity such as inflammation and alteration of glucose transporters in microglial cells of the central nervous system (CNS).2 Oral medicines, recently used for this pathological condition include glibenclamide and metformin, but it is known that glibenclamide can lead to some mutation in insulin genes which also induces fragmentation of DNA, leading to β cells apoptosis.3 Metformin has a high toxicity in the liver.4The effect of CSN1S2 protein is certainly known as a repairing agent for bone microstructure through cell proliferation 5, However, the influence of CSN1S2 in microglial cells is barely known. This study was designed to observe the effect of CSN1S2 protein on the structure of microglial cells in rats’ brains (Rattus norvegicus) as an animal model of T2DM.

EXPERIMENTAL DETAILS This study was conducted from September until December 2016, at the Animal Model Laboratory, Histopathology Laboratory, and Microscope Laboratory, Bioscience Institute, University of Brawijaya, Malang, East Java, under ethical certificate number 417-KEP-UB, 2015 from the ethical committee of University of Brawijaya.


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Animal model Twenty-four of animal models (Rattus norvegicus) were obtained from UPT LPPT Gajah Mada University, with twelve rats in the control group and the remainder in the T2DM model. The control group was nourished with comfeed pars, while the T2DM model group was treated with a high-fat diet for two months until the cholesterol level was >200 mg/dl.6 T2DM induction was intraperitoneally injected with 25 mg/kg BB of Streptozotocin (STZ) until the glucose level reached >250 mg/dl.7 Rats (Rattus norvegicus) were divided into eight groups: Control (CM) with 375 mg/kg Body Weight (BW) CSN1S2 (CM375); 750 mg/k BW CSN1S2 (CM750); 1500 mg/kg BW CSN1S2 (CM1500); T2DM (DMK); T2DM treated with 375 mg/kg BB CSN1S2 (DM375); 750 mg/kg BB CSN1S2 (DM750); and 1500 mg/kg BB CSN1S2 (DM1500) with three loops. CSN1S2 treatment was conducted over 28 days.

Histopathology Rats’ cerebra were stored in paraformaldehyde (PFA).8 Histological procedures of the brain were formed based on9 through dehydration with alcohol, clearing with xylene, and infiltration. Trimming processes were done with a rotary microtome where each tape thickness was 5 μm, the ribbon was put on a hot plate for 12 hours then stained with Haematoxylin-Eosin (HE). The staining procedure was begun with deparaffination with xylene and dehydrated with alcohol and rinsed with phosphate buffer saline (PBS) for 15 minutes, then cleared with xylene for 10 minutes, mounted with Entellan, and covered with a cover glass.

Counting cells and statistical analysis The cerebral were then observed with an Olympus BX53 microscope (M=1000X) and the cell count analysed using one-way ANOVA (α=0.05) using SPSS v16.

RESULT AND DISCUSSION

Jumlah Mikroglia (sel)

The influence of CSN1S2 protein is observed through the cell by cell count and cell observation. Secreted by monocytes in the CNS, they activate and are responsible for cell injury or inflammatory conditions.10 The microglial cell amount in rats with T2DM rises significantly compared with the control. This could be responsible for the inflammation that usually occurs in T2DM.11 Microglial cells are responsible for maintenance of immune conditions in the brain.12 Accumulation or activity of microglial cells is increased depending on inflammation that is stimulated by current cytokine-like TNFα IL-1, and IL-6.13 20 15 10 5 0

FIGURE 1. Amount of Microglial in each treatment These cytokines are also increasing through the T2DM pathway. After CSN1S2 treatment, there are fluctuations in microglial cell amounts depending on the treatment. After CM750 treatment, microglial cell amounts increased

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and were almost the same as the microglial cell amount in control rats that were treated with twice the current dosage, 1500mg/kgBW. This could be evidence that CSN1S2 protein can induce proliferation twice greater for repairing damaged tissue in T2DM. Proliferation is one of the regeneration methods of tissue when injury stimulation exists and is activated by immunocompetent cells.14 Based on the ANOVA analysis, treatment of CSN1S2 protein in a dosage of 750mg/kgBW significantly affected the microglial cell amount. Therefore, CSN1S2 with a dosage of 750 mg/kgBW is suitable for T2DM. In higher dosages, CSN1S2 treatments precisely cause a reduction of microglial cell amounts. This reduction could be caused by the high proliferate rate of the immunocompetent cells. The excess microglial cells in the brain would activate immunoregulatory activity to suppress the proliferation, as the surplus microglial cells will secrete more cytokine-like TNFα, NO, IL-1, ROS and could be neurotoxic.15 After CSN1S2 treatment, there are fluctuations microglial cell numbers depending on the treatment. After CM750 treatment, the microglial cell amount increased and was almost the same as that in the control rats treated with twice the current dosage, 1500mg/kgBW. This could be evidence that CSN1S2 protein can induce proliferation twice greater for repairing damaged tissue in T2DM. Proliferation is one of the regeneration methods of tissue when injury stimulation exists and activation of immunocompetent cells occurs.14 The cerebrum is the main control organ for many mechanisms in the body. In histological observation, in both control and T2DM rats, there are capillaries, microglial cells, astrocyte cells, neutrophils, and neurons. Astrocyte cells are found in control rat brains treated with 375 mg/KgBW. Neurons are the most frequent nerve cells observed in the cerebrum. These are suitable, based on their function to deliver impulses.16 Neutrophil cells can be found which look like red fibers among the neurons. CSN1S2 treatment could induce proliferation of microglial cells which are immunocompetent cells that are responsible for chronic inflammation and the adaptive immune system [5], found in rat brains treated with dosages of 375 and 750 mg/KgBW. This proves that CSN1S2 protein can induce proliferation of microglial cells in the CNS. CSN1S2 is known as an inducible molecule for proliferation17 of pre-osteoblast cells that form the microstructure of the femur.18 Protein CSN1S2 has not just the potential for proliferation of immunocompetent cells, however, it could provide potential nutrition for inflammation.

FIGURE 2. Histopathology of cerebrum in each treatment. Hematoksilin-eosin staining (M=1000X). K: Control; CM375:Control with protein 375 mg/kg CSN1S2 ; CM750: Control with protein 750 mg/kg CSN1S2; D: Control with protein 1500 mg/kg CSN1S2; DMK : T2DM ; DM375: T2DM with protein 375 mg/kg CSN1S2 ; DM750 : T2DM with protein 750 mg/kg CSN1S2; DM1500 : T2DM with protein 1500 mg/kg CSN1S2. Notes :(K) Capillary, (Mg) microglia cells, (A) Astrocytes, (Np) Neurophil, (N) Neuron cells.

