Agricultural chemicals end up in the environment as complex mixtures and it is their combinatorial effects that need to be evaluated, rather than the traditional single effect of the active ingredients. This study emphasises effects-directed analyses (androgen receptor (AR) activity) of such environmentally relevant mixtures. Soil, where glyphosate and 2,4-dichloro-phenoxyacetic acid (2,4-D) were sprayed on Bt maize, were extracted with rainwater. This allowed to test the bio-available fraction. AR effects were measured with an in vitro reporter-gene assay using MDA-kb2 cells. The cells were exposed to: single active ingredients; formulations; environmentally relevant concentrations of the active ingredients and formulations; as well as rainwater extracts. The AR was activated by rainwater extracts from soil that received a pre-and post-emergent Roundup application. The testosterone equivalents (TTEQs) derived from AR activation exceeded international drinking water trigger values. We conclude that (i) rainwater run-off from maize sprayed with Roundup and 2,4-D contained androgen active substances and (ii) the chronic exposure to this water may cause endocrine disrupting effects in humans and aquatic life which emphasise the need for intensified monitoring of environmental water resources. Copyright © 2019 Elsevier B.V. All rights reserved.