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mass spectrometry
Marine Chemistry 98 (2006) 121 – 130 www.elsevier.com/locate/marchem
Determination of ammonium regeneration rates in the oligotrophic ocean by gas chromatography/mass spectrometry Darren R. Clark *, Tim W. Fileman, Ian Joint Plymouth Marine Laboratory, Prospect Place, Plymouth, Devon, PL1 3DH, United Kingdom Received 14 December 2004; received in revised form 31 May 2005; accepted 19 August 2005 Available online 12 October 2005
Abstract A method has been developed for the determination of ammonium concentration and isotopic enrichment in seawater samples at the low nanomolar range (10–100 nmol/kg). It is based on the reaction of phenol/hypochlorite with ammonium to form indophenol, with subsequent solid phase extraction, derivatisation and analysis by Gas Chromatography Mass Spectrometry. The precision of the method was maximised by incorporating a deuterated indophenol internal standard. A system was developed which generated seawater with extremely low ammonium concentrations thus matching sample and standard matrices for quantitative analysis. Data are presented from a study of ammonium regeneration rates at three stations in the oligotrophic North–East Atlantic where ambient ammonium concentrations were b 21 nmol/kg. Results suggested that ammonium availability for phytoplankton was limited by the rate of ammonium regeneration. Efficient ammonium assimilation contributed to the very low ambient ammonium concentrations measured at these stations. The study highlights the need for the accurate determination of ammonium regeneration rates in studies of new production, particularly in extreme oligotrophic conditions. If not corrected for isotope dilution, f-ratio estimates may be overestimated by 10.7–13.7%. D 2005 Elsevier B.V. All rights reserved. Keywords: Ammonium; Regeneration;
15
N; GC/MS; Indophenol; f-ratio; North–East Atlantic
1. Introduction Ammonium regeneration is an important component of N-cycling, often providing the dominant form of nitrogen for N-assimilation by marine phytoplankton in many regions of the global ocean. Estimates of phytoplankton inorganic N demand (primarily as NH4+ and NO3) are usually determined by 15N tracer techniques that generate assimilation data. These rates are used to estimate the f-ratio (Dugdale and Goering,
* Corresponding author. Tel.: +44 1752 633100; fax: +44 1752 633101. E-mail address: [email protected] (D.R. Clark). 0304-4203/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2005.08.006
1967; Eppley and Peterson, 1979) and hence, to determine global-ocean carbon flux. However, many estimates of new production may be in error because most studies have not taken into account the regeneration of unlabeled 14NH4+ in tracer experiments (Glibert, 1982; Harrison and Harris, 1986). This omission creates uncertainties concerning the extent of isotope dilution and consequently may lead to underestimation of NH4+ assimilation rates. The majority of ammonium regeneration studies have been limited to marine provinces with relatively high NH4+ concentrations, usually in the low Amol/kg range, where the recovery of sufficient N for Emission Spectrometry or Isotope Ratio Mass Spectrometry (IRMS) analysis has rarely been an issue. Previous
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approaches have included the diffusion (Slawyk and Raimbault, 1995; Sigman et al., 1997) or distillation methods (Bremner and Keeney, 1965; Tupas et al., 1994; Haesgawa et al., 2000; Ward and Bronk, 2001), with detection limits generally in the order of 2 Amol/kg of ambient NH4+. In an adaptation of the diffusion method, Holmes et al. (1998) improved the limit of detection to 0.5 Amol/kg of ambient NH4+ (1 Amol of sample-N for IRMS), partly by increasing sample volume (and hence total sample N) and also by correcting for isotopic fractionation associated with the diffusion method. For the analysis of samples with yet lower ammonium concentrations, previous methods have perturbed samples by the addition of unlabelled N dcarrierT in order to provide sufficient sample N for IRMS analysis (Slawyk and Raimbault, 1995; Diaz and Raimbault, 2000). These methods have proven to be relatively labour intensive and time consuming, limiting the scope and replication of samples generated. In addition, the detection limit of IRMS makes studies of N flux in the oligotrophic oceans extremely challenging, because NH4+ concentrations are typically in the range of 10– 100 nmol/kg. In a departure from the IRMS approach, Preston et al. (1996) and Ko¨ster and Jo¨ttner (1999) presented methods based on the use of Gas Chromatography Mass Spectrometry (GC/MS) for marine and freshwater environments respectively. These methods employed either the phenol/hypochlorite or Berthelot reactions forming indophenol from dissolved NH4+, with subsequent collection and isotopic analysis of volatile derivatives. With injections of sample N for GC/MS analysis typically in the order of 200 pmol, the method of Preston et al. (1996) demonstrated the sensitivity that could be gained by the development of GC/MS methods, with measurements at ambient concentrations as low as 190 nmol/kg. The method presented here is a development of that of Preston et al. (1996) and combines the advantages of ammonium-specific derivatisation and pre-analysis concentration of sample-N by solid phase extraction, with the sensitivity of GC/MS analysis. A system is presented which removes ammonium and provides seawater with very low ammonium concentrations (~ 1 nmol/l) suitable for the generation of calibration standards. Greatly improved precision over the Preston et al. (1996) method was obtained by incorporating a deuterated indophenol internal standard, which minimised cumulative error during the preparation and analysis of samples. The procedures were tested in the oligotrophic waters of the North Atlantic Gyre. Regeneration rate data are presented from three stations in