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SUMMARY In summary, our findings demonstrate that CSN1S2 protein could induce or affect proliferation of microglial cells in the T2DM brain.

ACKNOWLEDGEMENT This study was sponsored by BOPTN-RU PTN UB decentralization research grant of 2015. The authors thank Bioscience Institute, University of Brawijaya for providing laboratories and microscope facilities.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

International Diabetes Federation, Idf Diabetes Atlas, 2015. H. W. Baynes, J. Diabetes Metab 6(541), 1–9 (2015). H. Su, X. Liu, L. S, L. Zhang, X. Liu, H. Ji, H. Rong, J. Diabetes Mellitus 3(3), 122–128 (2013). M. L. Francisco, P. F. Susana, M. R. B. Lopez, F. J. Tinahones, R. J. Andrade, G. H. Ricardo, Diabetes care 35(3), e21 (2015). A. L. Mescher, Junqueiras’s Basic Histology Text, and Atlas, 2013. M. J. Daniel, Am. Heal. drug benefits 4(5), 312–22 (2011). T. M. Dall, W. Yang, P. Halder, B. Pang, M. Massoudi, N. Wintfeld, A. P. Semilla, J. Franz, P. F. Hogan, Diabetes Care 37(12), 3172–3179 (2014). R. R. Bia, R. P. Virgirinia, B. Setiawan, A. Soewondo, and F. Fatchiyah, Biomarkers Genomic Med 7(2), 64– 71 (2015). K. S. Suvarna, C. Layton, J. D. Brancroft, Bancroft's Theory and Practice of Histological Techniques 7th Edition (Churchill Livingstone Elsevier, Oxford, 2013) M. C. Calle and M. L. Fernandez, Diabetes Metab 38(3), 183–191 (2012). S. A. Austin and C. K. Combs, Cent Nerv Sys Dis and inflam 2, 13–33 (2008). L. Dimou and M. Götz, Physiol Rev 94(3), 709–737 (2014). M. Tsuda, J. Diabetes Investig 7(1), 17–26 (2016). M. E. Lull and M. L. Block, Neurotherapeutics 7 (4), 354–365 (2010). G. Ramesh, A. G. MacLean, and M. T. Philip, Mediators Inflamm 2013, 1–20 (2013). E. Kolaczkowska and P. Kubes, Nat Rev Immunol 13(3), 159–75 (2013). C. Chotimah, G. Ciptadi, B. Setiawan, and F. Fatchiyah, Asian Pacific J Trop Dis 5(3), 219–223 (2015). F. Fatchiyah, B. Setiawan, S. Suharjono, and Z. Noor, Biomarkers Genomic Med 7(4), 139–146 (2015).

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Comparative method of protein expression and isolation of EBV epitope in E.coli DH5α Nadya V.M Anyndita1, b), Nurul Dluha1), Karimatul Himmah1), Muhaimin Rifa’i1), Widodo1, a) 1


Corresponding author: widodo@ub.ac.id b) nadyavero96@gmail.com

Abstract. Epstein-Barr Virus (EBV) or human herpes virus 4 (HHV-4) is a virus that infects human B cell and leads to nasopharyngeal carcinoma (NPC). The prevention of this disease remains unsuccessful since the vaccine has not been discovered. The objective of this study is to over-produce EBV gp350/220 epitope using several methods in E.coli DH5α. EBV epitope sequences were inserted into pMAL-p5x vector, then transformed into DH5α E.coli and overproduced using 0.3, 1 and 2 mM IPTG. Plasmid transformation was validated using AflIII restriction enzyme in 0.8% agarose. Periplasmic protein was isolated using 2 comparative methods and then analyzed using SDS-PAGE. Method A produced a protein band around 50 kDa and appeared only at transformant. Method B failed to isolate the protein, indicated by no protein band appearing. In addition, any variations in IPTG concentration didn’t give a different result. Thus it can be concluded that even the lowest IPTG concentration is able to induce protein expression.

INTRODUCTION Epstein-Barr Virus (EBV) is the main cause of Nasopharyngeal Carcinoma (NPC).1 This virus contributes to 0.6% of cancer disease in the world, which usually infects children and rarely causes any symptoms after infection.2–3 This disease still causes a serious threat since its prevention using vaccine has not been successful. The EBV viral structure consists of double-stranded DNA, a toroid shape, has tegument and envelope. This envelope consists of glycoprotein which encoded by BLLF1 gene and known as major antigen in EBV. The glycoprotein is commonly known as gp350/220, which is a glycosylated 907 polypeptide residue. Primary infection was mediated by binding of this glycoprotein to the B-cell human receptor CR2 and this interaction causes binding of the virus to the cell surface.4–7 The previous bioinformatics study resulted in a potential single epitope of EBV as a vaccine candidate.9 In this study, we aimed to define the best method of producing the EBV epitope using several IPTG concentrations in E.coli DH5α.

EXPERIMENTAL DETAILS Transformation to E.coli DH5α A bacterial colony was grown using 10 ml of LB broth (HiMedia, M1245) at 37°C until it reached OD600 ~0.4. It was incubated on ice for 10 min then centrifuged at 4100 rpm for 10 min. The pellet was resuspended in 1.5 ml cold MgCl2-CaCl2 then centrifuged at 4100 rpm for 10 min at 4°C and pellet were resuspended in 1 ml cold CaCl2. A total of 25 μL was removed to a microtube and 1 μL of plasmid DNA was added. It was incubated on ice for 30 min, then quickly removed to a water bath with a temperature of 42°C for 90 seconds, and back to the ice for 5 minutes. A total of 100 μL SOB medium was added and incubated in a shaker for 2 8th International Conference on Global Resource Conservation (ICGRC 2017) AIP Conf. Proc. 1908, 060002-1–060002-4; https://doi.org/10.1063/1.5012735 Published by AIP Publishing. 978-0-7354-1600-0/$30.00

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hours, then spread onto LB Agar with ampicillin. The transformed bacterial colony appeared after overnight incubation at 37°C.9

Plasmid DNA isolation and transformation validation using AflIII restriction enzyme A bacterial colony which contained recombinant plasmid DNA was inoculated into 10 ml of LB broth containing ampicillin overnight. Plasmid DNA was isolated using a mini preparation DNA-spinTM Purification Kit (iNtRON Biotechnology). The transformation result was validated using an AflIII restriction enzyme in 0.8% agarose (Invitrogen, 75510-019). Electrophoresis was running at 50 V for 85 min.

