Discriminating between intra‐ and extracellular metals using chemical extractions: an update on the case of iron

Iron influences the climate system by limiting primary productivity. It is therefore essential to accurately measure the iron fraction associated with phytoplankton in aquatic systems. A washing procedure using EDTA, being efficient for numerous trace metals, is not strong enough to remove iron adsorbed to the surface of microorganisms. Stronger washing solutions are used for iron, but these have only been assessed for a marine diatom. This study assesses the applicability of the oxalate washing procedure for both freshand seawater aquatic systems. We assessed iron solubilization as a result of oxalate washing in both synthetic and natural freshwater and seawater, and we tested it on several model phytoplankton and natural assemblages from Lake Champlain, the Southern Ocean, and the Derwent River estuary. We report the effects of the oxalate solution contact time, concentration, and amendment. Our study shows that 20-min washing provides an efficient measurement of the intracellular phytoplanktonic pool of iron in both freshwater and seawater. The direct amendment of oxalate in the experimental solution presents many advantages that are critical for the measurement of size-fractionated particulate iron. These include fine control of bioaccumulation termination, a significant gain in time, and homogeneity of the washing treatment. *Corresponding author: E-mail: christel.hassler@csiro.au Acknowledgments C. Hassler was supported by the National Science Foundation project (Chem Oce no. 0327730 of M.R. Twiss of Clarkson University, NY, USA, and R.M.L. McKay and G.S. Bullerjahn of Bowling Green State University, OH, USA); the Swiss National Science Foundation (no. PBGEA-104637); and a CSIRO postdoctoral fellowship. The Belgian Federal Science Policy Office (contract SD/CA/03A) and Belgian French Community (ARC contract no. 2/07-287) gave support to V. Schoemann. This study is also a contribution to the SOLAS international research initiative and the European Network of Excellence EUROCEANS (contract no. 511106-2). The authors thank J. de Jong and S.J. Phipps for useful comments on this manuscript; S.M. Havens, J. O’Sullivan, and R. Watson for technical assistance; L. Clementson and I. Jameson for pigments analysis and microscopic observations; M.R. Twiss, A.R. Bowie, and T. Remenyi for water sampling; and W. Howard, A.R. Bowie, B. Griffiths, and E.C.V. Butler for giving us the opportunity to join the SAZ-SENSE voyage on board the RV Aurora Australis. Limnol. Oceanogr.: Methods 7, 2009, 479–489 © 2009, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS Iron has two redox states: Fe(III) and Fe(II). Whereas Fe(II) is highly unstable under the oxic conditions prevailing in surface waters, Fe(III) is poorly soluble. Iron is mainly bound to organic ligands that increase its solubility and residence time in surface waters (Turner and Hunter 2001, Maranger and Pullin 2003, Chen et al. 2004). The affinity constant for organic ligands is typically several orders of magnitude higher for Fe(III) than for Fe(II) (Morel and Hering 1993, Turner and Hunter 2001). Phytoplankton are known to take advantage of these properties, using highly specific uptake transport systems to access Fe (e.g., Shaked et al. 2005). Generally, diatoms are known to use a surface reductase that reduces Fe(III) that is organically bound, thus favoring the subsequent dissociation of the Fe(II) complex and Fe internalization (Shaked et al. 2005). Under Fe-deficient conditions, cyanobacteria secrete and/or recognize siderophores (Wilhelm 1995) that efficiently compete with the organically bound Fe(III) present in the surrounding water (log K′Fe′L 11.5–13.9 in HNLC seawater; Boye et al. 2001, Croot et al. 2004). This is due to a high affinity constant of siderophores for Fe(III) (e.g., log K′Fe′L 11.8–16.5 for the hydroxamate siderophore desferrioxamine B in seawater; Maldonado et al. 2005). The Fe(III)–siderophore complex is then specifically recognized by surface receptors (e.g., Völker and Wolf-Gladrow 1999). Such high-affinity transport systems imply that Fe will be strongly bound to transporters; therefore strong washing agents are required to dissociate the Fe complex on the biological surface. Despite the fact that the EDTA washing technique is effective at reporting the intracellular pool of numerous trace metals (see Hassler et al. 2004), the technique is not strong enough to remove Fe adsorbed to the surface of