• Journal of Environmental Health Sciences
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• v.31 no.3
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• pp.227-233
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• 2005

This study was conducted to determine the endorcrine disruption effects of the several major pharmaceutical residues in water using adult Japanese medaka (Oryzias latipes). Four frequently used pharmaceuticals including caffeine, ketoconazole, acetaminophen, and diltiazem were investigated for the vitellogenin(Vtg) induction in the medaka using Western blotting and ELISA. $17\beta$,-estradiol was used as a positive control. Vtg was qualified and quantified through Western blot and ELISA. Following SDS gel electrophoresis, the dominant protein band was identified to molecular weight approximately 205 kDa in whole body samples of vitellogenic female. With female medaka exposed to $17\beta,-estradiol$, no significant difference in total protein induction was noted. In contrast, three to five day exposure of male fish to $17\beta,-estradiol$ resulted in $63.07\%o$, increase of total protein comparing to that of control males (p<0.01). Vtg induction in male fish was observed with all the test pharmaceuticals: At concentrations greater than 1ppm of diltiazem, 2 ppm of caffeine, 4 ppm of acetaminophen, and 10 ppm of ketoconazole, Vtg induction was monotonously increased in a dose dependent manner. This study is one of the first reports suggesting potential endocrine disruption mechanism of common human pharmaceutical products in aquatic ecosystem. Although the effect concentrations obtained from this investigation are environmentally unrealistically high, endocrine disruption should be considered as one of the important consequences of pharmaceutical pollution in aquatic environment, and warrants due attention in future researches.

• Environmental Analysis Health and Toxicology
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• v.23 no.3
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• pp.221-230
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• 2008

Chlorpyrifos (CPF) is a widely used organophosphate insecticide. Japanese medaka, Oryzias latipes was selected to investigate the effects of chlorpyrifos on survival rate, hatching rate, reproductivity and histopathological changes. Adult fish were exposed to CPF at concentrations of 20, 40, 80 and $160{\mu}g/L$, and the test duration was 4 weeks. An experiment with eggs was also performed under CPF exposure continued for 7 days covering from the fecundation to end-hatching stages. Survival rate of the adult medaka decreased in CPF $80{\mu}g/L$ and $160{\mu}g/L$ treatment groups compared with control, solvent control, $20{\mu}g/L$ or $40{\mu}g/L$L treatment group. Spontaneous start of feeding significantly decreased compared with the control (p < 0.01) when exposed to CPF at above 40 $\mu g/L$. Total spawning decreased by the 20 or $40{\mu}g/L$ CPF treatment compared with the control or solvent control in the test with eggs obtained from untreated adult medaka, and abnormal eggs increased in those CPF-treated groups. There were certain adverse effects at above $40{\mu}g/L$ determined from liver and gonad histological examinations. The results indicate that CPF has insignificant toxic effects in Japanese medaka at less than $20{\mu}g/L$ in long term exposure.

• Environmental Analysis Health and Toxicology
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• v.18 no.2
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• pp.145-153
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• 2003

The goal of this study is to develop a biomarker used in monitoring abnormal behaviors of Japanese medaka (Oryzias latipes) as a model organism caused by hazardous chemicals. Japanese medaka was treated by copper of appropriate sublethal concentrations after starvation for 48 hr. The untreated individuals showed common behavioral characteristics (i.e. , smooth and linear movements). Locomotive activity of the fish was monitored using an image processing and automatic data acquisition system. When treated with copper (100 ppb), the fish showed shaking patterns more frequently. As the concentration of copper increased to 1,000 ppb, activity decreated, and the fish showed an erratic movement. Fish were exposed to copper at various concentrations (0,100 and 1,000 ppb) for 24 hrs, and acetylcholine esterase (AChE) activity was observed. When fish were exposed to 1,000 ppb of copper, the body AChE activities appeared to decrease but the head AChE activities showed little change. Expressions of tyrosine hydroxylase (TH) protein in the different organs from both head (brain) and body (kidney) portions affected by the copper treatment were analyzed using immunohistochemical technique compared with control. Five organs of the fish (olfactory bulb, hyothalamus, optic lobe, pons and myelencephalon regions) showed a relatively strong TH protein expression in the control experiment. A differential expression of TH, however, was observed in the treatment (100 ppb and 1,000 ppb). The treatment (1,000 ppb) significantly suppressed TH protein production in the brain regions. In kidney, however, the same treatment caused little suppression compared with the control. Copper appeared to be less effective in suppression of TH than diazinon, a known TH suppressor. It was concluded that TH could be used at a potential biomarker to monitor the acute copper toxicity in Japanese medaka.

