Estrogenic potencies of four herbicides (triclopyr, 2,4-dichlorophenoxyacetic acid (2,4-D), diquat dibromide, glyphosate), two alkylphenol ethoxylate-containing surfactants (R-11 and Target Prospreader Activator (TPA)), and the binary mixture of surfactants with the herbicides were evaluated using an in vivo rainbow trout vitellogenin assay. Juvenile rainbow trout exposed to 2,4-D (1.64 mg/l) for 7 days had a 93-fold increase in plasma vitellogenin (Vtg) levels compared with untreated fish, while rainbow trout exposed to other pesticides alone did not show elevated vitellogenin levels compared to the control fish. When combined with surfactants, trends indicated enhanced estrogenicity for all combinations, but only 2,4-D and triclopyr caused significant induction of Vtg. Concentration-response studies demonstrated that the lowest observed effect concentrations (LOECs) for 2,4-D and triclopyr were 0.164 mg/l and 1 mg/l, respectively. In terms of measured 4-nonylphenol (4-NP), the LOECs of R-11 and TPA were 20 micro/l and 9.5 microg/l, respectively. Binary mixtures of TPA and 2,4-D showed a greater than additive estrogenic response at the lowest concentrations tested, but a less than additive response at the highest combined concentrations. Binary mixtures of TPA with triclopyr also caused greater than additive Vtg responses in two middle concentrations when compared to TPA or triclopyr alone. When trout were exposed to water collected from a site where triclopyr was used in combination with TPA, a concentration-dependent increase in Vtg expression was observed. Measured values of 4-NP were 3.7 microg/l, and triclopyr concentrations were below detection (<5 ng/l). Estradiol equivalents (EEQs) of the lake water were calculated from an estradiol concentration-response curve and were similar (8.5 +/- 7.7 ng/l) to the mean values for the combined triclopyr + TPA treatments (9.9-12.2 ng/l) in the laboratory, suggesting the estrogenicity of the water may have been due to the treatment. These results demonstrated the binary mixture of alkylphenol ethoxylate-containing surfactants with two aquatic pesticides possessed greater than additive estrogenic responses in fish under laboratory conditions and in a field setting.