The revised default guideline value for iron in fresh waters was derived from chronic toxicity data, and resulted in protective concentrations of total iron above its solubility limit. This indicated that colloidal forms contributed to the observed toxicity response. However, in nature these forms coexist with other iron mineral species of lower reactivity (e.g. iron oxides) which would be recovered as part of a total iron measurement. Previous evidence showed that a pH 2 nitric acid extraction was able to recover the colloidal fraction in synthetic freshwater samples without interference from low reactivity iron particles also present in the sample matrix (Balsamo Crespo et al. 2022). The aim of this new work is to assess whether the pH 2 extraction is an effective method to assess iron bioavailability in fresh waters. In this presentation, considerations in the development of the experimental setup will be discussed, particularly those regarding aggregation and adsorption which prevent particulates from remaining in suspension between medium renewals. A range of variations for a 7-day Ceriodaphnia dubia reproduction test was tested, including agitation, additional medium renewal, and different test chamber volumes. Continuous agitation caused inhibition of reproduction below the minimum acceptable rate (<15 neonates/adult). A design with intermittent agitation pulses was found to be satisfactory with reproduction rates above 15 neonates/adult. However, in the presence of suspended solids, a double feeding rate was needed to achieve reproduction rates above the minimum acceptable threshold. Additional details about other test variants will be discussed during the presentation. Iron exposure concentrations are measured according to three operationally defined fractions: filtered (0.45 μm), pH 2-extractable, and total iron. Two extraction times for the pH 2 method are being assessed (0.5 and 16 h), and results fitted to dose-response models to determine which operationally defined fraction best measures the bioavailable fraction.