JAVA MEDAKA (Oryzias javanicus) AS A POTENTIAL BIOMONITORING A GENT AND TESTING ORGANISM FOR MARINE COASTAL ENVIRONMENT POLLUTION

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1 JAVA MEDAKA (Oryzias javanicus) AS A POTENTIAL BIOMONITORING A GENT AND TESTING ORGANISM FOR MARINE COASTAL ENVIRONMENT POLLUTION AHMAD ISMAIL & SHAHRIZAD YUSOF Biology Department Faculty of Science Universiti Putra Malaysia

2 MINAMATA DISEASE Minamata disease is a neurological disorder caused by methylmercury poisoning. It is known as the first major environmental disaster from human causes in Japan.

3 First published in 1962, Silent Spring alerted a large audience to the environmental and human dangers of indiscriminate use of pesticides, spurring revolutionary changes in the laws affecting our air, land, and water Rachel Carson stated that the large number of chemicals (approximately 500, many were pesticides) introduced each year was possibly making the earth unfit for all life. Before World War II, inorganic chemicals were the main pest controls. Arsenical were greatly used, and toxicological problems occurred. Chlorinated hydrocarbons and organophosphates as the main problems leading to bird and fish kills, human nervous system disorders, and deaths. Possibility causes surface and ground water contamination problems. Water treatment plants did not remove chemicals because multiple chemicals in catch basins could interact to form toxic compounds, and thus cancer hazards from polluted waters would increase in the future. Chemicals treatment of soils led to the destruction of beneficial biological species, and resulted in imbalance to the ecosystem. Long-term persistence of chlorinated hydrocarbons in soil and the possible transfer of chemicals into plants grown in such soils. Scientists' and government officials' concerns addressed only classical toxicity of pesticides and that no testing was done on effects to wildlife. Exposure to or ingestion of various products, each at individually safe levels, taken together, could lead to health problems (e.g., mental problems and cancer), disruption of key metabolic pathways and mutations. What is safe today is not safe tomorrow. Tumors and leukemia brought on by carbamates, DDT, and aminotriazole as problems.

4 Quote from Rachel Carson in Silent Spring: "If we are going to live so intimately with these chemicals - eating and drinking them, taking them into the very marrow of our bones - we had better know something about their nature and their power"

5 Scientific Findings Revealed in Our Stolen Future For the past thirty years, fears about toxic chemicals have focused primarily on cancer. OUR STOLEN FUTURE explores an entirely different danger-interference with hormones and the delicate process of human development. Hormones are chemical signals that guide vital physiological processes in the body, including development from embryo to adulthood. Hormone-disrupting chemicals work in a variety of ways to garble the body's own internal message system.

6 Straits of Malacca one of the busiest shipping line in the world About 70,000 ships expected to navigate through this narrow straits in 2005 More 50% oil super tankers, more 25% cargos. Dominant users are Japan, China, Korea

7 Distribution of BTs in Southeast Asia 100 ng g -1 wet wt. 0 Sudaryanto et al. (2000)

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9 STATES JOHORE MALACCA NEGERI SEMBILAN SELANGOR PERAK KEDAH PERLIS LANGKAWI PENANG n Estradiol concentration (pg/ml) av erage max min > 1.2> > 4 Summary of 17 -estradiol concentration in surface water collected from main estuaries along the west coast of Peninsular Malaysia The data from this study indicate that the concentrations of estradiol in Malaysian water samples are 10 times higher than those of Japanese water environment. This result suggests the serious estrogen pollution in Malaysian water. The detail monitoring must be needed to confirm the status of the other chemical pollution and bioavailability, and degree of remediation necessity in Malaysian water environment.

10 Melaka and Negeri Sembilan with high pig farming activities, 0% exceeded standard?

11 Need a monitoring activities to assess hazardous chemicals in coastal marine environment Continuous work on intertidal mollusc as a monitoring agent for TBT and heavy metals Assessment on Java medaka as another monitorinng and testing organism for hazardous chemicals in coastal environment

12 Criteria for biomonitoring agent Widely distributed in the coastal waters A sedentary life style and therefore is easily sampled Long-lived and available at all periods of the year Large enough to provide sufficient tissue for metal analysis Low variability and easy to identify morphologically Can be easily maintained in laboratory conditions for experimental studies Have strong ability to accumulate heavy metals from ambient environment A good suspension-feeder with a high filtration rate Tolerant of heavy metal stresses Sensitive to heavy metal stresses A significant relationship can be found between heavy metal levels in the mussel and its environment Of public concern from the health point of view

13 Fish are an important monitoring indicators in aquatic environment They live in well defined habitat They can be used for assessing the threats of various pollutants to ecosystems Fish like japanese medaka (Oryzias latipes) are used internationally as test fish. Java medaka (Oryzias javanicus) for example have a wide range of salinity tolerance and can live in salt water as well as in freshwater. They have a unique osmotic adaptation mechanisms.

14 The use of medaka fish as a monitoring agent for waterborne contaminants such as metals been documented elsewhere. Now medaka have been use in toxicological studies for various types of chemicals at many levels from behavioral responses and biochemical and molecular biological processes. More information on the use of medaka in toxicological research has been documented in The Fish Biology Journal MEDAKA Volume 8, 1996.

