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Ammonium in micromole quantities may be concentrated directly, with a 65% yield, from 250ml seawater into 10pl 0.25 N sulphuric acid by diffusion for 6 h if the samples are heated and agitated. This makes it possible to measure ammonium regeneration in water containing heterotrophic microplankton by means of 1 5 ~ with a minimum expenditure of time, labour, and hazardous chern~cals, and a greatly diminished risk of contamination. A recommended diffusion procedure is described, together with tests for reproducibhty, recovery, and interference. Results of trials with a mixed alga-rotifer culture and a marine plankton sample are described. Ammonium is an important nitrogen source for phytoplankton growth (Glibert 1988, Paasche 1988). In general, ammonium is made available to phytoplankton by regeneration by heterotrophic organisms, microheterotrophs (organisms < 200 pm in size) frequently being responsible for most of this activity (McCarthy & Carpenter 1983, Glibert 1988). Regeneration by microheterotrophs is usually measured by isotope dilution (Blackburn 1979, Caperon et al. 1979, Glibert 1988). The dilution of added 15N ammonium by unlabelled ammonium is used together with net changes in the total amount of dissolved ammonium pool to calculate the regeneration rates. Before the ammonium can be subjected to isotope analysis, it must be stripped from the water and concentrated into a very small sample. This may be achieved in several ways. Distillation followed by evaporation (Glibert et al. 1982) or by microdiffusion (Paasche & Kristiansen 1982) is extremely time-consuming. More recent methods, in which ammonium is precipitated as a mercury salt (Fisher & Morrissey 1985) or extracted into an organic phase in the form of indophenol blue (Dudek et al. 1986, Selmer & Sorensson 1986), may be somewhat less cumbersome but necessitate the use of hazardous chemicals. All these procedures are prone to contamination due to extensive handling of the samples or for other reasons. O Inter-Research/Printed in F. R. Germany Blackburn (1979) used a diffusion technique in his experiments with ammonium-rich sediments. As originally described, the method is useful only with high ammonium concentrations in small volumes. If diffusion is to be applied directly to plankton samples, without prior distillation, it must be scaled up to permit ammonium to be collected from much lower concentrations in much larger volumes of water. This was attempted by Probyn (1987) who found, however, that recovery was insufficient unless diffusion was prolonged for up to 2 wk. In the direct diffusion method described below, designed to be used in coastal and oceanic water, a combination of heating and shalung is employed. This reduces the time needed for a satisfactory yield to 6 h, whereby a number of samples can be processed within reasonable time with a minimum of handling or noxious chemicals. Description of the direct diffusion method. Based on a series of trials, we recommend the following procedure. The diffusion is carried out in new 500ml polyethylene bottles that have been washed with 10 % HC1 and rinsed with deionized water. Before use, the inner surfaces of each bottle are wetted with 10 -4 M NaOH, and then by a portion of the seawater sample. The bottle is then filled with 250ml of the sample to be analysed. The total ammonium concentration in this sample, i.e. the sum of NH4+ initially present and that added as 1 5 ~ ~ 4 ' and carrier, should be 4 p M (i.e. 1 pm01 NH4+-N per bottle). Each bottle receives 4 m1 50 % NaOH (w/v) and is then quickly closed with a silicone stopper. A 40 mm needle of stainless steel projecting downwards from the stopper into the bottle carries (by means of a 'clamp' made of silicone rubber tubing) a 20 X 5 mm strip of glass fibre filter (precombusted at 450 "C for 4 h) to which l 0 p1 0.25 NH2S04 has been applied. The sulphuric acid on the filter must be the only acidic surface within the enclosed space. The 204 Mar. Ecol. Prog. Ser. 54: 203-207. 1989 bottles are placed upright in a temperature-controlled water bath at 40 ' C , provided with shaking at 90 strokes min-' wlth an amplitude of 4 to 5 cm. After 6 h, during which time ammonia diffuses from the alkaline samples into the acid on the filter strips, the latter are removed from the bottles and are dried at 60 to 70°C for 15 to 30 min while still attached to the needles. Finally the filter strips are transferred to test tubes and dried for another 1 to 2 h. The dried samples may be stored in VacutainersTM (Becton Dickinson) for at least 4 wk without any significant change in atom % "N. It is desirable to include some standard samples diffused from deionized water. These are needed to keep a check on contamination by unlabelled nitrogen. Methods used in the tests. Results reported in this note were obtained following the procedure described above unless otherwise noted. Ammonium chloride standards of varylng 15N content, added from concentrated stock solutions, were used in the hffusion experiments at a final concentration of 4 pM. The atom % 15N was measured by emission spectrometry (Knstiansen & Paasche 1982) using a Jasco Model N-150 N-15 Analyzer. The sample (0.6 to 0.7 pm01 NH4+-N on the filter strip assuming 65 % recovery) was ground with a glass pestle in a small test tube (both precombusted at 450°C), together with 0.1 g Ca0-M2O3 (1 : 1) mixture (precombusted at 1000°C) and 0.05 g CuO (precombusted at 700°C). The ground mixture was sealed into a 5mm OD Pyrex discharge tube (precombusted at 450°C) and processed for isotope analysis as described by Kristiansen & Paasche (1982). The isotope ratios were adjusted by means of appropriate standard curves prepared from commercial gas samples of known atom % '=N. The amount of contaminating nitrogen was calculated according to the formula where C = contamination in ymol sample-'; P = amount of (labelled) nitrogen intentionally added, in pm01 N sample-'; Asrd and Adilf = atom % I5N in the added nitrogen and in the diffused sample, respectively; and A,,, = natural abundance of 15N (0.37 atom % 15N). Deionized distilled water (DDW) was used throughout in the preparation of standard solutions and blanks. Ammonium concentrations were measured according to Solorzano (1969). The recovery of ammonium in the diffusion experiments was checked chemically after extracting it from the filter strips with DDW. The initial pool of ammonium was measured chemically before isotope addition and diffusion, and the analytical values were used to correct the measured isotope ratios for isotope dilution attributed to this ammonium. Recovery of total ammonium. The effects of heating Table 1. Effect of heating and shalung on the recovery, expressed as %, of 1 btmol N in the form of ammonium diffused from seawater. Recovery is given either as mean -t standard error (n = 3) or as mean or single value (n = 2 or n = 1) Temperature Shalung No. of Recovery "C samples ("/.l 20 No 3 15.6 2 1.3 20 Yes 3 36.2 2 2.0 40 No ! 18.6 40 Yes 3 64.7 f 2.7 55 No 3 15.2 f 2.1 55 Noa 52.9 a Bottle oriented sideways and shaking on the chemically measured yield are shown in Table 1. Heating alone, either to 40°C or to 55 "C, did not produce an acceptable yield. Shaking at room temperature improved the recovery, while the best yield, of about 65 %, was obtained when shaking was combined with heating to 40°C. By placing the diffusion bottle sideways, thus doubling the surface/ volume ratio, the recovery in the absence of shalung was improved from 15 % to 53 %. For practical reasons, 40°C and shaking as described above were adopted as standard diffusion conditions. At higher temperature and/or increased shaking speed, the filter strips tended to collect moisture, and the silicone stoppers sometimes popped off during diffusion. The resulting 15N-values were erratic or not measurable. Chemical analyses (data not shown) indicated that diffusion for 6 h , with heating and shaking, was required for a recovery of 60 % or better. A time course of isotope recovery, shown in Fig. 1, confirmed that the optimum diffusion time was 6 to 10 h.

An improved method for determining relative 15N abundance in ammonium regeneration studies by direct diffusion