Protein over-expression using IPTG and protein isolation Transformant colony were inoculated into 10 ml LB broth with ampicillin until OD600 reached 0.5–0.6. The optimum concentration of IPTG was analyzed by adding various concentrations of IPTG: they are 0.3, 1 and 2 mM for 2 hours in 37°C. In addition, 2 methods were also used to isolate the protein to show the best method. Method A begins by centrifuging 10 ml of an induced culture at 4,500 rpm, 20 min, RT. The pellet was resuspended in hyper-osmotic solution 30 mM Tris-Cl, 20% sucrose, pH 8.0 with 1 mM EDTA and incubated at RT for 10 min using a shaker. A centrifuge was used at 9,000 rpm, 20 min, 4°C. The pellet was resuspended in 4 ml 5mM MgSO4, then incubated in ice using a shaker for 10 min. The centrifuge was set at 9000 rpm, for 20 min, at 4°C. Supernatant which contains periplasmic protein was collected and 160 μL Tris-Cl pH 7.4 was added 10. Method B began with 10 ml of induced culture centrifugation at 4,500 rpm, 20 min in 4°C. The pellet was resuspended in 2.5 ml 20% sucrose, EDTA 0.1 M, and 0.2 M Tris-Cl pH 8.0. It was incubated in ice for 20 min and inverted at regular intervals. The centrifuge was set at 13,000 rpm, for 20 min at 4°C. The pellet was resuspended in 2.5 ml 0.01 M Tris-Cl pH 8.0, 0.005 M MgSO4, 0.2% SDS and 1% Triton. It was incubated on ice for 20 min and inverted at regular intervals. The centrifuge was used at 13,000 rpm, for 20 min at 4°C and the supernatant was saved.11 The protein isolation result was confirmed using 10% SDS-PAGE, which runs at 60 A for 25 min.

RESULT AND DISCUSSION Transformation validation using AflIII restriction enzyme The transformation result was confirmed after plasmid isolation using AflIII restriction enzyme. The restriction enzyme is one of the endonuclease enzymes that is produced by bacteria as a defense mechanism to destroy foreign DNA such as a virus by cleaving it into some parts. This enzyme was specific for a DNA sequence that cut between 2 nucleotides, for example, AflIII which cut in 5’-A↑CRYGT-3’12.

FIGURE 1. Transformation validation by electrophoresis gel 0,8%. Lane 1 = marker, 2 = uncutted plasmid, 3 = plasmid after cutted by AflIII

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Based on Snapgene simulation, the plasmid will finally cut into 2 parts with 4,450 and 1,500 bp. It is confirmed by Figure 1 on lane 3 which shows 2 precise sizes of DNA band. It can be concluded that transformation was successfully done to E.coli DH5α.

Protein confirmation using SDS-PAGE Protein expression was characterized using SDS-PAGE. Based on protein prediction size using Protparam (www.bioinformatics.org/sms/prot_mw.html), our protein has molecular weight around 50 kDa. This prediction is suitable with the band appearing on SDS-PAGE using method A, which shows a thin band below the 52 kDa marker and only appears at transformant, while no band appears when using method B. Thus it can be concluded that the best method to isolate our periplasmic protein is method A. In addition, any variations in IPTG concentrations also didn’t show any significant result, but IPTG positively stimulates protein expression, indicated by no band appearing in uninduced samples. Thus it can be concluded that 0.3 mM IPTG is the optimum concentration for producing our protein.

FIGURE 2. Protein analysis of periplasmic fraction in E.coli DH5α by SDS-PAGE. A. Method A. Lane 1 and 6 = Marker, 2 = Induction by 0.3 mM IPTG, 3 = Induction by 1 mM IPTG, 4 = No IPTG induction, 5 = Wildtype, 7 = Induction by 2 mM IPTG. B. Method B. Lane 1 = Wildtype, Lane 2 = No IPTG induction, Lane 3 = Induction by 1 mM IPTG

SUMMARY Method A produces a protein band around 50 kDa and appears only at transformant, which was predicted as our protein band. Method B failed to isolate the protein, indicated by no protein band appearing. In addition, any variations in IPTG concentration didn’t give a different result. Thus, it can be concluded that even the lowest IPTG concentration is able to induce protein expression.

ACKNOWLEDGEMENT Authors would like to thanks, The Ministry of Research, Technology and Higher Education, The Republic of Indonesia for supporting and funding this research.

REFERENCES 1. 2. 3. 4.

P. J. Farrell. Epstein-Barr virus. In Genetic Maps (Cold Spring Harbor, New York, 2009), pp.133. C. Gullo., W.K. Low., G. Teoh, Ann Acad Med Singapore 37(9), 769-77 (2008). I. A. R. C. Globocan, "Estimated cancer incidence, mortality, and prevalence worldwide," World Health Organization, America, 2016. J. M. Middeldorp, Crit Rev in Oncology Hematology 45(1), 1-36 (2003).

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5.

S. J. Molesworth., C. M. Lake., C. M. Borza., S. M. Turk., L. M. Hutt-Fletcher, Journal of Virology 74 (14), 6324–6332 (2000). 6. G. Szakonyi, M. G. Klein, J. P. Hannan, A. Y. Kendra, Z. M. Runlin, A. Rengasamy, V. M. Holers, S. C. Xiaojiang, Nature Structural and Molecular Biology 13(11), 996-1001 (2006). 7. M. P. Thompson and R. Kurzrock. Clinical Cancer Research 10(3), 803-821 (2004). 8. S. L. Sitompul, Widodo, M. S. Djati, D. H. Utomo, Bioinformation 8(10), 479-482 (2012) 9. Sambrook, Russel, Molecular Cloning Third Edition (Spring Harbour Laboratory Press, New York, 2011). 10. New England BioLabs, PROTEIN EXPRESSION, AND ANALYSIS: pMAL protein fusion & purification system (New England BioLabs, USA, 2015), pp. 10-12. 11. H.C. Neu and L.A. Heppel, J Biol Chem 240 (9), 3685-92 (1965). 12. Lodge. J., Lund. P., Minchin. S. Gene cloning (Taylor & Francis Group, New York, 2007), pp. 38-39

