Wastewater effluents have been documented as major contributors of hormone endocrine disrupting compounds (EDCs) in to the aquatic ecosystem. The need for rapid, simple and cost effective methods to detect these EDCs has increased. The use of Radio-immunoassays (RIA) were assessed to determine the fate of estradiol in a laboratory batch test and the three natural estrogens (estrone (E1), estradiol (E2) and estriol (E3)) in wastewater treatment plants (WWTPs) with different types of configurations. Precision of the RIAs were done using intra-assay and inter-assay validations. The E2 intra-assay variation was <8% and inter-assay variation was <11% for standards 1 to 6. E1 RIA showed less than 8% for both the intra-assay and inter-assay variations. E3 RIA showed extremely good variations with both the intra and inter-assay variations being below <8% for all standards. The lab scale investigation showed a 94% reduction in E2 after 5 h and after 10 h both E2 and E1 were no longer detected. The simple activated sludge process, the biological nutrient removal (BNR) activated sludge process and the oxidation pond had final effluent concentrations of 10.75, 5.96 and 25.48 pg E2/mL respectively; 20.80, 9.30 and 46.55 pg E1/mL, respectively, and 0.12, 0.07 and 0.17 ng E3/mL, respectively. Thus far findings indicated that the RIA can be employed as a rapid technique for detection of natural estrogens in water. Results indicate that these potential problematic hormone EDCs are still present in final wastewater effluents that are discharged in to South African aquatic sources.