Marine Pollution Bulletin 63 (2011) 347–349

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Marine Pollution Bulletin

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Effect of mercury and cadmium on early life stages of Java medaka(Oryzias javanicus): A potential tropical test fish

Ahmad Ismail ⇑, Shahrizad YusofDepartment of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Keywords:Java medakaTest organismTropicalEarly life stage

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⇑ Corresponding author. Tel.: +60 389466617; fax:E-mail addresses: aismail@science.upm.edu.my (A

edu.my (S. Yusof).

Several organisms have been used as indicators, bio-monitoring agents or test organisms in ecotoxicolog-ical studies. A close relative of the well established Japanese medaka, the Java medaka (Oryzias javanicus),has the potential to be a test organism. The fish is native to the estuaries of the Malaysian Peninsula,Thailand, Indonesia and Singapore. In this study, newly fertilised eggs were exposed to different concen-trations of Cd and Hg. Observations were done on the development of the embryos. Exposure to lowlevels of Cd and Hg (0.01–0.05 ppm) resulted in several developmental disorders that led to death. Expo-sure to P1.0 ppm Cd resulted in immediate developmental arrest. The embryos of Java medaka showedtolerance to a certain extent when exposed to P1.0 ppm Hg compared to Cd. Based on the sensitivity ofthe embryos, Java medaka is a suitable test organism for ecotoxicology in the tropical region.

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1. Introduction

Fish, particularly small sized ones have been the most popularchoice for vertebrate test organisms because they are presumablythe best-understood organism in the aquatic environment(Buikema et al., 1982). The medaka fish are important model spe-cies among bony fish. It is a diverse group of small fish distributedin large areas in Asia. They occupy the freshwater, brackish waterand saltwater. The most established among the medaka fish is theJapanese medaka (Oryzias latipes). It has been widely used in exper-imental vertebrate biology for over a century. It is distributed in thefreshwater of Japan, Korea and China (Naruse et al., 1993; Naruse,1996). One of its relatives, the Java medaka ( Oryzias javanicus(Bleeker, 1854)) is distributed in the brackish waters of PeninsulaMalaysia, Singapore, Indonesia, Thailand and Western Borneo(Iwamatsu et al., 1982; Magtoon and Termvidchakorn, 2009;Roberts, 1998). This specie was found to be fully adaptable to fresh-water and seawater (Inoue and Takei, 2002), making it suitable torepresent the coastal region. This fish has the potential to be devel-oped as test organism as discussed by Imai et al. (2005, 2007). Javamedaka is a small tropical fish which has many characteristicssimilar to the well known laboratory fish. They have a wide geo-graphical range and availability, short life span and life cycle, fastgrowth rate, hardy, easily identified and easy to culture. Anotherimportant characteristic is their large, transparent eggs that allowease of observation on the developmental stages. Fertilisation in

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the fish occurs externally soon after spawning. The spawning eventtakes less than 1 min. With this precise time of spawning collectionof eggs for further tests could be done easily especially in the exper-iments that involve very early stage of embryo development untilfurther stages. Java medaka is an important component of estuarineecosystems in the tropical region. As there is no well establishedfish model for assessing the marine or estuarine ecosystem healthparticularly for the tropical region, Java medaka can fill the gap.Reproducible laboratory culture of Java medaka has successfullybeen carried out at ambient temperature in our laboratory and thusit can support continuous testing activities.

The early life stages of fish have long been recognized as verysensitive biological material (Marchetti, 1965). The sensitivitiesof embryo-larval and early juvenile stages are, in most cases, com-parable with those of full life-cycle tests (McKim, 1977). Tests of afew days or a few weeks duration could be a good substitute forlife-cycle tests with a much longer duration (Persoone et al.,1990). A number of researchers have reported the relevance of uti-lizing early life stages of fish for assessing the ecological risk posedby pollutants in the aquatic environments (Eaton et al., 1978;Ward et al., 1982; Shazili and Pascoe, 1986; Strmac et al., 2002;Wadekind et al., 2007). Moreover, fish embryo tests are neitherbetter nor worse than acute fish toxicity tests and provide strongscientific support for it to surrogate the acute fish toxicity testwhich is not compatible with most current animal welfare legisla-tion (Lammer et al., 2009). This study utilized the early life stagesof Java medaka in order to assess their sensitivity to Cd and Hgexposures. A test organism for ecotoxicological studies shouldshow excellent responsiveness to chemical contaminant exposure.Thus this study is undertaken to assess the observable effects of

348 A. Ismail, S. Yusof / Marine Pollution Bulletin 63 (2011) 347–349

cadmium and mercury exposures to early life stages of Javamedaka.