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Tapak Liman (Elephantopus scaber L) Extract–Induced CD4+ and CD8+ Differentiation from Hematopoietic Stem Cells and Progenitor Cell Proliferation in Mice (Mus musculus L) Muhammad Sasmito Djati1, a), Hindun Habibu2), Nabilah A Jatiatmaja3), Muhaimin Rifa’i1, b)

1

Laboratory of Animal Physiology, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia

2

Master Program of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia 3

Faculty of Dentistry, Airlangga University, Surabaya, Indonesia

a)

Corresponding author: msdjati@ub.ac.id b) rifa123@ub.ac.id

Abstract. Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant containing several active compounds that potentially affecting hematopoietic stem cells, such as epifrieelinol, lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol, lupeol acetate,deoxyelephan-topin, isodeoxyelephantopin, polyphenol luteolin-7, as well as various flavonoids and glucosides. The aim of this study was to elucidate the effect of leaf extract of Tapak Liman on hematopoietic stem cells in mice BALB/c, by observation of the relative number of cells expressing CD4/CD8, CD4/CD62L, and TER119/B220 in the spleen, and TER119/B220, TER119/VLA-4 and TER119/CD34 in bone marrow, after being administered leaf extract for 2 weeks. This experiment used 12 female mice, which were divided into three treatment groups, P1= 0.5 g·g bw-1·day-1, P2= 1.0 g·g bw-1·day-1 and P3 = 2.0 g·g bw-1·day-1 Tapak Liman leaf extract as well as a control. The relative numbers of cells expressing surface molecules were analyzed by flowcytometry and quantitative data were tested using one-way ANOVA. The results showed that the leaf extract of Tapak Liman has no significant effect on erythrocyte proliferation; on the other hand, it had a significant effect on both proliferation and differentiation of B lymphocytes (B220+) in bone marrow (p=0.044) and increased the expression of CD4+, CD8+ molecule in B cells (p=0.026) and erythroid cells in spleen and bone marrow, based on the estimation of cells that expressed TER119+VLA-4+, identified as important in the development pathway of erythrocytes. An increased cell percentage of TER11+VLA-4+ occurred for treatment P2, 12% higher than the control. The increased expression of TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functioned to stimulate the progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119 + VLA-4+). There was an increasing number of cells expressing the surface molecules TER119+ and VLA-4+. This indicated that the Tapak Liman leaf extract with a dose of 1.0 g·g bw-1·day-1 could stimulate the proliferation of hematopoietic stem cells into the lymphoid and erythroid pathway, in spleen and bone marrow.


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INTRODUCTION Traditional medicines with an herbal component have many potential indications as antioxidants, immunomodulators, hormonal therapies, etc.1-5 Tapak Liman (Elephantopus scaber L) is a traditional medicinal plant that has been known for centuries, especially among pharmacologists in China. Tapak Liman contains epifrieelinol, lupeol, stigmasterol, triacontane-1-ol, dotriacontane-1-ol, lupeol acetate, deoxyelephan-topin, isodeoxyelephantopin, polyphenol luteolin-7, as well as various flavonoids and glucosides. Tapak Liman is used fresh, dried, or even extracted and put into capsules. Types of diseases that can be treated with Tapak Liman include various inflammatory diseases such as inflammation of the tonsils, eyes, and kidneys, acute and chronic inflammation of the uterus or vaginal discharge, as well as influenza and sore throat. Additionally, Tapak Liman also serves as a laxative of urine, an antioxidant, blood booster, and decreases fever and clears phlegm. It is also useful to overcome chicken pox and anemia.6 Anemia is a disease caused by a deficiency of red cell volume and hemoglobin levels (Hb), thus the body is hypoxic as a result of the reduced oxygen-carrying capacity of the blood.7 Anemia can be caused by autoimmune diseases such as hemolytic and aplastic anemia, due to delays in the process of erythropoiesis.8 Until now, the treatment of anemia that is caused by erythropoiesis barriers solely depends on the provision of artificial hematopoietin. This is commonly called ESA (erythropoiesis-stimulating agent), a synthetic compound that can stimulate the production of blood cells. However, ESA has harmful side effects such as cardiovascular complications9 and retinopathy.10 Thu, this provides an opportunity to investigate and search for safe drugs that could stimulate and affect erythropoiesis with no side effects, and/or boost the immune system by inducing the erythropoiesis and lymphopoiesis proliferation and differentiation processes. Thus, our research focused on the effects of Tapak Liman leaf extract on hematopoiesis in female mice (Mus musculus) BALB/c. It was expected that chemical substances contained in extracts of Tapak Liman leaves could potentially induce and influence the process of hematopoiesis differentiation and proliferation, especially lymphopoiesis and erythropoiesis.

EXPERIMENTAL DETAILS Preparation of Leaf Extract and Treatment Extraction of Tapak Liman leaves was conducted in a sterilized distilled water solvent.11-12 A total of 13.8 g of dried leaves of Tapak Liman were ground by a mortar into a fine powder. Then, 100 mL sterilized dH2O was added to the powder and stirred at 50 °C overnight and then filtered through filter paper. The final concentration was calculated, and the extract solution was ready for administration. The experimental animals used in this study were mice (Mus musculus) which were randomly sampled from a population of female mice strain BALB/c, six weeks old and healthy (actively moving and with intact fur). The experiment used 12 female mice, which were divided into three treatment groups, P1 = 0.5 g·g bw-1·day-1, P2 = 1.0 g·g bw-1·day-1 and P3 = 2.0 g·g bw-1·day-1 Tapak Liman leaf extract, as well as a control. The administration of Tapak Liman leaf extract was given orally. the body weight of the mice was monitored for two weeks before administration. Each mice group was given the same standard feed and drink. The main dependent variable measured was the relative number of cells in hematopoiesis phase with the following parameters: 1. The number of cells that expressed CD4/CD8, CD4/CD62L, and TER119/B220 in the spleen 2. The relative number of cells that expressed TER119/B220, TER119/VLA-4, and TER119/CD34 in bone marrow Spleen isolation was done by cutting part of the spleen with surgical scissors. The spleen sample was then washed with PBS and cleaned of attached fat. Homogenates were filtered and collected in a sterile microtube and stored at a temperature of 4°C. The bone marrow was obtained from the femur bone of the left and right legs of mice. Muscles were separated from the femur, and both ends of the joints were cut with surgical scissors.13

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Bone marrow was isolated using a flushing method, injecting PBS with a 1 ml syringe (26½ size needle) at one end of the bone. The results of the injection were collected in a sterile petri dish, filtered and stored in a sterile microtube at a temperature of 4°C.

