Synthetic chemicals are frequently detected in water bodies, and their concentrations vary over time. Water monitoring programs typically employ either a sequence of grab samples or continuous sampling, followed by chemical analysis. Continuous time-proportional sampling yields the time-weighted average concentration, which is taken as proxy for the real, time-variable exposure. However, we do not know how much the toxicity of the average concentration differs from the toxicity of the corresponding fluctuating exposure profile. We used toxicokinetic–toxicodynamic models (invertebrates, fish) and population growth models (algae, duckweed) to calculate the margin of safety in moving time windows across measured aquatic concentration time series (7 pesticides) in 5 streams. A longer sampling period (14 d) for time-proportional sampling leads to more deviations from the real chemical stress than shorter sampling durations (3 d). The associated error is a factor of 4 or less in the margin of safety value toward underestimating and an error of factor 9 toward overestimating chemical stress in the most toxic time windows. Under- and overestimations occur with approximate equal frequency and are very small compared with the overall variation, which ranged from 0.027 to 2.4 × 10¹⁰ (margin of safety values). We conclude that continuous, time-proportional sampling for a period of 3 and 14 d for acute and chronic assessment, respectively, yields sufficiently accurate average concentrations to assess ecotoxicological effects.