2. Materials and methods

Java medaka is cultured in the Department of Biology, UniversitiPutra Malaysia. Newly-spawned Java medaka eggs clusters werecollected gently from the female’s body by hand. The clusters ofeggs were separated using forceps and then surface sterilized usinga mixture of NaCl, KCl, CaCl�2H2O, MgSO4�7H2O and methyleneblue for 5 min (Kinoshita et al., 2010). The eggs were then incu-bated in separate test solutions containing notional concentrationsof 0.01, 0.02, 0.03, 0.04, 0.05, 0.10 and 1.00 ppm Cd and Hg.Analytical-grade HgCl2 and CdCl2 were used (Merck, Damstadt,Germany). Stock solutions of 100.0 ppm were prepared in distilledwater, from which small aliquots were added to saline water inorder to obtain desired concentration. The metal concentrationswere chosen based on the Malaysian Interim Standard for Hg(0.001 ppm) and Cd (0.1 ppm). Concentrations higher and lowerthan the interim standard were chosen. The saline water was madeusing artificial sea salt with salinity 20.0 ppt. This salinity was cho-sen based on the average salinity of the localities of the fish weinvestigated during our survey on their habitat. Control is a salinewater of 20.0 ppt. One hundred and twenty eggs were exposed toeach metal concentration. Exposure was done under semi-staticsystem where the test solutions were renewed every 24 h. Obser-vations on the development of the embryos were made every24 h using stereomicroscope. Exposure was done till the eggshatch. When hatched, the juveniles were cultured in 20.0 pptsaltwater.

3. Results and discussion

Table 1 shows the percentage of developmental consequenceswhen fertilised eggs of Java medaka were exposed to different con-centrations of Cd and Hg. Six consequences were recorded, namely,death of embryo, death of hatched embryo, slow developing em-bryo, premature hatching, short-lifespan juvenile and juvenilereaching adulthood. Death of embryo refers to the death that oc-curred throughout the developmental course, before hatching.Death of newly hatched larvae refers to the death that occurredimmediately after hatching. Normal incubation period for the em-bryos is 12–14 days. Embryos that took more than 15 days to hatchare considered as slow developing embryo and hatching that occur

Table 1Percentage of developmental consequences of the exposure of Java medaka eggs to differe

Conc.(ppm)

Metal Death ofembryo

Death of newly hatchedlarvaea

Juvenile hatcheslow developingembryoa

0.01 Cd 80.0 14.2 0Hg 37.5 50.0 20.0

0.02 Cd 47.5 37.5 0Hg 28.3 49.2 3.6

0.03 Cd 57.5 20.0 0Hg 32.5 41.7 32.3

0.04 Cd 55.0 35.0 0Hg 25.8 41.4 15.4

0.05 Cd 47.5 27.5 0Hg 21.7 32.9 9.1

0.10 Cd 85.0 10.0 0Hg 43.3 28.3 5.9

1.00 Cd 100.0 0 0Hg 64.2 27.5 10.0

Control Nil 10.0 3.3 0

a Calculated from all hatched embryos that lived till at least 2 weeks.b Lifespan = 2 weeks.c Fish aged 4 weeks or more.

before the 12th day is considered as premature hatching. In Table1, percentages for death of embryo and death of newly hatched lar-vae were calculated based on the total number of embryo (N = 120)exposed in each treatment. The percentages for juvenile hatchedfrom slow developing embryo, premature hatching, short-lifespanjuvenile and juvenile reaching adulthood were calculated based onthe number of all hatched embryos that lived till at least 2 dayspost-hatch.

