Control of Cd Concentrations in a Coastal Diatom by Interactions

Pierre-Jean Superville , Anastasia Ivanovsky , Pratima Bhurtun , Jean Prygiel ... Travis Mellett , Matthew T. Brown , P. Dreux Chappell , Carolyn Duck...
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Environ. Sci. Technol. 1998, 32, 2961-2968

Control of Cd Concentrations in a Coastal Diatom by Interactions among Free Ionic Cd, Zn, and Mn in Seawater WILLIAM G. SUNDA* AND SUSAN A. HUNTSMAN Beaufort Laboratory, NMFS, NOAA, 101 Pivers Island Road, Beaufort, North Carolina 28516

concentrations ([Cd2+]) but are also inversely related to Mn and Zn ion concentrations ([Mn2+] and [Zn2+]) (2, 7). The repressive effect of Mn appears to be linked to the uptake of Cd by the cell’s Mn uptake system (8). Here we report the results of long-term culture experiments in trace metal ion buffer systems that investigate the relationships between Cd:C ratios in a coastal diatom Thalassiosira pseudonana and important independent and dependent controlling variables: free ion concentrations of Cd, Zn, and Mn; cellular zinc concentration; and specific growth rate. In addition, short-term experiments were conducted to examine the antagonistic effect of Zn on cellular Cd uptake rates.

Experimental Section Cadmium and phosphate concentrations in seawater are closely correlated, suggesting that Cd distributions, like those of PO4, are controlled by algal uptake and regeneration. But the factors controlling Cd levels in phytoplankton are poorly known. Experiments in metal ion buffer systems with a coastal diatom Thalassiosira pseudonana revealed that cellular Cd:C ratios within the Cd ion concentration ([Cd2+]) range (10-13 to 10-10 M) in seawater were generally proportional to [Cd2+] and inversely related to concentrations of Zn and Mn ions ([Zn2+] and [Mn2+]) and specific growth rate. The effects of Mn and Zn reflect cellular uptake of Cd by two inducible transport systems: the Mn system whose capacity (Vmax) is enhanced at low [Mn2+] and a separate system induced at low cellular zinc. At the low [Zn2+] of surface oceanic waters (e10-11.0 M), Cd uptake is controlled by this latter system and, therefore, is inversely related to ionic zinc levels. However, at the higher [Zn2+] range of coastal waters, Cd uptake by this system is strongly suppressed and Cd instead is taken up by the Mn system; as a result it is inversely related to [Mn2+] and largely independent of variations in [Zn2+]. Because of the suppression of Cd uptake by high [Zn2+] and [Mn2+] in coastal waters, algal Cd concentrations may be lower in these waters than in the ocean despite the presence of higher coastal [Cd2+].

Introduction Cadmium is normally considered to be a nonessential or toxic metal, but recent evidence indicates that it can nutritionally substitute for zinc in at least some phytoplankton species (1-3). Like zinc, cadmium is strongly depleted in surface seawater, apparently due to uptake by phytoplankton (4, 5). Cadmium concentrations are closely correlated with those of phosphate and nitrate, and in the central North Pacific, cadmium and phosphate concentrations both increase by ∼500-fold between the surface and 800 m (6). The close relationship between Cd and PO4 concentrations in seawater is believed to result from the uptake of both constituents by phytoplankton in surface waters and remineralization of sinking biogenic particles at depth, but the factors controlling algal uptake of Cd and resulting ratios of Cd to major nutrient elements (C, N, and P) are poorly understood. Previous studies have shown that algal Cd concentrations are not only controlled by aqueous Cd ion * Corresponding author telephone: (252)728-8754; fax: (252)7288784; e-mail: [email protected]. S0013-936X(98)00271-5 Not subject to U.S. Copyright. Publ. 1998 Am. Chem. Soc. Published on Web 08/21/1998

