Yohei Shimasaki

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.

Two-generation reproductive toxicity study of tributyltin chloride in female rats

A two-generation reproductive toxicity study of the effects of tributyltin chloride (TBTCl) was conducted in female rats using dietary concentrations of 5, 25, and 125 ppm TBTCl. Reproductive outcomes of dams (number and body weight of pups and the percentage of live pups) and the growth of female pups (the day of eye opening and body weight gain) were significantly decreased in the 125 ppm TBTCl group. A delay in vaginal opening and impaired estrous cyclicity were also observed in the 125 ppm TBTCl group. However, an increase in anogenital distance was found in all TBTCl groups on postnatal d 1. A dose-effect relationship was observed in TBTCl-induced changes in anogenital distance. These results indicate that the whole-life exposure to TBTCl affects the sexual development and reproductive function of female rats. In addition, the TBTCl-induced increase in anogenital distance seems to suggest it may exert a masculinizing effect on female neonates. However, the concentrations of TBTCl used in this study are not environmentally relevant.

Early–life‐stage toxicity in offspring from exposed parent medaka, Oryzias latipes, to mixtures of tributyltin and polychlorinated biphenyls

The present study examined the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and mixtures of both chemicals on reproduction in Japanese medaka, Oryzias latipes. For 21 d we gave groups of medaka freeze-dried brine shrimp flakes contaminated with a mixture of either 0, 1, 5, or 25 microg TBT g(-1) plus 0 or 25 .micro PCBs g(-1). We measured the fecundity and fertility of the parent fish and assessed the deformity, hatchability, time-to-hatching, and swim-up failure rate of the next generation. Fertilization success in the third week of the administration period was significantly decreased by administration of 25 microg TBT g(-1) (77%) compared with the control group (87%). Both TBT and PCBs were transferred maternally into the eggs of the next generation, causing early life-stage toxicity. Administration of 1 microg TBT g (-1) was not toxic to embryological development, but abnormal eye development (i.e., small eyes or no eyes) occurred when TBT at the same concentration was mixed with PCBs (6.4%). Administration of TBT alone significantly decreased hatchability and increased swim-up failure, and administration of PCBs alone significantly increased time-to-hatching. Statistical analysis by two-way analysis of variance detected an interaction between TBT and PCBs in these three parameters. TBT induces abnormal development of the eyes, reduced hatchability, and increased swim-up failure, whereas PCBs delay time-to-hatching. Administration of mixtures of TBT and PCBs has more adverse effects on the developmental stage of medaka than does that of each chemical alone.

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.

Extracellular polysaccharide-protein complexes of a harmful alga mediate the allelopathic control it exerts within the phytoplankton community.

The goal of this study was to examine the significance of allelopathy by the raphidophyte Heterosigma akashiwo in a multispecies phytoplankton community in the field. Towards this aim, we sought allelochemicals of H. akashiwo, which had allelopathic effect both in laboratory experiments and in the field. As an initial step, we showed that the allelopathic effects of H. akashiwo filtrate were both species-specific and dependent upon the cell density of the target species. Secondly, we found for the first time that extracellular, high-molecular weight allelochemicals [that is, polysaccharide-protein complexes (APPCs)] were produced by a marine phytoplankton species, H. akashiwo. Thirdly, we indicated that the purified APPCs selectively inhibited the growth of the diatom Skeletonema costatum that is a major competitor of H. akashiwo, and thereby tended to promote the formation of monospecific H. akashiwo blooms. Furthermore, we demonstrated that the inhibitory effect of APPCs on the growth of the diatoms was determined by binding to the cell surface of the target species. Finally, we succeeded in the detection of APPCs in the field samples at concentrations exceeding their experimentally determined action threshold during the H. akashiwo bloom. Strategies for ecosystem control, including mitigation of harmful algal blooms (HABs), should take into account that red-tide organisms like H. akashiwo are already part of complex webs involving inter-specific allelopathic inhibition and ecosystem control during their dense blooms.

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.

