Due to their unique properties, nanomaterials (NMs) are being incorporated in several applications including consumer products, electronics, pesticides and the pharmaceutical industry. As such, the rapid development and large-scale production of NMs has in‐ spired concerns regarding their environmental health risks. In order to address these con‐ cerns, there has been a rapid development in the methods of toxicity testing of NMs, specifically in aquatic organisms. Understanding the unique properties of nanoscale ma‐ terials has proven to be a particular important aspect of their toxicity. Properties such as surface area, surface coating, surface charge, particle reactivity, aggregation and dissolu‐ tion may affect cellular uptake, in vivo reactivity and distribution across tissues. The be‐ haviour of NPs is influenced by both the inherent properties of the NP as well as environmental properties (such as temperature, pH, ionic strength, salinity, organic mat‐ ter). As such, this chapter describes methodologies of NM characterization in exposure media and NM in vivo toxicity experimental procedures under variable environmental conditions (with special emphasis on temperature).