Uptake of ⁶⁴Cu−Oxine by Marine Phytoplankton

Abstract

Short-term uptake experiments using five phytoplankton species (Synechococcus clone DC2, Amphidinium carterae, Chrysochromulina polylepis, Ditylum brightwelli, and Prorocentrum micans) demonstrated rapid uptake of the lipophilic complex 64 Cu-oxine, presumably by diffusion of the complex across the plasma membrane. This passive uptake mechanism was extremely rapid and significantly faster than facilitated uptake by the free metal ion. Measured values of the observed permeability, P obs , ranged from 0.55 to 18.6 × 10 -4 cm s -1 , showing only small differences between the various algal species. Removal rate constants, k bio , varied much more widely, 0.009-570 × 10 -9 L cell -1 h -1 , between the algae, indicating the influence of surface area on the uptake kinetics. Maximum internal Cu levels were reached after approximately 2 h, showing that a major limiting factor in the uptake of Cu from Cuoxine is the concentration of intracellular Cu binding sites. IntroductionIt is now well established that low molecular weight lipophilic organic complexes can be taken up by microorganisms via passive diffusion across the plasma membrane of the cell (1-3). The presence of a lipid bilayer acts as a barrier for aqueous hydrophilic molecules, but small lipophilic molecules can readily diffuse through the membrane. The diffusion of lipophilic organic metal complexes across a cell membrane is a process that has, until recently, been neglected in studies of the uptake mechanisms for metals by phytoplankton. The free metal ion model of metal uptake has been used extensively for assessing the criteria of metal bioavailability and toxicity to phytoplankton over the past 20 years (4-6). In the free ion model, it is assumed that an equilibrium is established between the inorganic metal species in solution and the specific cell surface transport sites. Subsequently, metal complexed to the surface sites is transported across the cell membrane and into the cytosol. Only the labile inorganic species are considered to be directly utilized by the cell, with the metals chelated to organic ligands being essentially inert and instead acting as a buffer for the free metal ion in solution. The early laboratory studies that addressed the free ion mechanism used organic ligands such as ethylenediamine-tetraacetate (EDTA 4-) or nitriliotriacetate (NTA 3-); these form low molecular weight anionic hydrophilic complexes such as CuEDTA 2-and CuNTA -.In contrast to uptake via a free metal ion mechanism, only a few studies have shown that lipophilic organic metal complexes can be directly assimilated by microorganisms (see review by Campbell (7)). Carlson-Ekvall and Morrison (8) showed that the Cu toxicity of sewage sludge to the bacterium Photobacterium phosphoreum was greatly enhanced in the presence of organic complexes such as oxine that could form lipid-soluble Cu complexes. Similarly Florence and co-workers showed that the complex Cu(Ox)2 0 is toxic to the marine diatom Nitzchia closterium (9-11). Recently it has also been shown in work by Phinney...

Uptake and efflux of 64Cu by the marine cyanobacterium Synechococcus (WH7803)

“…In the present study, we assumed that Cu uptake was related to the free copper concentration, although direct uptake of lipophilic copper complexes is also possible (Croot et al 1999). Observed Cu uptake rates were similar to that predicted by the diffusion of free or weakly complexed Cu to the cell (Table 1), indicating the validity of our assump-Uptake and efflux of Cu by Synechococcus tions.…”

“…Because the K S values are similar, it may indicate a similar transport pathway for Cu in both the cyanobacterium and the dinoflagellate. In our earlier work with 64 Cu (Croot et al 1999), we found a linear relationship between uptake rate and phytoplankton surface area, which might indicate similarity in the uptake systems. Alternatively, this linear relationship may simply reflect the convergent evolution of kinetic properties in response to the same selective pressures.…”

“…We do not believe that such phytoplankton uptake rates are supported or representative of ambient natural seawater. Because the concentration of lipophilic Cu complexes, which could pass directly through the cell membrane (Croot et al 1999) at the diffusion rate, are typically only a small fraction of the total Cu-binding ligands present in seawater (Donat et al 1986;Yoon et al 1999), we reject this possible explanation of the high uptake rates. Application of the data of Vasconcelos and Leal (2001) to phytoplankton in ambient seawater should therefore be treated with caution.…”

“…For copper, the radioisotopes are difficult to prepare ( 67 Cu) or have relatively short half-lives ( 64 Cu). We recently used 64 Cu to investigate the uptake of lipophilic 8-hydroxyquinoline (oxine) complexes by five phytoplankton species (Croot et al 1999). This work showed great potential for using 64 Cu for short-term laboratory uptake experiments examining the mechanisms and processes involved in Cu regulation by phytoplankton.…”

“…In the present study, we examined the kinetics of Cu uptake and how internal Cu concentrations are regulated in the Cu-sensitive cyanobacterium Synechococcus. The present work is part of an overall project to examine the kinetics of processes by which Cu speciation in seawater may be affected by phytoplankton responses to toxicity (Croot et al 1999(Croot et al , 2002. .)…”