Engineered nanoparticles (ENPs) will be released to the environment during use or following the disposal of ENP-containing products and concerns have been raised over the risks of ENPs to the environment. Many studies have explored the toxicity of ENPs to aquatic organisms but these studies have usually been performed with little understanding of the ENPs' behaviour in the test media and the relationship between behaviour in the media to behaviour in natural waters. This study evaluated and compared the aggregation behaviour of four model gold nanoparticle (NP) types (coated with neutral, negative, positive and amphoteric cappings) in standard ecotoxicity test media and natural waters. The effects of humic acid (HA) and test organisms on aggregation were also investigated. In standard media, positive and neutral NPs were stable, whereas amphoteric and negative NPs generally showed substantial aggregation. In natural waters, amphoteric NPs were generally found to be stable, neutral and positive NPs showed substantial aggregation while negative NPs were stable in some waters and unstable in others. HA addition stabilised the amphoteric NPs, destabilised the positive NPs and had no effect on stability of negative NPs. The presence of invertebrates generally lowered the degree of particle aggregation while macrophytes had no effect. Given the dramatically different behaviours of ENPs in various standard media and natural waters, current regulatory testing may either under- or overestimate the toxicity of nanomaterials to aquatic organisms. Therefore, there is a pressing need to employ ecotoxicity media which better represent the behaviour of ENPs in natural system.