Experiments were conducted with the estuarine diatom, Thalassiosira pseudonana (clone 3H), obtained as an axenic culture from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton, West Boothbay Harbor, ME. It was maintained in f/8 medium (9) using sterile technique until needed. Both long-term metal uptake and growth experiments and a short-term uptake kinetic study were conducted utilizing methods similar to those in previous algal studies with Cd, Mn, and Zn (7, 10, 11). Cells were grown at 20 °C and pH 8.2 ( 0.1 in 450-mL polycarbonate bottles containing 250 mL of 36‰ seawater medium. They were grown under fluorescent light at an intensity of 500 µmol quanta m-2 s-1 on a 14:10 h light:dark cycle. Experiments were conducted in filtered, enriched natural seawater containing added trace metal ion buffer systems. The seawater was collected from the Gulf Stream with a peristaltic pump and stored in the dark before use. To prepare media, the seawater was passed through 0.4-µmpore Nuclepore filters and enriched with 32 µM NaNO3, 2 µM Na2HPO4, 40 µM Na2SiO3, 10 nM Na2SeO4, 0.074 nM vitamin B12, 0.4 nM biotin, and 60 nM thiamin. Trace metal ion buffer systems were added to quantify and control free ion concentrations of relevant trace metals. Long-Term Growth and Uptake Experiments. Longterm experiments were conducted in 0.1 mM EDTA buffer systems. To initiate these experiments, cells were transferred from f/8 medium to experimental medium containing the lowest concentrations of the metals that would be varied. The cells were acclimated for 4-7 d (depending on the culture growth rate) and then inoculated into radiolabeled media at biomass levels of 0.1-0.2 µmol of cell carbon/L of medium. The algae were grown for 10-11 cell generations to the end of the exponential phase. During this time, total cell concentrations and volumes were measured daily with a Coulter (model TA-2) multichannel electronic particle counter. Specific growth rates of cultures were computed from linear regressions of ln cell volume vs time for the exponential phase of growth. Cellular concentrations of Cd and Zn were measured with radiotracers (109Cd and 65Zn) in exponentially growing cultures, 8-9 cell divisions after inoculation (7, 11). The fraction of radiotracer in the cells was multiplied by the total metal concentration and divided by the total cell volume to yield cellular metal concentrations in units of mol (L of cell volume)-1. These values were converted to molar metal: carbon ratios by dividing them by the mean cell C:volume ratio (14 ( 1.5 mol L-1, ( SD) determined for T. pseudonana cultures (n ) 25) grown at rates of 0.24-1.8 d-1 under various levels of metal (Fe or Zn) or light limitation (11, 12, and unpublished data). Net steady-state Mn and Cd uptake rates VOL. 32, NO. 19, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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[in units of mol (mol of C)-1 d-1] were computed by multiplying the cellular metal:carbon ratios by the specific growth rate. Short-Term Metal Uptake Kinetic Studies. A short-term experiment was conducted to examine the effect of [Zn2+] on the uptake kinetics of Cd and Zn and to examine interrelationships between Cd and Zn kinetics. The methods used were similar to those described previously (7, 10). In this experiment, cells were grown for 6-10 generations in 0.1 mM EDTA-metal ion-buffered media without radiotracers at a [Zn2+] of 10-11.30 M and [Cd2+] of 10-12.00 M. They were gently filtered onto 3-µm-pore Nuclepore filters, washed with nonlabeled medium at a [Zn2+] of 10-11.5 M, and resuspended in radiolabeled uptake media at [Zn2+] ranging from 10-11.5 to 10-9.5 M. The uptake media contained 65Zn and 109Cd and 0.03 mM EDTA buffer systems with the same computed free ion concentrations of Cd, Mn, Co, and Cu as the acclimation media. Uptake of 65Zn and 109Cd was measured periodically over 4 h by filtering the cells onto Nuclepore filters and washing them with Gulf Stream seawater to remove any residual dissolved radiotracer. During this time, the total cell volume in the uptake cultures was determined by Coulter counter. The concentration of Zn and Cd taken up by the cells at each sampling time was determined by the same procedures used for the long-term metal uptake experiments. Free Metal Ion Composition of Media. Free ion concentrations of Zn, Co, Cd, Mn, and Cu in the experimental media were computed from the total metal concentration and the extent of metal complexation by EDTA and inorganic ions. Total metal concentrations were computed from the sum of the concentration added as the metal salt and added with the radiotracer solution plus the measured or estimated background concentration in the seawater media. Cu and Mn were analyzed directly in the background media without EDTA, and their low measured concentrations (2 nM for Mn and 1.0 nM for Cu) show no evidence of metal contamination. Mn was measured by chelate extraction with 8-hydroxyquinoline preabsorbed on C18-Sep-Pak cartridges followed by atomic absorption spectrometric analysis (13). Copper was determined by chemiluminescence analysis (14). Background concentrations of Zn (0.9 nM), Cd (∼0.1 nM), and Co (∼0.1) nM were estimated from measured concentrations of metal added with stock solutions of EDTA and major nutrients (0.7 nM for Zn and