Flowcytometry Flowcytometry was performed on homogenate from spleen and bone marrow that was centrifuged at 3200 rpm, 4 °C for 2 minutes.14 The supernatant was removed and the pellet resuspended in 1 mL of sterilized PBS and then homogenized. Homogenates were taken in aliquots of 100-200 μL by using a micropipette and inserted into new sterile microtubes that were covered with aluminum foil. Homogenates were recentrifuged at 3200 rpm, 4 °C for 2 minutes, then the supernatant was removed and the microtube put into the icebox. Antibody BD BioscienceTM DC4 FITC anti-mouse conjugated and PE-CD8, DC4 FITC anti-mouse conjugated and PE-CD62L was added to all pellets in the microtubes. BD BioscienceTM antimouse TER-199/Erythrocyte Cell FITC conjugated and PE- B220 was added to pellets isolated from spleen. To pellets from bone marrow were added antibody BD Bioscience antimouse TER-199/Erythrocyte Cell FITC conjugated PE- B220, TER-199/Erytrocyte Cell FITC conjugated CD49d/(VLA-4), and antimouse TER-199/ Erythrocyte Cell FITC conjugated PE-CD34 and then incubated for 15 minutes. Next, a flow cytometer (BD Bioscience FACS CaliburTM) was connected to a computer in the condition Acquiring, and a program run on the computer according to expected parameters, including setting instrument (Detector, Threshold, and Compensation) on the number of cells to be analyzed (Acquisition and storage), label of antibody and laser excitation power, simple name and determining the grated area (R1) on the plot of histogram. The setting plot on Acquiring mode, according to the label of antibody on the axis of Y and X (FITC or PE) and grating area (G1=R1). Flowcytometry was ensured in the set of Low-Run. After the instrument was ready, pellets to which had been added antibody were put into the cuvette of the flow cytometer by micropipette, to which was added 1000 μL of sterile PBS and homogenized by pipetting. The cuvette was attached to the nozzle of the flow cytometer and data measurements acquired using the acquisition control module. Data from flow cytometry were processed with the software BD CellQuest ProTM and displayed in the form of a histogram.

Data Analysis Quantitative data included the relative number of progenitor and precursor cells in the development of lymphoid and erythroid cells in the bone marrow and spleen, which was obtained from the flow cytometer. Data were statistically analyzed by the normality test and homogeneity of variance test. Data that had a homogeneous variance were tested by one-way ANOVA with p= 0.05. If p>0.05 then there was no significant treatment effect, whereas if the p0.05) upon treatment with Tapak Liman (groups P1-P3). The cell population of CD4+CD8- and cells of CD4-CD8+ in treatment P2 (1.0 g·g bw-1·day-1 Tapak Liman extract) increased, compared to control (Figure 1). P2 appeared to increase the total number of cells isolated from spleen. We assume that it happened due to several factors. First, the concentration of leaf extract of Tapak Liman at

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dose P2 increased lymphocytes proliferation; second, the population of P2 exposure to the disease; last, the population of P2 had a bigger spleen (splenomegaly), i.e. the spleen ability to get and keep the erythrocytes will be increased. Splenomegaly causes a decrease of erythrocytes, leukocytes, and thrombocytes in the blood circulation. Figure 1 shows that the populations of T cells CD4+ and CD8+ increased in the P2 group, 7.92 x 106 cells and 8.712 x 106 cells, respectively. Otherwise, the number of T cell CD4+ and CD8+ tended to decrease with treatment P3. It is presumed that this was negative feedback from a high dose Tapak Liman leaves extract. It may be caused by an abundance of lupeol and flavonoid compounds in the extract, which is known as anti-inflammatories. This dose-dependent phenomenon is in accordance with a previous study, which mentioned that herbal medicines will stimulate or conversely suppress the degree of immunity.16

FIGURE 1. Profile of T Cell CD4 and CD8; the cell expression of CD4 +CD8-, CD4+CD8+, and CD4+CD8+ in the spleen. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

The results showed that mice administered leaf extract of Tapak Liman for two weeks compared to the control group showed no significant effects on erythropoiesis in female BALB/c mice. This was most likely caused by several factors, e.g. the extraction method was not appropriate to obtain the active substance, or a longer extraction time was needed. However, the study indicates a significant effect on the production of B lymphocytes in the bone marrow and increases in the number of B cell (CD62L+) in the spleen.

Profile of Cell Express CD4 and CD62L Molecules in Spleen CD62L is an antigen expressed by B cells, T cells, monocytes and natural killer cells (NK cells). It binds CD34 which functions as a leukocyte adhesion molecule (LAM/L-selectin), GlyCAM, and has roles in the interaction of rolling over endothelial cells.3 Analysis of the flow cytometry results (Fig. 2) showed that the treatment groups had no significant differences in all treatment (p>0.05) in the percentage of cells that express CD4 +CD62L and CD4+CD62L+, whereas the average percentage of cells that express CD4CD62L+ (B cell) increased for treatment P1 and P2, 6% and 3% respectively, over the control, and decreased in treatment P3 by 5%. Statistical analysis showed a significant effect (p0.05). Whereas the number of cells of TER119+ and B220+ increased in P2 (Fig. 3), compared to other treatments and control. This indicates that the flavonoid compound increased the production of IL-2, thus increasing the proliferation of B lymphocytes.

