Vitellogenin protein (Vtg) in Oreochromis niloticus plasma has been indirectly quantified through protein-bound phosphate groups also known as alkali-labile phosphates (ALP) using a recently modified method. Such method as described in the literature was originally applied to Crucian carp and resulted in lower detection limits (3.2 mu g PO(4)(3-)per mL). In this study, O. niloticus males were exposed to intermittent doses of oestrogens for 15 days using different concentrations (converted to loads) of 17 alpha-ethinyloestradiol (EE2) (two different aquarium volumes), oestrone (E1) and 17 beta-oestradiol (E2) individually and in combination (1:1:1). The induction of physiologic and genotoxic effects in erythrocytes was investigated. For the tested oestrogen (EE2), load proved to be more relevant than concentration in determining the oestrogenicity. O. niloticus males proved to have lower ALP baseline (4.11 mu g PO43-/mL plasma, IQ(25)=3.38; IQ(75)=5.18) than other fish species, including Crucian carp, which makes it suitable for oestrogenicity detection in water. Exposure to E2, EE2 separately and in combination (1:1:1) all induced significant increases in the ALP levels at loads >= 0.72 mu g/fish. This load was three times lower than the E1 load required to increase ALP (>= 2.2 mu g/fish). All oestrogens with loads >= 0.072 mu g/fish caused significant increase in micronucleus frequency (>= 2 parts per thousand) compared with the control (0.1 +/- 0.4 parts per thousand). The study highlighted the importance of taking into account not only concentration and dose regime but also the mass load and therefore, the volume used in the experimental units, which is rarely addressed in ecotoxicity assays. Considering the good sensitivity of O. niloticus exposed to relatively low concentrations of oestrogens, the combination of the ALP method with auxiliary biomarkers (particularly micronucleus) can be used as a protocol for oestrogenicity and genotoxicity detection in different contaminated waters as part of water environmental monitoring programmes.