microorganisms (Tang and Morel 2006). To date, two different washing solutions have been reported as being successful in discriminating total particulate and intracellular Fe in seawater (Hudson and Morel 1989, Tovar-Sanchez et al. 2003): Ti-citrateEDTA (referred to as Ti-EDTA here) and oxalate-citrate-EDTA (referred to as oxalate here). Ti-EDTA is a strong washing agent (reducing and chelating) requiring only a 2-min contact time to efficiently remove extracellular biogenic, organic, and inorganic colloidal Fe (Hudson and Morel 1989; Tang and Morel 2006); however, the reagents used to prepare this solution cause significant Fe contamination (ca. 7000 nM; Tang and Morel 2006). This is problematic when assessing particulate Fe in natural waters, where dissolved Fe concentrations can be as low as 5 nM in freshwater (e.g., Lake Superior; Sterner et al. 2004) and <0.1 nM in seawater (e.g., HNLC Southern Ocean; de Baar and de Jong 2001). In addition, this washing solution has to be freshly prepared, as it is stable for only 24 h (Hudson and Morel 1989). The oxalate washing solution relies mainly on synergistic surface chelation and light-induced dissolution processes (Sulzberger et al. 1989, Borer et al. 2005). It has the advantages of exhibiting lower Fe background contamination (500 and 17 nM for the basic and trace metal clean solutions, respectively; Tovar-Sanchez et al. 2003) and of being stable for 8 weeks. Recently, Tang and Morel (2006) further optimized the oxalate washing procedure; they showed that sodium citrate was not required, that Fe washing efficiency was higher at pH 7 than at pH 8 (as initially recommended by TovarSanchez et al. 2003), and that an increase in the contact time with the oxalate solution from 5 min (Tovar-Sanchez et al. 2003) to 10 min (two successive 5-min washes) was required to measure accurate intracellular Fe in T. weissfloggi compared with Ti-EDTA washing. Following a 5to 10-min oxalate wash, no effect on cell integrity could be seen for a wide range of eukaryotic and prokaryotic plankton (TovarSanchez et al. 2003) or on the growth rate (Tovar-Sanchez et al. 2003) or membrane integrity (Tang and Morel 2006) of Thalassiossira weissflogii. To date, the use of the oxalate wash has been limited because optimal contact time with the oxalate solution has not been clearly defined, and it was tested only on a single marine diatom in seawater under chemical conditions failing to reflect natural conditions. Tang and Morel (2006) reported a significant decrease of intracellular Fe when contact time was prolonged from 10 to 20 min. The oxalate Fe dissolution efficiency was studied only for Thalassiossira weissflogii with 84–3340 nM Fe buffered by excess EDTA (Tang and Morel 2006) or shortterm pulse exposure to high Fe concentrations, ca. 100–1000 nM range (Tovar-Sanchez et al. 2003). Because the ability to efficiently solubilize particulate/colloidal Fe will depend on Fe chemistry (e.g., colloidal forms; Sulzberger and Laubscher 1995) and also Fe biological ligands, it is essential to study dissolution efficiency for varied organisms and environments under natural conditions. This is critical for determining the extent of Fe limitation in both freshand seawater systems. In this study, we assessed the washing efficiency of the oxalate solution in both synthetic and natural waters for freshand seawater model phytoplankton, as well as for natural assemblages. We also report the effects of oxalate concentration and contact time. Therefore, this study represents an update of Hassler et al. (2004), as well as a further optimization of the oxalate washing procedure given in Tovar-Sanchez et al. (2003) and Tang and Morel (2006). A 20-min contact time with the oxalate solution was efficient in discriminating extracellular from intracellular iron for both freshwater and marine plankton. Given that a 20-min total contact time implies a significant increase of experimental time, constraining the amount of data that can be gathered, especially during field expeditions, an alternate direct dilution of the oxalate solution (1:10 vol:vol) in the sample before the filtration was successfully used to measure intracellular iron, thus providing a significant gain of time during experimentations. This study describes a standard procedure that can be successfully used on natural (and culture) marine and freshwater plankton, representing a critical step toward the determination of the extent of Fe limitation in aquatic systems. Hassler and Schoemann Intracellular iron in aquatic systems