FIGURE 3. Profile of TER119 and B220 cells; relative cell number of TER119 + B220, TER119B220+ and TER119+B220+ in the spleen. Description: Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

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Profile of Cell Expression of TER119 and B220 Molecules in Bone Marrow Bone marrow is the primary or central lymphoid organ where the process of hematopoiesis occurs. It includes the lymphopoiesis for maturation, differentiation, and proliferation of T cells and B cells into lymphocytes that recognize the antigen. Bone marrow is also where erythropoiesis takes place. In this study, isolated cells from the bone marrow to which was added antibody antimouse TER-119/Erytrocyt Cell conjugated to FITC and antimouse B220 conjugated to PE (BD Bioscience©). TER-119 is a marker for the erythroid cell development in erythropoiesis from proerythroblasts to mature erythrocytes. Otherwise, B220 (CD45) is a molecular marker for B cell and all hematopoietic cells, which function for phosphatase thyroxine, increasing signal through the antigen receptor of B and T cells. Flowcytometry results (Figure 4) showed a difference in the average percentage of B220 + (CD45) between treatment groups compared to the control. The percentage of cells that expressed TER119B220+ increased 9% over the control for P1, while for treatment P2, significantly increased 16% compared to the control. Otherwise, the number of cells expressing TER119+B220 was not significantly different, similar to TER119+B220+. Referring to these data, the administration of doses of Tapak Liman leaf extract increased the B cell population (B220 +/CD45+).

FIGURE 4. Profile of TER119 and B220 cells; relative cell number of TER119+B220, TER119B220+ and TER119+ B220+ in bone marrow. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

The highest cell population of TER119B220+ was found in P2 (24.546666 x 106 cells). This demonstrated proliferation and differentiation of hematopoietic stem cells had occurred, which tend to the production of B cells (B220+/CD45+). The increase in B cells is assumed to be due to the flavonoid and lupeol compounds contained in Tapak Liman.

Profile of Cell Expression of TER119 and VLA-4 Molecules in Bone Marrow VLA-4 (Very Late Integrin Antigen-4) or CD49d is an antigen expressed by B cells, thymocytes, granulocytes and dendritic cells. It functions as an integrin a4, connected to CD29 (leukocytes), and binds fibronectin, MadCAM1, and VCAM-1 (Vascular Adhesion Molecule-1). The number of cells that expressed the TER119VLA-4+ and TER119+VLA-4+ are presented in Figure 5. The percentage of cells that expressed TER119VLA-4+ decreased by 11% for P2, but increased by 12% in TER11+VLA-4+, compared to the control. As shown in Fig. 5, the highest cell population of TER119+VLA-4+ was in P2, i.e. 7.536 x 10 6 cells. The increased expression of TER119+VLA-4+ in the P2 group treatment is assumed to be due to the iron content in Tapak Liman, which functioned to stimulate the progenitor hematopoietic cells to proliferate and differentiate into the precursors of erythroid and lymphoid cells into TER119+VLA-4+, which express nucleated erythrocyte cells, and

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other cells. Besides that, the increased expression of TER119VLA-4+ may be caused by adhesion molecules that interact between cells with the extracellular matrix, which is needed for the activation of T cells for leukocytes maturation. The increasing number of cells expressing surface molecules of TER119 + and VLA-4+ indicates that the leaf extract of Tapak Liman at 1.0 g·g bw-1·day-1 was adequate to stimulate the proliferation of hematopoietic stem cells into lymphoid and erythroid cells. The protein VLA on the surface of T cell functions to channel activation signals through the T cell receptor. Under normal conditions, leukocyte only attaches to endothelial cells, but under inflammation stimuli, adhesion between leukocyte and endothelial cells is enhanced.13

FIGURE 5. Profile of TER119 and LA-4 cells; relative cell number of TER119 VLA+ and ER119+VLA-4+ cells bone marrow. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

Profile of Cell Express the TER119 and CD34 Molecules in Bone Marrow A cluster of differentiation 34 (CD34) is an antigen expressed by hematopoietic precursor cells and in capillary endothelial cells functions as Ligand CD62L (L-selectin). The average cell percentage of TER119CD34+, TER119+CD34, and CD34+TER119+ in the bone marrow, as measured by flow cytometry, is presented in Figure 6. The percentage of cells expressing TER119D34+ decreased compared to control, as well as the percentage of TER119D34+ cells. While the percentage of cells that expressed CD34+TER119+ increased by 4% in treatment P1, it tended to decrease in treatment P2 and P3, compared to control.

FIGURE 6. Profile of TER119 and CD34 cells; relative cell number of TER119 +CD34+, TER119CD34+ and TER119+ CD34+ in bone marrow. Control (K), P1: 0.5 g·g bw-1·day-1, P2: 1.0 g·g bw-1·day-1, P3: 2.0 g·g bw-1·day-1 Tapak Liman extract.

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Based on the absolute number of cells, the TER119+CD34cell population did not increase after being treated with Tapak Liman leaf extract. However, it tended to increase in P 2, compared to the other two treatments. Otherwise, the number of TER119CD34+ and CD34+TER119+ cell population tended to increase in treatment P1 compared to control (Figure 6). Statistical analysis showed no significant difference (p>0.05) between treatments. This is assumed to be due to several factors: 1. The solvent for leaf extraction was not optimal to obtain sufficient active compound, and 2. the time of samples extraction was not sufficiently long. The treatment dose used in P2 (1.0 g·g bw-1·day-1) showed a significant effect (p=0.026) on increasing the percentage of cell CD62L+ in the spleen and B220+ (p=0.044) in the bone marrow, compared to the 0.5 and 2 g·g bw-1·day-1 doses as well as the control group. It indicates that there was increased proliferation and differentiation of hematopoietic cells into lymphocytes, i.e. CD4+, CD8+ and B220+ T cells. This may have been caused by flavonoid compounds present in the extract that potentially stimulate the activity of IL-2, thus increasing the proliferation of lymphocyte cells. Based on the cell numbers from all of the treatment groups, it is shown that the cell populations increased with the addition of Tapak Liman leaf extract dose of 1.0 g·g bw-1·day-1 (P2) compared to the control group. Otherwise, the cell population decreased with the 2.0 g.g bw.-1day-1 dose (P3). It appears the flavonoid compounds present, besides having an effect as an immunostimulant, it also has the effect of immunosuppressant at higher doses.12 Moreover, it appears the cytotoxic effects of compounds in Tapak Liman, which serve as an immunosuppressant, also allow the resistance progenitor cells in the lymphoid and erythroid pathway to proliferate and differentiate into lymphocytes and erythrocytes. The increased cell expression percentage of TER11+VLA-4+, 12%, seen in treatment P2, compared to control, was important because it identifies the pathway for development of erythrocytes. The increased expression of TER119−VLA-4+ is likely caused by adhesion molecules interacting with the extracellular matrix, which is needed in the activation of T cell in the maturation process for leukocytes. Furthermore, the increased expression of TER119+VLA-4+ was assumed to be due to the iron content in Tapak Liman, which functions to stimulate progenitor hematopoietic cells to proliferate and differentiate into a precursor of erythroid cells (TER119+VLA-4+), which further develop into nucleated erythrocyte cells and others. There was an increasing number of a cell expressing the surface molecules TER119+ and VLA-4+, with doses of 1.0 g·g bw-1·day-1 Tapak Liman leaf extract, stimulating the proliferation of hematopoietic stem cells into lymphoid and erythroid cells.