15 Medaka such as Java medaka and Indian medaka are well distributed in the Asian countries and can be developed as an experimental tool for toxicological studies. Unfortunately many more information on these medakas are not well documented including biology, ecology and possibilities of culture them for toxicological studies.

16 More studies is needed to understand their distribution, ecological parameters of their habitat, genetic variations, biology and physiology in order to establish a standard system to culture them in the laboratory.

17 Java medaka Found in abundance in brackishwater and saltwater of Malaysian Peninsula, Thailand, Indonesia, Viet Nam. Available all year round Possess the basic vertebrate plan for organogenesis

18 Medaka Oryzias have 14 (17) species All endemic in Asia Each species inhabits a characterestic osmotic environment Some are endemic to fresh water, brackish water or sea water. Interesting to study the wide range of habitat and their ecological and behavioural respons

19 Inoue and Takei, 2003 suggest a general advantage of medaka Relative small size and hardy nature and easy to maintain in small aquaria Male and female are easy to distinguish by morphology fins They spawn eggs everyday under the control photoperiod The large and transparent eggs are suitable for embryonic manipulation The short generation time (2-3months) make them possible to obtain five generations a year A number of inbred strain are available A various mutants have observed and easily induced Popular species for genomic research

20 Java medaka one of the 14 (17) species belonging to the genus Oryzias Distributed in estuarine waters of East to Southeast Asia Java medaka can be cultured easily and inexpensively, and has a short life cycle It only take 100 days to mature from hatching High adaptability to different salinities

21 Java medaka High sensitivity to chemicals Toxicity test using F2 generation are possible in one year Its characteristics are suitable for assessing the environmental risk, toxicity test for new pollutants in various environment, biological responses testing and sensitive molecular biomarkers testing.

22 Imai et al and 2007 Successfully culture the java medaka to be used as a testing organism to study the effects of estrone on full life cycle of java medaka (Oryzias javanicus) Effects of 17beta-estradiol on the reproduction of java medaka (Oryzias javanicus)

23 Java medaka easy to collect in the coastal and estuary Available all year round

24 habitat for java medaka Not expose to direct wave and easy to collect during high tide

25 Ecological parameters in Kuala Linggi and Kapar Temperature ( o C) Conductivity (µmhos) Salinity (ppt) 5-25

26 Frequency (%) of Java Medaka body length collected from Kuala Linggi, Negeri Sembilan and Kapar, Selangor in three months Length Class September 2001 Linggi/Kapar October 2001 Linggi/Kapar November 2001 Linggi/Kapar <20mm 34/34 42/41 34/ mm 43/43 50/42 33/ mm 23/23 8/17 33/25 Total number 150/ / /160

27 Copper and Zinc in Java Medaka, Sediment and Surface water

28 Cu and Zn in surface sediment Zinc Copper Kapar, Selangor Resistant:75% Nonresistant:25% Resistant:90% Nonresistant:10% Kuala Linggi, Negeri Sembilan Resistant:86% Nonresistant:14% Resistant:86% Nonresistant:14%

29 Cu and Zn in surface water Zinc Copper Kapar, Selangor Kuala Linggi, Negeri Sembilan

30 Cu and Zn in tissue (excluded stomach and head) Zinc Copper Kapar, Selangor Kuala Linggi, Negeri Sembilan

31 Ratio of Cu and Zn in fish, water and sediment Zinc Copper Ratio Kuala Linggi, Negeri Sembilan Kapar, Selangor Kuala Linggi, Negeri Sembilan Kapar, Selangor Sediment:Fish Tissue 2.3:1 1.1:1 10:1 3.5:1 Water:Fish tissue 0.001: :1 0.02:1 0.01:1

32 Java Medaka in Laboratory

33 Laboratory culturing

34 Acclimatisation to laboratory condition: 1 week in natural brackish water (15 20 o / oo ) Temperature o C Photoperiod: 12 h dark: 12 h light The fish adapted well to laboratory condition

35 Acclimatisation to freshwater: Salinity was reduced by o / oo each day until it reaches 0.0 o / oo by replacing some amount of the saltwater with dechlorinated tap water. Takes 1 1½ months The fish could tolerate the descending salinity and remained active

36 Feeding: Post-hatch 1 month: Ground guppy feed (2x/day) Adult : Dried Tubifex worm (1x/day) Artemia (1x/day)

37 Reproduce freely under culture condition Has a rapid and well-characterized period of embryo larval development A fertilised egg takes approximately 25 days to hatch in freshwater % of harvested fertilised eggs hatched successfully

38 By having a uniform and standard systems of culturing Java Medaka, then a uniform experimental design can be developed and use by all interested scientists from Asian countries. Therefore, a series of expeditions are needed to study on the distributions, ecology and genetic variations of medakas in Asian countries.

39 Suggestion for Future Directions 1. more studies on ecology, biology and genetic studies of medaka in response to environmental changes and water quality 2. establish a common tools for toxicological studies and environmental monitoring of hazardous chemicals in the coastal waters 3. develop networking in Asian countries on medaka as a tool for conservation and environmental protection strategies 4. joint another bigger group such as JSPS to strengthen the collaboration and contributing to science and environment

40 Acknowledgement Thanks to the organiser for inviting me to joint this meeting Thank you Prof Miyazaki and Dr Koji Inoue for encourage me to study Java Medaka and Transgenic Medaka Thank you to Universiti Putra Malaysia for allowing me to participate in this meeting and providing space for research

41 Thank you for your attention