Multigenerational exposure of Daphnia magna to tetracycline was carried out through four consecutive generations. The effects of tetracycline on the survival, reproduction and growth of D. magna were assessed over a period of 21 days per generation. The evaluated endpoints were overall fecundity (total mean neonates per female over 21 days), time to first reproduction, longevity, molting number and somatic growth, such as body weight and body length. Using the results obtained for reproduction and survival rates, the intrinsic population growth rate (PGR) was calculated and compared throughout the generations. Reproductive impairment was observed in all generations and magnified with increasing generation number. The value of no observed effect concentration on D. magna also markedly decreased with increasing generation number. This subsequently resulted in a reduction of the PGR value. In addition, the PGR value was decreased with increasing exposure concentration, decreasing by about 30 and 60% at 0.1 and 5.0 mg/L tetracycline, respectively. On the other hands, somatic growth increased with increasing generation number, because the remaining input energy from the reduced reproduction was mainly used for body maintenance. As a result, the somatic growth and reproduction showed reversed trends on continuous exposure of tetracycline to four generations. In conclusion, multigenerational exposure of tetracycline can induce overall responses on reproduction and the somatic growth of D. magna. Moreover, the PGR value of D. magna exposed to tetracycline was reduced with increasing generation number; thereby, inhibiting the long term propagation of D. magna.

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The object of this study was to improve pre-concentration techniques using hollow fibre protected-liquid phase micro-extraction (LPME) for its application on biotic samples and limited environmental samples provided in small volumes. Five parameters, including extraction time, type of solvent, extents of agitation, fibre length and salt content of HF-LPME were optimised for the yeast twohybrid assay and gas chromatography-mass spectrometer (GC/MS) chemical analysis. The results of chemical analysis were compared to the results of a bioassay using the yeast two-hybrid system to analyse endocrine-disrupting chemicals (EDCs). For the yeast two-hybrid assay, the optimised parameters comprised of an extraction time of 40 min, toluene as the extraction solvent, a stirring rate of 500 rpm, a fibre length of 2.0 cm and a salt concentration of 20% (w/v). For GC/MS analysis, HF-LPME conditions were optimised with an extraction time of 40 min, toluene: dichloromethane at a ratio of 1 : 1 (v : v) as the extraction solvent, a stirring rate of 700 rpm, a fibre length of 2.0 cm and a salt concentration of 30% (w/v). The results indicated that fibre length, extraction time, and degree of agitation were major contributing parameters for HF-LPME in EDCs analysis. Consequently, bioassays such as yeast two-hybrid assay could be easily, rapidly, and inexpensively conducted with small volume of sample using HF-LPME for the measurement of oestrogenic activity.