SUMMARY The administration of 1.0 g·g bw-1·day-1 of Tapak Liman leaf extract stimulated proliferation of lymphocytes and erythrocytes lineage (TER119+VLA-4+), in spleen and bone marrow.

ACKNOWLEDGEMENT The author would like to thank Prof. Dr. Ir. Siti Chuzaemi, MS, the head of Institute of Research and Community Services (LPPM), University of Brawijaya and also the Project Leader of Graduate Grant, Directorate General of High Education DEPDIKNAS.

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M. S. Djati, “Implementing“bio-prospecting of Reproductive Knowledge”: an Effort scenario to conserved Indonesian biodiversity and endemicity toward a post-modern globalized world,” in International Conference on Global Resource Conservation 2016, AIP Conference Proceedings 1844, 020002 (Brawijaya University, Indonesia, 2017), doi: http://dx.doi.org/10.1063/1.4983413. D. R. Dwijayanti, M. S.Djati, M. Rifai, ASIAN Journal of Cell Biology 10 (2), 43-45 (2015). D. R. Dwijayanti, M. S.Djati, M. Ibrahim, M. Rifai. American Journal of Immunology 11 (2), 56-60 (2015).. S. R. Lestari, M. S. Djati, A. Rudijanto, F. Fatchiyah, Asian Pacific Journal of Tropical Medicine 5 (10), 852857 (2015).

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R. Roffico and M. S.Djati, Biotropika 2 (3), 174-180 (2014). S. Dalimartha, Atlas tumbuhan obat 1st -3rd Ed. (Puspa Swara, Jakarta, 2003). P. J. Haen, Iron deficiency anemia. In: Principles of hematology. (Brown Publishers, Wangshinton, 1995), pp. 117–125. K. G. Baratawidjaja, Imunologi Dasar 7th Ed. Medical Faculty (University of Indonesia, Jakarta, 2006). T. B. Drueke, F. Locatelli, N. Clyne, K.U. Eckard, I. C. Macdougall, D. Tsakikis, H. U. Surgery, A. Scherhag, N. Engl. J. Med. 355(20), 2071-2084 (2006). S. Gedik, L. Erkanli, G. Yilmaz, I. Özcebe, Y. A. Akova, Turkiye Klinikleri J. Ophthalmol 16(2). 136-140 (2007). Widodo, K. Khaur, B.G. Shrestha, Y. Takagi, T. Ishii, R. Wadhwa, S. C. Kaul. Clin. Cancer Res. 13(7), 2298306 (2007) S. W. Lestari, M. S. Djati, A. Rudiyanto, F. Fatchiyah, Asian Pac. J. Trop. Dis. 4, S780-S785 (2014). E. Kurnianingtyas, M.S. Djati, M. Rifai, J. Exp. Life. Sci. 3(1). 25-30 (2013). M. Farsely, M. S. Djati, M. Rifai, J. Exp. Life. Sci. 3 (1). 20-24 (2013). R. G. Murthy, S. J. Greco, Brain Behav. Immun. 22(4). 442-450 (2008). S. Rahimi, Z. T. Zadeh, M. A. K. Torshizi, R. Omidbaigi, H. Rokni, J. Agri. Tech. 13. 527-539 (2011).

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Field Study Learning Model to Introduce Environmental Health Problems to Medical Students at the Faculty of Medicine, University of Brawijaya, Malang, Indonesia Lilik Zuhriyah1, a), Nanik Setijowati1), Sri Andarini1) 1

Department of Public Health, Faculty of Medicine, University of Brawijaya, Malang, Veteran Street, Malang, East Java, Indonesia 65145 a)

Corresponding author: lilikz.fk@ub.ac.id

Abstract. Some diseases in the community have a relationship with the environment. Therefore, medical students need to be exposed early to environmental problems in the community. The aim of this paper is to explain the role of field studies for medical students in introducing environmental health problems at an early stage. Field studies were applied by the Department of Public Health in 2005–2006 and 164 students from Semester II, which come from two classes, were required to join it. The portion score of the field study was 10%. Each class consisted of ten groups. Each group consisted of approximately eight students. Each group took different topics/targets of observation. These included ecological farming, household waste management, communal waste management, family medicine plants, food home industry, food street vendors, slaughterhouses, traditional markets, management of communal waste water, and recycling home industry. Each group observed in a community and interviewed related informants. Students were required to make a report and present it in their class. At the end of the exam, students were required to assess the benefit of this activity using a range of 1 (minimal) to 5 (maximal). The students considered the benefits of the field study method, giving an average score of 3.9 and 3.95 for presentation and discussion in class. Some students proposed to maintain field studies and discussion, and to conduct this method every semester with more time. Other students suggested that a lecturer accompany them in the field. Several students regretted unpunctual discussion time that reduced lecture time. The learning model of field study increased the students’ interest in the subject of public health.

