Can the choice of endpoint lead to contradictory results of mixture-toxicity experiments?

Theoretically, the effect of two independently acting compounds in a mixture will depend on the slope of the doseresponse curves of the individual compounds if evaluated in relation to the model of concentration addition (CA). In the present study, we explored development of the shape of the dose-response relationship for four different recommended endpoints (surface area, frond number, fresh weight-specific, and dry weight-specific relative growth rates [RGR(A), RGR(F), RG(FW), and RGR(DW), respectively]) and for two differently acting herbicides (metsulfuron-methyl and terbuthylazine) over time (3-15 d) on the standard test plant Lemna minor to identify endpoints and experiment times for which predictions of independent action (IA) would depart the most from those of CA. After a test time of 6 d, predictions of IA based on RGR(A) and RGR(FW) showed antagonism in relation to CA. Based on RGR(DW), synergy was predicted, whereas IA based on RGR(F) was indistinguishable from CA. To test the prediction of choice of endpoint giving different results in mixture-toxicity experiments, three endpoints and six combinations of independently acting herbicides were evaluated using isobolograms. The experiments showed that in four of six herbicide combinations, different conclusions were reached depending on endpoint. The contradictory isoboles did not follow the theory of IA and, therefore, are more likely to be related to differences in susceptibility of the physiological processes affecting each endpoint than to the shape of the dose-response curve.