Exposure to low levels of Cd (0.01–0.10 ppm) resulted in severaldevelopmental impairments that led to death while exposure to1.0 ppm Cd resulted in immediate developmental arrest. Prema-ture hatching was significantly observed in eggs exposed to0.01–0.05 ppm Cd (up to 20.0%). Similar results were obtained byWoodworth and Pascoe (1982) with rainbow trout. Exposure tothe same concentrations of Hg caused higher rate of prematurehatching (up to 34.0%). Jezierska and Witeska (2001) discussedthe possibilities of metal-induced hypoxia, disturbances of meta-bolic processes and damage to the cell membrane that lead to pre-mature hatching of metal exposed eggs.

Newly hatched Java medaka larvae were more sensitive to Hgcompared to Cd. Exposure of the eggs to 0.01–0.1 ppm of the stud-ied metals resulted in the mortality of 10.0–37.5% newly hatchedlarvae for Cd and 28.5–50.0% for Hg. Nevertheless the larvae of pre-viously Cd-exposed eggs did not live long where 5.0–25.0% of themdied before reaching 2 weeks old. For larvae from Hg-exposed, only5.0–20.8% were short lived. Many of the embryos are weakened bymetal exposure and do not survive hatching which is an energyconsuming process (Jezierska et al., 2009).

The embryos were very sensitive to low levels of Hg (0.01–0.02 ppm). They are more tolerant to slightly higher level of Hg(0.03–0.05 ppm) and become sensitive again to higher level of Hg(0.1–1.0 ppm). Every concentrations of Hg resulted in slow devel-oping embryos. Mercury was found to delay the incubation periodof killifish, Fundulus heteroclitus (Perry et al., 1988) and estuarinecrab (Sanchez et al., 2005). The delay could be due to the effectof the metals on mitotic divisions (Perry et al., 1988) or suppres-sion of embryogenesis at particular developmental stages (Kapurand Yadav, 1982). It was noticed that both premature hatchingand delay in incubation period can occur in the same Hg concen-tration. Fig. 1 shows both developmental abnormalities of embryowhen exposed to 1.0 ppm Hg.

Java medaka embryos are more sensitive to Cd compared to Hg.At every concentration of Cd and Hg used, reduction of hatchingsuccess was more pronounced in Cd exposed eggs. This is seen

nt concentrations of Cd and Hg (N = 120).

d from Prematurehatchinga

Short lifespanjuvenilea,b

Juvenile reachingadulthooda,c

0 100.0 066.5 13.5 050.0 50.0 060.2 12.1 24.137.0 63.0 029.0 38.7 050.0 50.0 051.3 25.6 7.716.7 83.3 030.9 32.7 27.30 100.0 038.2 29.4 26.50 0 060.0 30.0 00 3.8 96.2

Fig. 1. Delayed incubation period (A) and fast-developed embryo (B) after 11 daysof exposure to 1.0 ppm Hg.

Fig. 2. Teratogenic effect of 0.2–1.0 ppm Hg.

A. Ismail, S. Yusof / Marine Pollution Bulletin 63 (2011) 347–349 349

from the percentage of death of embryo. Under optimum condi-tions the hatching success is 90%. Hatching success for Cd-exposedeggs is between 0% and 52.5% while hatching success for Hg-exposed eggs is between 35.7% and 78.6%. The reduced hatchingsuccess of metal-exposed embryos is attributed to structural andfunctional disturbances during embryonic development (Jezierskaet al., 2009).

4. Morphological impairments

Teratic juveniles exhibiting permanently bent tail were pro-duced by Hg treatment (0.02–1.0 ppm) (Fig. 2). Birge et al. (1983)observed a backward and lateral curvature of the spine (lordosco-liosis) in rainbow trout larvae after exposure of the fertilised eggsto 2–25 ppm of inorganic mercury while Weis and Weis (1977)found the same effect in killifish, Fundulus heterocilitus after expo-sure to as low as 0.03 ppm of inorganic mercury. Lower concentra-tions of Hg were found to cause teratogenesis in this studyshowing high sensitivity of Java medaka to Hg.

5. Conclusion

Based on the sensitivity of the embryos, Java medaka is a suit-able candidate to be established as test organism for ecotoxicolog-ical studies in the tropical region. The successful laboratory cultureof Java medaka could supply the required early life stages of Javamedaka for testing purposes. Exposure to early life stages of Javamedaka can be an effective way of assessing the toxicity of Cdand Hg in water. Since Java medaka is native to the tropical region,results of testing using Java medaka will reflect the natural envi-ronment of the tropical region.

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