Measuring total dissolved Fe concentrations in phytoplankton cultures in the presence of synthetic and organic ligands using a modified ferrozine method

Low concentrations and complex speciation can present major challenges for measuring Fe in natural waters easily and accurately. This study describes an optimized ferrozine method for measuring total dissolved Fe in the nanomolar range in seawater samples containing various concentrations of organic matter, as well as the commercially available Fe ligands ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFB), which are widely used in phytoplankton cultures. The method involves sample acidification to liberate Fe from strong complexes and/or colloids, followed by reduction of Fe(III) to Fe(II) using sulfite, and finally the measurement of Fe(II)-ferrozine complexes using long optical path spectrophotometry. The performance of each step was improved in such a way as to achieve the most efficient dissociation rate of Fe from the given ligands in the least possible time, regardless of their type or concentration. Storage of samples at pH 1 for 15 days enabled full recovery of Fe in the presence of 50 μmol/L DFB, while the duration required for full Fe recovery in the presence of the same concentration of EDTA was only about 1 h. Addition of ferrozine after a brief (about 15 min) reduction step with subsequent incubation for 24 h resulted in stable color development in the samples over time. The approach was successfully applied to determine total dissolved Fe in samples from coastal waters containing 10.5 μmol/L of particulate organic carbon (POC), and also in samples containing 2 μmol/L EDTA and 800 μmol/L of POC collected from laboratory cultures of the marine cyanobacterium, Trichodesmium erythraeum.