In the present study, the authors investigated and defined development stages of Daphnia magna eggs that could be potential endpoints for sublethal toxicity tests with in vitro parthenogenetic egg cultures. Such an "egg test" could be a cost-effective alternative to the classic 21-day test with D. magna. Three main stages of embryonic development were considered: release of the egg external membrane, release of the internal membrane, and separation of the caudal spine. The first embryonic stage was attained approximately 30 h after transfer of eggs from ovaries to the brood chamber (considered as time zero), the second stage at 48 h, and the third stage at 68 h. Embryonic development was considered completed with the caudal spine separation. Thereafter, juveniles were able to swim in the water column. Egg mortality, duration of each egg stage, egg diameter, and egg abnormalities were investigated as potential endpoints. In vitro tests were carried out with several toxicants (DBS, 3,4-DCA, cadmium, and copper) and with acid mine drainage, sensitivity generally being higher than with the accepted chronic 21-day test with D. magna.