We evaluated differences in the sensitivity of Daphnia of different ages to an insecticide in acute toxicity tests. The sensitivity was higher in younger individuals and lower in older ones, and was correlated with body length. However, first- instar individuals < 12 h old were less sensitive than those 12 to 24 h old. This phenomenon seems to be related to the timing of molting. Our results suggest that the sensitivity of a Daphnia population to toxic chemicals depends on its life stage composition, which should be considered in evaluating the effects of chemicals on populations in the natural environment.

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In order to develop the acute toxicity test using marine zooplankton, applicability of Tigriopas japonicas (Copepoda, Harpacticoida) as test organisms was investigated from the view point of sensitivities to pollutants and the rearing procedures.
T. japonicus survived for 14 days under the experimental conditions feeding Tetraselmis tetrothele as diet, and nauplius larvae were spawned from the experimentally reared female adult.
The nauplius larvae used for the acute toxicity test were separated effectively from the female adults by filtration with the plankton net of 200 &mico;m mesh size. The nauplius larvae can swim actively in the filtered seawater during 24 hours from the collection without feeding. EC₅₀ based on the suppression of swimming activity changed by the variation of water temperature and salinity, and appropriate value was 20-25°C and 32-34, respectively. EC₅₀ values of PCP-Na and organotin compounds to T. japonicus were similar to those values of freshwater zooplankton Daphnia magna (Branchiopoda, Cladocera ), therefore T. japonicus was a sensitive to hazardous chemicals. From these results, it is considered that the nauplius of T. japonicus is appropriate test organisms for acute toxicity test. The procedure of acute toxicity test using T. japonicus was established by summarizing the results of present research.

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An enzyme-linked immunosorbent assay (ELISA) was developed to quantify hepatic vitellogenin (VTG) in medaka (Oryzias latipes). VTG quantification was performed by a direct sandwich method using specific monoclonal antibodies to medaka VTG and biotinylated polyclonal antibodies. The ELISA's performance was optimized and characterized. The assay's working range was 0.488 to 500 ng/mL; intra- and inter-assay variations were 3.0% to 7.1% (n=4) and 0.7% to 8.3% (n=3), respectively. Recovery and extraction studies with liver homogenate samples showed that this assay is well applicable to the determination of the hepatic VTG in medaka. An exposure study with 17β-estradiol demonstrated that this ELISA assay was available to quantify the VTG in the livers of individual medaka. These results indicate that the ELISA assay with liver homogenate samples is suitable for use as an in vivo screening assay for determination of the hepatic VTG in medaka exposed to xcno-estrogens.

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Males of the medaka (Oryzias latipes), FLF (female leucophore-free) strain in which genetic gender can be discerned even at the embryonic stages, were exposed to 17β-estradiol (E2) for 6 days, from the 3rd day after hatching, at concentrations ranging from 10 to 160 ng/L. Sex reversal rates were checked at the adult stage. The concentration that induced sex reversal in 50% of the male medaka was calculated to be 37 ng/L by a probit regression equation. Vitellogenin concentrations in the livers of adult males increased from 0.67 to 11.4 ng/mg wet weight (mean values) depending on the E2 exposure concentration. Vitellogenin concentrations in the livers of sex-reversed males (functional females) increased to levels between 1200 and 3600 ng/mg wet weight, irrespective of the E2 concentration.
The growth stage during which males were most vulnerable to sex reversal was determined by shifting the start of E2 exposure (200 ng/L, for 2 days) from the 1st day after hatching to the 11th day. Sex reversal rates in cohorts of 15 males exposed to E2 for 2 days between the 1st and 4th day after hatching was ca. 60 to 70%, and sex reversal rates among those exposed to E2 between the 3rd and 6th day was around 40%. However, with few exceptions, sex reversal was not observed among the males exposed to E2 for 2 days during the 6th to 13th day after hatching. The reproduction potential of males, as determined by fertilization rates of eggs produced by mated control females, was more than 90% and was not impaired regardless of whether the males were selected from cohorts in which ca. 70% or 40% sex reversal had occurred. On the other hand, the reproduction potential decreased to ca, 60% in the eggs produced by sex-reversed males (functional females) mated with control males.

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