Many of the herbicides used in tropical agriculture have limited ecotoxicology data available for setting guidelines for ecosystem protection. This lack of data reduces the capacity to assess the risk of herbicide run-off to non-target species in freshwater systems. The National Environmental Science Program (NESP) program Project 3.1.5 - ”Ecotoxicology of pesticides on the Great Barrier Reef for guideline development and risk assessment” supported research into assessing toxicity of ten emerging or alternate herbicides to seven autotrophic species under tropical freshwater conditions. The selection of herbicides for the project were determined from data-gap assessments by the Queensland Department of Environment & Science (DES) from a priority list of herbicides used in Great Barrier Reef catchments. Microalgae, cyanobacteria, aquatic macrophytes and a freshwater fern were used to measure a range of sub-lethal responses to herbicide exposure. Response measures included photosynthetic efficiency, growth rates, changes in biomass and surface area. Toxicity among herbicides varied and was dependent on measured endpoint, species and herbicide mode of action. Some interesting responses were observed in different taxonomic groups. As part of the program, developments and improvements were made to some endpoint assessments to reduce operator bias and automate assessment processes. The outcomes from this project are now being used to develop water quality guidelines for these herbicides.