Aquatic herbicides with active ingredient 2,4-dichlorophenoxyacteic acid (2,4-D) are commonly used to control and combat aquatic non-native species that cause detrimental impacts including habitat destruction, strained resources among biota, and biodiversity loss. While many (eco)toxicology studies are performed in the laboratory under highly controlled circumstances, these studies may disregard the nuances and disorder that come with the complexity of natural aquatic ecosystems. Therefore, we conducted a series of laboratory experiments using laboratory system water, different lake waters, and different water parameters to determine the effects of ecologically relevant concentrations of 2,4-D (0.00–4.00 ppm a.e.) on the development and survival of two freshwater game species (Sander vitreus and Esox lucius). For 2,4-D exposures using different water sources, there were significant main effects of 2,4-D concentration and water source on walleye embryo and larval survival, however, there was no significant interaction between 2,4-D exposure and water source. For 2,4-D exposures and pH (5–9 pH), there were significant main effects of 2,4-D concentration and pH on walleye and northern pike embryo survival and a significant interaction between 2,4-D exposure and pH. Our results indicate that 2,4-D exposures in controlled laboratory system water can predict similar outcomes as 2,4-D exposures in natural lake water. Moreover, individual water parameters, such as pH, play a significant role in the toxicity of 2,4-D. Taken together, these results suggest that highly controlled laboratory studies are a useful tool for predicting impacts on survival of non-target fish in natural waters, but it is crucial for management agencies to consider individual water sources and specific lake water parameters in herbicide risk assessments to minimize the impacts to non-target organism.