Ik Joon Kang

Effect of 17β-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Abstract Estrogenic compounds such as 17β-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis–ova. Males treated with E2 concentrations=55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis–ova in male medaka exposed to environmentally relevant concentrations of E2.

Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes)

The toxicity of three pharmaceutical and personal care products (PPCPs)--carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS)--was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish (Oryzias latipes). Medaka were exposed to 6.15 mg L(-1) CBMZ, 1.0 mg L(-1) DCF, 0.17 mg L(-1) TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5-9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms.

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17β-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.

Effects of 4‐nonylphenol on reproduction of japanese medaka, Oryzias latipes

We exposed mature Japanese medaka (Oryzias latipes) to 4-nonylphenol (4-NP) to elucidate its reproductive effects and estrogenic responses. For each treatment, eight breeding pairs were exposed for 3 weeks at measured 4-NP concentrations of 24.8, 50.9, 101, and 184 micrograms/L. Egg production of the pairs during the third week of exposure (15-21 d) was significantly decreased in the > or = 101-microgram/L treatment group, and fertility was significantly decreased in the 184-microgram/L group. Induction of testis-ova was observed in male fish at all 4-NP concentrations, whereas abnormality of spermatogenesis was found only in those receiving the 184-microgram/L treatment. The gonadosomatic index of the males in the 184-microgram/L group was also remarkably reduced. Hepatic vitellogenin (VTG) levels were significantly increased in both sexes receiving 4-NP treatments of > or = 50.9 micrograms/L. Overall, the lowest-observed effective concentrations (LOECs) of 4-NP for decreasing reproductive capacity (fecundity and fertility) and eliciting estrogenic responses (induction of testis-ova and VTG) were 101 and < or = 24.8 micrograms/L, respectively. The LOEC for reduced reproduction in medaka was at least four-fold greater than that required to elicit estogenic responses in male fish. Thus, we suggest that the estrogenic responses expressed in male fish are not responsible for the reduction in reproductive capacity.

In ovo nanoinjection of triclosan, diclofenac and carbamazepine affects embryonic development of medaka fish (Oryzias latipes)

We examined the toxicity of three pharmaceuticals and personal care products (PPCPs) - triclosan (TCS), diclofenac (DCF), and carbamazepine (CBMZ) - on embryonic development of Japanese medaka (Oryzias latipes) using in ovo nanoinjection. Medaka eggs (8h post-fertilization; late blastula stage) were injected with 0.5nL of triolein (vehicle control) or 0.5nL of PPCPs, using different doses of TCS (1, 5, or 9ng), DCF (1, 5, or 12ng), or CBMZ (1, 5, or 12ng) per egg in triolein, in addition to uninjected control. Following injection, we recorded survival, embryonic lesions, delay in embryonic development (eye, embryonic body and internal organs), heart beat rate, hatchability, and hatching time of embryos and upward swimming of larvae. As a result, injected PPCPs caused toxic responses to medaka embryos during embryonic development and around the day of hatching. Based on estimated EC(50) values of PPCPs doses on survival of injected embryos at hatching, TCS (at a dose of 4.2ngegg(-1)) was generally more toxic to medaka embryos, followed by DCF (6.0ngegg(-1)), and CBMZ (13.1ngegg(-1)). We conclude that the nanoinjection medaka embryos model is a valuable tool for analyzing the effects of chemicals on the development of fish embryos and feasibility of nanoinjecting PPCPs into small fish eggs perhaps mimicking early exposure resulting from oocyte uptake of contaminants from maternal extra gonadal tissues.

The effects of methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias latipes)

We examined the effects of methyltestosterone (MT) on the reproduction of medaka (Oryzias latipes). Six mating pairs of medaka were exposed for 3 weeks to MT at the measured concentrations of 22.5, 46.8, 88.1, 188, and 380 ng/L. The fecundity and fertility of paired medaka were significantly decreased in the 46.8, 88.1, 188, and 380 ng/L MT groups compared with those of controls. The gonadosomatic indices of female fish exposed to >or=46.8 ng/L were increased significantly. Gonadal histology showed degeneration of oocytes in the ovaries of fish in all MT treatment groups. Hepatic vitellogenin concentrations were significantly decreased in female fish treated with MT at 188 and 380 ng/L, and the hatchability and survival rate of the offspring were decreased in all MT treatment groups. These results clearly demonstrate that MT at >or=46.8 ng/L inhibited gonadal development and adversely affected the reproduction of medaka.