INTRODUCTION Medical education in Indonesia has evolved from the enactment of the Indonesian physician competence standard (SKDI) 2006 to SKDI 2012. Medical education graduates will be expected to play a major role in primary health care. Therefore, the medical education graduates are expected to be able to plan health programs to improve public health including environmental health. The ability to communicate effectively and manage health problems is part of the competency of medical graduates [1]. In order to provide candicate physicians with applied science in the community, medical students need to learn the determinants of public health. Blum and Lalonde grouped the factors into four principal areas: (1) environment, (2) heredity, (3) lifestyle, and (4) health care services. This is a model of the determinants of health or a global concept of health. This is an ecologic or systems theory approach which evolved in the early 1970s [2,3]. One aspect of public health science is environmental health. The role of environmental health in health has received attention for several years. The evolution of public health thinking in Canada and elsewhere, based on documents published between 1974 and 1994, always placed the environment as one factor that must be considered in health [3]. However, the delivery of theory alone is not sufficient for students’ understanding. Therefore, students need to be exposed early to the condition of the surrounding environment by observing it [4]. Medical students must be exposed to the sources of disease as evidence. In order to understand the evidence of disease, medical students


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require skills in communication, information retrieval, and formulating and answering focused public health questions. Medical students need increasing exposure to the principles of Evidence Based Medicine in order to have a better understanding of health problems [5]. How to provide interesting and effective learning of environmental health subjects for medical students is challenging. The city of Malang is the second largest city in East Java Province, Indonesia which is surrounded by mountains. The city with a population of 851 298 inhabitants, has an area of 110.06 Km2 which is divided into five sub districts. The city is a high area, as it is located at an altitude of 440–667 m above sea level, with temperatures of 19–30˚C, and humidity of about 65–90%. The economic activities in this city vary from trade, tourism, services and social sectors, processing industries, buildings, agriculture, plantations, and fisheries [6]. Thus, the opportunity to study the health problems of the surrounding environment that has a variety of activities becomes greater. The model of field study is expected to be an appropriate method of study for medical students. The model begins with a public lecture followed by field visits, reporting, presentations, and discussions. However, as the curriculum for medical education has been changed, evaluation of this learning model is needed to improve the next learning model. This paper aims to explain the role of the field study learning model for medical students in introducing environmental health problems at an early stage.

EXPERIMENTAL DETAILS This is a descriptive observational study which evaluated the implementation of a field study. The field study was applied by the Department of Public Health, Universitas Brawijaya, Malang, Indonesia during 2005–2006. The portion score of the field study was 30%, including its report and discussion, while for the mid test it was 25%, and for the final test it was 45%. A total of 164 students from Semester II, which come from two classes, were required to join it. Each class consisted of ten groups, while each group consisted of approximately eight students. Each group took different topics/targets of observation. These were ecological farming, household waste management, communal waste management, family medicine plants, food home industry, food street vendors, slaughterhouses, traditional markets, management of communal waste water, and recycling home industry. Each group observed in the community and interviewed related informants. Students were required to make a report and present it in their class. At the end of the exam, students were required to assess the benefit of this activity ranging from 1 (minimal) to 5 (maximal) and answer an open ended questionaire.

RESULT AND DISCUSSION Field study is a task that must be done in groups. However, each member is asked to rate the contribution of their peers individually. This is to ensure a fairer evaluation. The results of the student learning evaluation obtained are shown Table 1. Individual scores seem to always be lower than group scores, both for field values and report scores. This difference in scores indicates that the contribution of each student in his/her group assignment varies from a lesser contribution to a greater contribution. In addition, the results of the open questionnaires can be grouped into several themes as follows:

Course material Most students assume that the course material was easy to understand. But some students claimed that the material was less good and required a college book.

Time allocation Some students said that the time for college was still lacking and needs to be added.

Presenters lecture Lecturers are considered to be good at delivering the material, but some assume that the delivery is boring so that the lecture feels longer.

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Field observation sessions This activity is considered helpful and enjoyable by most students. However, some students said it was still not good because the material is not appropriate and is time consuming. In addition, the lecturers need to go to the field to accompany the students. Here are some student’s quotes: “Helpful, fun. It should be conducted more often, conducted and maintained” (11 students) “First semester is confusing, second semester is exciting” “The public health subject is fun” “The lecturer needs to supervise students in the field” (2 students) “Less appropriate, need more time” (2 students)

Discussion sessions and presentations of field activities Students are said to like the discussion and even asked for extra time. However, there are also students who feel that the time for discussion is too long, and are less satisfied with the discussion because some students tend not to pay attention Improvements to discussions need to be made so that the it provides equal opportunities for all groups to present their group work. Here are some student’s quotes: “Good, helpful, effective, attractive” (6 students) “Not effective” (4 students) “Need more time” (3 students) “Too much time and boring” (9 students) “Topics and method need to be improved” (7 students) “We also want to present our field studies” In general, students stated that the lectures were good, sufficient, tasty, fun, and useful. However, improvements were still needed to make college more interesting, student attendance can be 100%, and bureaucratic licensing of field studies was no longer an obstacle. Introducing common disease and health problems in the first semesters by direct observation and grouping students into the various fields of observation were the most important. A similar result was found in Iran [7]. Early introduction of disease or health problems related to the environment will open the students’s mind about what next to be learned and what next to be done to overcome the problems. Direct observation in the community also provides experience for students on how to adhere to bureaucracy, how to communicate with people, and how to compose a solution. This model makes the subject of public health fun and enjoyable [4]. The expansion of materials tailored to up-to-date issues is needed to enable students to be sensitive to public health concerns. Technical improvements, such as time allocations for lectures and discussions, technical discussions, licensing, and technical assessment also need to be done. The challenge now is that a reduction of hours for public health materials has occurred since 2006 compared to previous years due to the large amount of material that must be provided. However, although generally the medical school curriculum is fully scheduled, there are sufficient times for sincere incorporation of environmental health into both pre-clinical subjects and the clinical stage [8]. However, the problem-based learning (PBL) format and the ecological model paradigm may be one solution to integrate environmental health materials with clinical health problems. PBL can be a strong method to introduce medical students to public health problems, especially environmental health. By alocating the public health problems initially in the pre-clinic medical curriculum, we hope that students have a strong foundation to consider the contribution of population health to other health problems later in their studies. Finding creative methods to deliver public health ideas is necessary for medical educators [9]. The limitation of this report is that not all of the students completed the open ended questionnaire. However, in this experience, the learning model of field study can increase students’ interest in public health subjects and needs to be improved. However, due to the reduction of public health subject hours and the use of the PBL model, the integration with PBL becomes very important. This can be achieved if the case is discussed using an ecological paradigm rather than a clinical one.

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SUMMARY Early exposure to public health problems in the community, especially those related to the environment is important for medical students. The aim of this paper is to explain the role of field study for medical students in introducing environmental health problems at an early stage. Field studies were applied by the Department of Public Health in 2005–2006. The objects of observation were ecological farming, household waste management, communal waste management, family medicine plants, food home industry, food street vendors, slaughterhouses, traditional markets, management of communal waste water, and recycling home industry. Each group observed in the community and interviewed related informants. The learning model of field study can increase students' interest in the public health subject and needs to be improved by integration with PBL and using an ecological paradigm.

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