Effects of polychlorinated biphenyls on the schooling behavior of Japanese medaka (Oryzias latipes)

The effects of polychlorinated biphenyls (PCBs; Kanechlor 400) on the schooling behavior of Japanese medaka (Oryzias latipes) were tested after feeding with various concentrations of PCBs (0, 1, 5, 25, and 125 microg/g). To test schooling, three PCB-exposed medaka and three untreated fish were placed in one chamber, and their swimming trajectories were recorded. Swimming velocity, turning angle, and nearest-neighbor distance (NND) were analyzed individually, and the polarization and expanse of each schooling group were analyzed. Fractal dimension analysis was performed for trajectory, swimming velocity, and turning angle. Six behaviors (school formed by six swimming medaka, school formed by six stationary medaka, school formed by three to five swimming medaka, school formed by three to five stationary medaka, swimming without schooling, and stationary without schooling) were quantified with time. Behavioral parameters were the same for PCB-exposed and unexposed medaka in the same schooling group. Swimming velocity decreased in a dose-dependent manner, and NND in the group exposed to 25 microg/g of PCBs was significantly longer (126%) than that in the controls. The fractal dimensions of trajectory and turning angle increased significantly in the highest PCB-exposure group, indicating that schools containing PCB-exposed individuals frequently changed direction. The PCBs shortened the time of school formed by six swimming fish, which was 23.7% of the control value in the group exposed to 25 microg/g of PCBs. The frequency of collisions between individuals was significantly greater in the highest-exposure group (31.3) than in the controls (13.3). Thus, PCB exposure influences the behavior of unexposed fish in the same school, consequently affecting schooling behavior.

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.

Effects of Light Quantity and Quality on the Growth of the HarmfulDinoflagellate, Cochlodinium polykrikoides Margalef (Dinophyceae)

해양의 기초생산자인 식물플랑크톤은 태양에너지를 이용 하여 무기물로부터 유기물을 합성한다. 식물플랑크톤의 광 합성과 성장은 빛의 성질에 따라 크게 영향을 받게 되며, 특 히 빛의 파장과 조성은 광합성 생물 및 비광합성 생물의 신 진대사에까지 영향을 미친다(Sanchez-Saavedra and Voltolina 1994). 특히, 와편모조류를 포함하는 식물성 편모 조류는 미약하지만 운동능력을 보유하고 있어, 광학적으로 성장할 수 있는 심도까지 내려가 저층에 풍부한 영양염을 섭 취하여 성장할 수 있다(Honjo et al. 1989). 따라서 보다 낮은 광 조건에서도 성장할 수 있는 종은 유영심도가 더욱 깊어져 다른 종보다 유리한 생존 전략을 가진다. 또한, 수심에 따라 빛의 파장은 크게 다르기 때문에 여러 파장대 빛을 이용할 수 있는 종이 보다 효율적으로 성장할 수 있다. 한편 1995년 이후 매년 남해안과 동해안에서 대규모로 적 조를 발생시켜 막대한 경제적 손실과 해양생태계의 황폐화 를 초래하고 있는 유해성 와편모조류(NFRDI 1999, 2000), Cochlodinium polykrikoides Margalef는 Caribbean Sea에 위치 하는 Puerto Rico에서 처음으로 명명되었다(Margalef 1961). 더욱이 C. polykrikoides에 의한 적조는 우리나라에만 국한되 지 않고, 2005년에는 일본 Tottori현(동해에 위치한 현)에서 이 종에 따른 적조로 대량의 어패류 폐사가 있었다. 이곳의 Algae Volume 21(3): 311-316, 2006

Effects of irradiance of various wavelengths from light-emitting diodes on the growth of the harmful dinoflagellate Heterocapsa circularisquama and the diatom Skeletonema costatum

We investigated the effects of specific light wavelengths from light-emitting diodes (LEDs) on the growth of the dinoflagellate Heterocapsa circularisquama, which kills bivalves, and the diatom Skeletonema costatum, which is an important food source for bivalves. Growth of H. circularisquama was obviously inhibited at 590 nm and a photon flux density less than 75 μmol quanta/m2/s. However, growth of S. costatum was not suppressed by irradiance from any LEDs tested from near-ultraviolet to near-infrared wavelengths at 75 μmol quanta/m2/s. The growth rate of H. circularisquama in an experimental treatment group with irradiance provided by both cool-white fluorescent lamps (12∶12 h L:D cycle) and a 590-nm LED (continuous irradiance) was 0.43/day. In the control group with irradiance provided only by cool-white fluorescent lamps (12∶12 h L:D cycle), the growth rate was 0.63/day, indicating that growth of H. circularisquama was suppressed by 590 nm (less than 75 μmol quanta/m2/s) irradiance from the LED and the continuous irradiance. The use of 590-nm LEDs in bivalve culture at irradiance levels less than 75 μmol quanta/m2/s might encourage the growth of the useful diatom S. costatum without stimulating growth of the harmful dinoflagellate H. circularisquama.