• Korean Journal of Environmental Biology
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• v.23 no.3 s.59
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• pp.293-303
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• 2005

Japanese medaka, Oryzias latipes is widely distributed in the North East Asia including Korea, Japan and east China, and commonly used for freshwater toxicity tests and cytotoxicological studies worldwide. In this study, a series of experiments were conducted to identify the potential of the fish as a standard test species for saltwater toxicity evaluation such as marine receiving waters, ocean-dumped materials and sediment pore waters etc. Hatching, growth and mortality rates of the fish were estimated with the wide ranges of salinity from freshwater to seawater (35 psu). Direct exposure of the fertilized eggs in freshwater to the wide ranges of salinity (from 0 to 35 psu) without pre- acclimation to the saltwater revealed no significant differences in hatching rates by salinities (p =0.24). On the other hand, medaka larvae hatched in freshwater and exposed to saltwater directly showed high mortality at > 25 psu treatment groups (p < 0.0001). However, there was no significant difference in mortality of medaka larvae hatched in 13.8 and 14.2 psu at the wide ranges of salinities ($0\~35$ psu). Growth rates of medaka larvae hatched in the above two salinities showed no differences in body length either from 0 to 35 psu treatment groups (p =0.64 for 13.8 psu group and p=0.32 for 14.2 psu group). The number of gill chloride cell in medaka larvae sharply increased when the larvae were exposed to high salinity. Reference tests with zinc chloride revealed 96h $LC_{50}=8.84(7.19\~10.87)mg\;L^{-1}$ using 7~10 day old medaka larvae. These were comparable or better sensitivity in comparison with the other standard test species such as North American sheepshead minnow Cyprinodon variegatus. Based on the results of these experiments, hatching rates and larvalmortality of medaka must be good toxicity parameters for seawater bioassay and the species seems to be a good standard species for both the freshwater and seawater toxicity test.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.170-170
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• 2003

• Environmental Analysis Health and Toxicology
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• v.12 no.3_4
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• pp.55-59
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• 1997

Furfural, an organic solvent, is widely used as synthetic component material in producing chemical products. However, furfural has been reported that it shows strong toxicities to human being showing intense stimulus to skin, eyes, mucous membrane and nerve system. It is also known to cause anemia, liver cirrhosis, kidney failure and genetic toxicity in the human being working in the exposed area. LD$_{50}$ of furfural for peritoneal injected mouse has been known around 20mg/kg, but the acute toxicity on aquatic organisms such as fish, daphnid or algae are not well known, compared to those on rodents. In this experiment, we studied on the fish toxicity of furfural using Japanese Medaka (Orvzias latipes) and Common Carp (Cvprinus carpio). We also observed histological changes in the fish organs. The LC$_{50}$ were 12. Smg/L in Japanese Medaka and 21.8 mg/L in Common Carp, respectively. When Common Carps were exposed to 120mg/L of furfural concentration for 30 minutes, blood congestion in gills and lysis of secondary lamella were shown. Though the muscle of caudal fin was not completely eroded, its epidermic cells were shown to be necrotic in various parts. Tissue atrophy and cell necrosis were also shown in the liver of Common Carps exposed to furfural. From these results, furfural seems to cause histological damages on liver, an internal organ as well as on external organs such as gills and fins eventhough the fish were exposed for a short-term.

