The issue of phosphate limitation is a growing concern and highly topical, but also requires a considera-

	A strong relationship exists between P concentration and the presence of reduced and methylated As forms in surface Sargasso Sea samples taken in 2003.

tion of arsenic cycling in these same waters. There are close geochemical and biochemical linkages between P and As cycling (see Figure) that suggest that in low phosphate waters, the products of As detoxification, reduced and methylated As, may act as indicators of the P-status of resident phytoplankton. Furthermore, under P stress or limitation, the differing abilities of phytoplankton species to detoxify As (“arsenic stress”) may influence the structure
of the autotrophic community as has been demonstrated in coastal waters. We (Jim Sanders and Liz Mann at SkIO, Fritz Riedel at the Smithsonian Institution) are beginning a series of laboratory experiments using cultured isolates to help better define the linkage between the biogeochemistries of these two elements, and the role of As (and interactions with P) in the regulation of autotrophic communities in oceanic waters. The aim is to evaluate our hypotheses in the laboratory and then to apply them in the field at a future date.
The overall objectives of this research will be:

1) To evaluate the potential role of arsenic stress in affecting/controlling phytoplankton community structure in oligotrophic waters.

2) To establish a quantitative link between the phosphorus status of phytoplankton and the speciation of arsenic, and thereby develop arsenite and methyl arsenic as tracers of phosphorus stress or limitation on a variety of temporal and spatial scales.

The first objective will be met by investigating the resistance to As as a function of the As/P/N ratio in a suite of cultured isolates representing several important members of the phytoplankton community (Prochlorococcus, Synechococcus, larger eukaryotes).

To address the second objective, we will (1) determine if reduction and methylation rates of As increase under conditions of high As:P ratios and P stress or limitation using selected cultured isolates and (2) identify the As detoxification mechanisms of specific species.

Approach

Culture studies for both objective 1 and 2 will be done using batch culture, so a large number of isolates can be screened for As resistance. These cultures will be kept very dilute, with low nutrient concentrations, so growth of each species will be followed using flow cytometry. In vivo chlorophyll fluorescence will be used in addition, if possible. During the first year we will attempt to isolate representative species, particularly eukaryotic phytoplankton, from low phosphate regions of the Sargasso Sea. These will form a link between any future field studies and the laboratory experiments. Previously cultured isolates that are of particular interest include oceanic diatoms such as Thalassiosira oceanica, the coccolithophore E. huxleyi and the cyanobacteria Synechococcus and Prochlorococcus. If possible, species with sequenced genomes will be selected. Axenic cultures will be obtained from the CCMP or other investigators as appropriate (see letter of support from S. W. Chisholm). A few coastal species with already characterized As resistance will be included as a link to previous work. Media will be based on trace metal clean artificial seawater to exclude background levels of nutrients, including organic N and P sources, and inorganic nutrients will be added at both nitrogen and phosphate limiting N:P ratios. Phosphate limitation or stress will be measured using alkaline phosphatase activity. In some cases, the enzyme-labeled fluorescence (ELF) molecular probe will also be used in conjunction with flow cytometry to evaluate the P status of individual cells.

In order to evaluate objective 1, a range of prokaryotic and eukaryotic phytoplankton will be screened for As resistance at various As:P:N ratios during year 1. Prochlorococcus strains that can utilize organic P will be used to determine if adding DOP to the media will increase the As:P ratio required to reduce cell growth or yield. We expect that as the As:P ratio increases, so will AKA and cell division rates will decrease. One aim of this study is to confirm the hypothesis that cyanobacteria are more resistant to high As:P ratios than eukaryotic species.

To evaluate objective 2, cultures utilized for objective 1 (which will include Prochlorococcus, Synechococcus and representative eukaryotes) will be screened for the production of reduced and methylated As. Selected isolates will be used to measure the rates of arsenate reduction and methylation in cultures with different As:P:N ratios and degrees of nutrient limitation and stress. Samples for As speciation will be taken during exponential and stationary phases of growth, thus examining whether phytoplankton in log phase tend to reduce rather then methylate As. In order to relate these results to the field, the particulate As (per cell and per carbon) at the point where growth rate is half of µmax will be determined. These cultures will also be used to determine if certain species, like Synechococcus, are unable to methylate As. Preliminary samples for developing an assay for the expression of As methylation genes (putative arsenate reducers, arsenite transporters and the arsenical resistance operon repressor ArsR) in specific species will also be taken. Ideally, expression levels of these genes will correlate with increased As:P ratios and lower cell division rates.

Selected background references:

Sanders, J.G. 1985. Arsenic geochemistry in Chesapeake Bay: dependence upon anthropogenic inputs and phytoplankton species composition. Mar. Chem. 17:329 340.

Sanders, J.G. 1986. Alteration of arsenic transport and reactivity in coastal marine systems after biological transformation. Rapp. P. v. Reun. Cons. int. Explor. Mer 186:185 192.

Sanders, J.G., G.F. Riedel and R.W. Osman. 1994. Arsenic cycling and impact in estuarine and coastal marine ecosystems. Pages 289-308 in: J.O. Nriagu, (ed.) Arsenic in the Environment, Part I: Cycling and Characterization. John Wiley and Sons, NY.

Riedel, G.F. and J.G. Sanders. 2003. The interrelationships among trace element cycling, nutrient loading, and system complexity in estuaries: A mesocosm study. Estuaries 26: 339-351.