AQUATIC MICROBLAL ECOLOGY Published October 1 Aquat Microb Ecol Elemental composition of depth samples of Ceratium hirundinella (Pyrrophyta) within a stratified lake: an X-ray microanalytical study David C. Sigee*,Eugenia Levado, Andrew J. Dodwell School of Biological Sciences, The University of Manchester, 3.614 Stopford Building, Oxford Road. Manchester M13 9PT, United Kingdom ABSTRACT: The elemental composition of Ceratium hirundinella cells was investigated in mixed phytoplankton samples collected from the water column of a stratified lake. X-ray rnicroanalysis (XRMA) routinely detected Mg, Si, P, Cl, K and Ca with occasional peaks of Na. Al and Fe. Cell con- centrations of most elements showed no significant variation within the epilimnion, but Mg, P, C1 and K were significantly lower (and Si higher) in cells from the metalirnnion. Ratios of Mg/K, P/K, Mg/P, monovalent/divalent cations and diffusible anions/cations were constant throughout the sampled water column. Within depth populations of Ceratium, concentrations of each element varied consider- ably, approximating (except Si) to a normal distribution. Approximately 5 % of all cells had abnormally low concentrations of K and were possibly undergoing senescence. Correlation and factor analysis demonstrated a major statistical association between Mg, P, S and K in Ceratium cells throughout the depth samples. The XRMA results suggest an underlying homogeneity in the population of Ceratium throughout the sampled water column, consistent with the known high mobility and rapid migration of these cells within the water body. KEY WORDS: Phosphorus . X-ray rnicroanalysis . Ceratium . Dinoflagellates . Water column . Anions . Cations INTRODUCTION 1998) as well as lake bacteria (Booth et al. 1987).Other single-cell techniques that have been used to deter- Studies on the elemental composition of freshwater mine the chemical composition of phytoplankton cells phytoplankton have normally involved the use of bulk include proton-probe analysis (Brook et al. 1988) and analysis techniques, such as atomic absorption spec- cherniluminescence (Villareal & Lipschultz 1995). trophotometry (Behrendt 1990), providing mean data The present study was carried out on the dinoflagel- for the whole plankton sample. The recent develop- late Ceratium hirundinella (O.F. Mull.) (hereafter Cer- ment of electron-probe X-ray microanalysis (XRMA) atium). This alga is frequently encountered as a major as a routine analytical technique (Sigee et al. 1993) constituent of freshwater lakes, where it is found over now permits a more detailed study of the elemental a wide range of trophic conditions (H011 1928). Studies composition of freshwater biota, since it combines high were carried out at Rostherne Mere (Cheshire, UK), a spatial resolution (single cell analysis) with simultane- deep eutrophic lake (Carvalho et al. 1995, Moss et al. ous detection of a range of elements (determination of 1997) that regularly stratifies during summer months. elemental ratios and correlations). XRMA has been At this site, Ceratium typically CO-dominates with used for determining the elemental composition of a Microcystis aeruginosa in late summer (Reynolds & range of phytoplankton species (Clay et al. 1991, El- Bellinger 1992),occasionally reaching population lev- Bestawy et al. 1996, Sigee & Holland 1997, Sigee et al. els in excess of lo3 cells ml-'. The elemental composition of Ceratium is of particu- lar interest, since recent XRMA studies (Sigee et al. O Inter-Research 1999 178 Aquat Microb Ecol 19: 177-187, 1999 1998) have demonstrated marked temporal variations Water analysis and chl a determination. Filtered between micro-populations of this alga in relation to lake water samples were analysed for soluble inor- changes in water chemistry. In addition to major differ- ganic nitrate and soluble inorganic phosphate concen- ences between populations, considerable variation trations using a Skalar mass flow autoanalyser (Skalar also occurred in the elemental composition of individ- Analytical B.V.). Elemental concentrations were deter- ual Ceratium cells within each phytoplankton sample, mined by inductively coupled plasma atomic emission possibly relating to the different depths sampled with- spectroscopy (ICP--S, Fison VG Elemental Horizon in the integrated sample. spectrometer) from lake water samples that had been The major purpose of the present study was to deter- filtered through a 0.45 pm Nuclepore filter membrane, mine variation in the elemental composition of this then acidified with Aristar quality nitric acid. alga with depth, analysing micro-populations of Cer- Chl a determinations were carried out according to atium within phytoplankton samples collected at dif- the cold ethanol extraction procedure of Jespersen & ferent points within the water column. As far as we are Christoffersen (1987). A known volume of sample was aware, this is the first published investigation of single- filtered onto a 47 mm Whatman