• Environmental Analysis Health and Toxicology
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• v.15 no.4
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• pp.131-137
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• 2000

The release of hazardous waste materials into the environment poses serious risks in humans and ecosystems. The risk assessment of environmental pollutants including hazardous chemicals requires a comprehensive measurement of hazard and exposure of the chemicals that can be achieved by toxicity evaluation using a biological system such as biomarkers. In this report we have tried to develop a biomarker used to elucidate a molecular basis of, and to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. First, an attempt was made to clone tyrosine hydroxylase gene from Japanese medaka that would be a candidate for a biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon and their behavioral responses were observed . In this study we have successfully cloned a partial TH gene from the medaka fish through PCR screening of an ovary cDNA library. DNA sequencing analysis revealed that the amplified fragment was 327 bp encoding 109 amino acids. Comparing the DNA sequence of medaka TH with other species, TH gene revealed the DNA sequence was completely identical to that of rat TH. In the RT-PCR, 330 Up of mRNA was consistently amplified in all the treated samples including control There were no significant differences in the TH expression level regardless of treating concentrations (1∼5,000 ppb) and time (0∼48 hr) The reason appeared to be that RT-PCR was not performed using through a quantitative analysis normalized against an actin gene expression. Organ or tissue - specific detection of TH activity and mRNA as biomarkers will be a useful monitoring tool for neurobehavioral changes in fish influenced by toxic chemicals. Furthermore, quantitative analysis of locomotive patterns and its correlation with the neurochemical and molecular data would be highly useful in measuring toxicity and hazard ofvarious environmental pollutants.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.13 no.2
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• pp.140-146
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• 2008

Marine ecotoxicological standard method using fish larvae was established with the standard test species of Oryzias latipes(Japanese Medaka) and Paralichthys olivaceus(flounder) and with the 7 day $LC_{50}$ as endpoint. Test method referred to the USEPA(1994) with the replacement of test species found in the Korean water. Standard test species were selected in terms of the species supply and ecological importance in Korean waters. Japanese medaka can be reared with small tanks in the lab and has wide tolerance on salinity, and flounder eggs can be easily obtained from commercial fish hatcheries. General conditions for larval fish toxicity test are as follows. The possible salinity ranges for toxicity test were $0{\sim}35\;psu$ for medaka and >20 psu for flounder. Test type was designated as static non-renewal test if the dissolved oxygen in the test chamber does not fall below 4.0 mg/L. Ages of test species were selected as 7 days after hatched for medaka(about 5 mm TL) and 25 days for flounder(about 10 mm TL) because of the low natural mortality after these periods. Test can be accepted when the survival rates are over 80% in control. Also, species sensitivity on standard reference materials(copper, cadmium or zinc) must be provided with the toxicity test results.

• The Korean Journal of Pesticide Science
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• v.16 no.2
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• pp.187-193
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• 2012

Acute toxicity, water resolvability and short term reproduction test on Japanese medaka (Oriyzias latipes) for evaluating alachlor susceptibility to endocrine system were studied. Alachlor is known for suspected endocrine distruptors. As the results of tests, $LC_{50}$ (Median lethal concentration) was determined as 2.36 (1.994~2.805) mg/L, and test water replaced at 7 day intervals as its water resolvability was less than 20% in 7 days. The short term reproduction tests on Japanese medaka (Oriyzias latipes) were performed with a solvent control group, a treated group (alachlor concentrations of 0.02, 0.04, 0.11, 0.27, 0.68 ppm) and a positive control group (17 ${\beta}$ estradiol, 0.01, 0.1, 0.5 ppb). The number of spawning and embryo rates were declined in a alachlor-dose dependent manner, and the number of unfertilized eggs rates were in contrast increased depending on the concentrations. Further study should be needed to confirm whether the adverse effects may be effected by the concentrations. Additionally, alachlor was evaluated as a non-vitellogenin by the result of a test of significance of the vitellogenin content test for determination of the effect of estrogen among the endocrine disruptors.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2000.12a
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• pp.88-88
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• 2000