In situ on-line toxicity biomonitoring in water: Recent developments

In situ on-line biomonitoring is an emerging branch of aquatic biomonitoring. On-line biomonitoring systems use behavioral and/or physiological stress responses of caged test organisms exposed in situ either in a bypass system or directly in-stream. Sudden pollution waves are detected by several existing single-species on-line biomonitors, which until now have been placed mostly in streamside laboratories. However, recent achievements have been multispecies biomonitors, mobile biomonitors for direct in-stream use, development of new instruments, new methods for data analysis and alarm generation, biomonitors for use in soil and sediment, and scientific research supporting responses as seen in on-line biomonitors by linking them to other biological and ecological effects. Mobile on-line monitoring platforms containing an array of biomonitors, biosensors, and chemical monitoring equipment might be the future trend, especially in monitoring transboundary rivers at country borders as well as in coastal zones.

Alterations in social behavior of Japanese medaka (Oryzias latipes) in response to sublethal chlorpyrifos exposure.

The behavioral and biochemical responses of Japanese medaka (Oryzias latipes) to acute and subacute (sublethal) levels of chlorpyrifos were studied. In the acute exposure test, medaka were exposed to 0.018, 0.055, 0.166, or 0.500 mg L(-1) chlorpyrifos for 4 d. As a result, fish showed hypoactivity compared to the control (at 0.018, 0.055, and 0.166 mg L(-1), swimming speeds were 55.6%, 39.0%, and 27.3% those of the control), Brain acetylcholinesterase activity and swimming speed were significantly correlated. In the subacute toxicity test, medaka were exposed to 0.012 mg L(-1) chlorpyrifos (10% of LC(50)) for 8 d. On day 4, there were no significant differences in behavioral and biochemical endpoints in exposed fish as compared to the control. On day 8, exposed fish became hyperactive, and the swimming speed of the social group increased to 2 times that of the control, whereas acetylcholinesterase activity was decreased to 68% that of the control. In addition, fish exhibited significant alterations in social behavior (schooling duration increased to 2.6 times and solitary duration decreased to 28% that of the control). Our findings clearly demonstrate a subacute effect of chlorpyrifos on the social behavior of medaka, which may pose a risk at population level because of the disturbance of social behavior. In addition, the recorded behavioral alterations may provide a useful tool for assessing the toxicity of organophosphorous pesticides to aquatic organisms.

The application of entropy for detecting behavioral responses in Japanese medaka (Oryzias latipes) exposed to different toxicants

Growing concern over environmental pollution by chemicals that may directly or indirectly impact human and ecosystem health make it increasingly necessary to establish a biological early warning system to minimize these impacts. To achieve this, simple and reliable approaches and criteria for environmental risk assessment must be developed. We propose a new approach for detecting behavioral responses to toxicants in flowing water. We conducted a series of tests to determine toxic responses of Japanese medaka (Oryzias latipes) to lethal concentrations of potassium cyanide (KCN; 10 mg L−1) and phenol (25 mg L−1). Fish movements were tracked by three‐dimensional (3D) biomonitoring under flow‐through conditions. The 3D data were used to evaluate behavioral responses in terms of swimming speed, vertical position in the water, and entropies of these parameters. Fish exposed to KCN surfaced more frequently than unexposed fish, whereas fish exposed to phenol stayed near the bottom. Maximum swimming speed significantly increased after exposure to both test chemicals, whereas median swimming speed significantly decreased after exposure. Despite large variations in swimming behavior between individual fish, toxic behavioral responses could be identified by the significant decrease in the entropy of vertical position after 10–30 min of exposure to the test chemicals. We conclude that the use of entropy can contribute to the development of a biological early warning system for detecting toxicants at early stages of pollution. To use the present approach to detect lower concentrations of toxicants, further studies should be conducted using long‐term monitoring of fish exposed to sublethal concentrations.

Swimming behavioral toxicity in Japanese Medaka (Oryzias latipes) exposed to various chemicals for biological monitoring of water quality

We conducted a short-term behavioral toxicity test in medaka (Oryzias latipes). Fish were exposed to toxicants (potassium cyanide [1 or 5 mg/L], phenol [12.5 or 25 mg/L], fenitrothion [10 or 20 mg/L], or benthiocarb [10 or 20 mg/L]), and swimming behavior was recorded and evaluated for 1 h. The medaka were placed in an exposure chamber with a continuous flow-through system. Two cameras tracked the fish at positions to the front and side of the test chamber, and images from the cameras were used to calculate the positions of the medaka in three dimensions (3D); the 3D data were processed by computer and analyzed as swimming activity (swimming speed and surfacing behavior).The swimming behavior of medaka was affected by exposure to toxic chemicals. The frequency of fast swimming was remarkably increased in medaka treated with potassium cyanide (5 mg/L), phenol (25 mg/L), or fenitrothion (10 or 20 mg/L). An increase in the time spent close to the water surface was observed in fish exposed to potassium cyanide (1 or 5 mg/L), fenitrothion (10 or 20 mg/L), or benthiocarb (20 mg/L). We concluded that pollution of water with these toxic chemicals at high concentrations can be detected by monitoring the swimming behavior of medaka for 1 h.