GF/C filter, and stored species depth variation in elemental composition at -20°C prior to processing. Filters were then ground within the lake environment. Such variation in cell into small pieces, placed in 5 m1 of 96% ethanol in chemistry might arise due to variation in physical and 25 m1 universals and maintained for 20 h at 5°C. Uni- chemical properties of micro-environment with depth, versal~were then centrifuged for 10 min at 3000 rpm and would clearly relate to both lake stratification and (1800 x g). UV absorption of the extract (supernatant) the mobility of Ceratium within the water body. Depth was measured at 665 nm (chl a) and 750 nm (turbidity) variations in the elemental composition of phytoplank- using a Pye Unicam ultraviolet spectrophotometer. The ton species have significance in relation to biodiversity chl a concentration was subsequently determined within the freshwater environment and also have using the standard formula of Jespersen & Christof- implications for modelling studies on lake nutrient fersen (1987). cycling (Krivtsov et al. 1998). X-ray microanalysis. Membrane phytoplankton samples were transferred from liquid nitrogen to the pre-cooled stage (-60°C) of an Edwards tissue-drier MATERIALS AND METHODS and freeze-dried over a 12 h period at 10-2 torr. Sam- ples were subsequently mounted on SEM stubs and Collection and processing of samples. Sampling carbon coated (NanoTech coating unit) according to was carried out at midday on August 5, 1997, at a standard procedures. central point (total depth 30 m) in Rostherne Mere, XRMA was carried out in a Cambridge 360 scanning Cheshire, UK, as part of a regular sampling pro- electron microscope, with Kevex detector and LINK gramme at this site. 2 1 samples of lake water were col- AN10000 analyser. X-ray emission spectra were col- lected at the surface (0 m) and at depths of 1, 2, 5 and lected from the central region of individual Ceratium 8 m. Physical parameters were also noted at these cells (see Fig. 2), using a raster of approximately 30 X depths, including temperature, pH, conductivity and 20 pm in size. X-rays were collected over a livetime of oxygen concentration. Secchi depth was also recorded. 100 S, at an accelerating voltage of 15 kV and magnifi- The 2 l depth samples were subdivided for collection cation of 1.4 K. The detector-specimen distance was set of phytoplankton samples, cell population counts, at 25 mm, with a take-off angle of 35". Probe current chlorophyll a (chl a) determination and water analysis. was typically about 500 pA and was adjusted to give an Phytoplankton samples for XRMA were immediately X-ray count of 103 cps. collected by filtering 50 m1 of lake water through a Quantification was carried out using the LINK ZAFI 5.0 pm polycarbonate (Nuclepore) filter membrane PB programme for elements within a protein matrix, contained in a small filtration apparatus (Swinnex disc with cobalt as the reference element. Stored spectral filter holder, Millepore Corp.). Excess lake water was information from inorganic standards is used in this removed by passing air through the filtration system. programme for gain calibration and for determination Filter membranes were immediately removed and of goodness of fit of elemental peaks in cell spectra. plunge-frozen in liquid nitrogen (-196°C) while on the Analyses with a fit index greater than 2 were dis- boat. Samples were transported back to the laboratory carded. Elemental peaks within Ceratium spectra in liquid nitrogen for further processing For cell were considered significant when: counts, Lugol's iodine was added to 200 m1 of lake (P'- B) > 2J(P' + B) water, and counts subsequently made in the laboratory using a Sedgwick rafter slide, according to standard where P' = total peak integral, and B = estimated con- procedures. tinuum component of peak integral. Sigee et al.: Depth samples of Ceratium hlrundlnella Table 1. Concentrations of major anions, cations and algal biomass in the water column. Ion concentrations are expressed as mg I-', chlorophyll a (chl a) as pg 1-' and Ceratium hirundinella counts as cells ml-' N, Si, P and S are soluble inorganic nitrogen, sil- icon, phosphorus and sulphur, respectively Depth (m) Anions Cations Biomass N Si P S M g K C a Chl a C. hirundinella 0 0.81 0.83 0.103 26.85 12.19 4.79 57.51 13.19 20 1 0.81 0.84 0.083 27.72 12.11 4.78 58.36 13.91 50 2 0.83 0.82 0.103 28.77 12.06 5.02 58.87 13.79 30 5 0.86 0.88 0.098 31.57 12.46 4.96 61.05 15.47 40 8 1.24 1.02 0.146 31.00 12.37 5.21 61.57 4.20 20 Further details of the XRMA quantitation pro- to 4.20 pg 1-' at the top of the hypolimnion. Secchi depth gramme and procedures are given in Sigee & Holland was 1.9 m. Population counts of Ceratium (20 to 50 cells (1997).Elemental concentrations within cells were ini- ml-l) indicated that this alga was present as a major tially determined as percentage dry weight and subse- constituent of the phytoplankton throughout the sam- quently converted to mm01 kg-' dry wt.