which the ambient ammonium concentrations were in the range of approximately 10–20 nmol/kg. The impact of the measured NH4+ regeneration rates upon estimations of N-assimilation rates and f-ratio values is discussed. 2. Materials and methods 2.1. Low-ammonium seawater generation for standards—the PTFE–HCl system Studies of ammonium cycling in the sea are often frustrated by the problem of obtaining seawater with sufficiently low NH4+ concentrations for the preparation of standards. On this study, we developed a method to obtain seawater with extremely low NH4+ concentrations. The basis of the method is the diffusion of ammonium from seawater through a PTFE membrane into acid, building on the principles of Jones (1991). A 2 l volume of low nutrient seawater, collected from oligotrophic regions, typically containing 10–100 nmol/kg NH4+, was filtered through a 0.2Am membrane filter and placed in an acid-cleaned 2 l borosilicate reservoir vessel containing a magnetic stirrer bar. The vessel was sealed with a gas tight cap fitted with a 3-valve adaptor. Water was pumped at a flow rate of 10 ml/min from this reservoir through 8 1 m lengths of PTFE tubing (2 Am porosity, 1 mm internal diameter, 0.4 mm thick, W.L. Gore and Associates U.K.), immersed in 10% HCl. The treated seawater was returned to the 2-l vessel, completing a continuous loop. Seawater was recirculated through this system for N 24 h to obtain essentially ammonium-free seawater. It was noted that the pH of seawater produced with new PTFE tubing decreased, typically in the order of 0.1–0.5 pH units. However, this effect became less significant with successive seawater batches without affecting system performance, i.e. the PTFE tubing in the system required dagingT. Seawater which had been processed with the PTFE– HCl system was used for the generation of calibration standards. 2 l of processed seawater was split into 4 500 ml volumes and placed in 550 ml amber glass bottles. A spike of NH4Cl was added to three of the standards to give a final concentration within the range of 5–50 nmol/kg. No addition was made to the fourth standard which served as a zero. During the application of the method at sea calibration standards were generated throughout the duration of the fieldwork. A minimum of 3 measurments was made for each calibration concentration.
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Indophenol was developed, collected and analysed as described in Sections 2.2 and 2.4. After normalisation (see Section 2.7), a calibration curve was constructed by plotting integrated peak area (m/z 345.2 plus m/z 346.2 corresponding to 14N and 15N, respectively) from a calibration standard against the standard concentration. 2.2. Indophenol development All chemicals were supplied by Sigma-Aldrich Co. Ltd., unless otherwise stated. Acid-cleaned borosilicate glasswear was used exclusively. The use of plastics was avoided and all experimental work was undertaken while wearing nitrile gloves. Reagents were made fresh daily and stored in gas-tight borosilicate bottles at 4 8C until use. The first reagent was made by dissolving 2.35 g phenol in 100 ml of milli-Qk water (MQ) followed by 0.16 g sodium nitroprusside. The second reagent was made by dissolving 0.6 g sodium dichloro-isocyanurate in 100 ml of MQ followed by 1.4 g sodium hydroxide. Standard and sample volumes (500 ml) were placed in 550 ml amber glass bottles. Indophenol synthesis was initiated by adding 3 ml of phenol/sodium nitroprusside reagent to the sample or standard, which was thoroughly mixed and left for 5 min. 3 ml of sodium dichloro-isocyanurate/sodium hydroxide reagent was added to the sample or standard which was again thoroughly mixed. Samples and standards were placed in the dark at 18 8C. Indophenol development was complete after 12 h. Indophenol generated in samples and standards was collected by solid phase extraction (SPE) using 6 ml/ 500 mg C18 cartridges (Kinesis Ltd., UK) that required a preparatory cleaning procedure. It was important to ensure that the SPE matrix did not dry between the conditioning and sample collection stages. After soaking the SPE in methanol for 5 min, it was rinsed sequentially with 10 ml methanol, 10 ml MQ and 10 ml 1% NaCl in MQ. Samples and standards were acidified by the addition of 700 Al of 1 mol/l citric acid, resulting in a pH of approximately 5.5. A deuterated indophenol internal standard (see Section 2.3) was added prior to the collection of samples and standards by SPE under low vacuum (120 mm Hg). The SPE cartridges were then rinsed with 50 ml of MQ and allowed to run dry. The SPE matrix was then rigorously dried under high vacuum (360 mm Hg) for 10 min. At this stage, indophenol could be eluted from the C18 as described below, or the cartridge could be capped and stored at 20 8C. It was found to be imperative that the
123
SPE matrix was pH-neutral and completely dry prior to storage. If this was not the case, matrix degradation would occur resulting in analytical complications. Indophenol was eluted from the SPE cartridges by first allowing 7 ml of methanol to soak into the SPE matrix for 5 min, before drawing the solvent through the cartridge under low vacuum for collection in a 10ml test tube. Sample volume was decreased to approximately 200 Al using a Zymark Turbo-vap (high capacity evaporating unit using oxygen-free nitrogen). Samples were quantitatively transferred to GC vials with 300-Al inserts (Chromacol Ltd., UK) and blown to dryness. Sample tubes were rinsed with a further 200 Al methanol which was also transferred to GC vials and blown to dryness. Once dry, samples could be analysed as described in Section 2.4, or capped and refrigerated until analysis. 2.3. Deuterated indophenol internal standard The accuracy and precision of the method was greatly improved by including an internal standard, which in this case was a deuterated form of the sample molecule. 1 g of phenol-3,5-d2 (Qmx Laboratories Ltd., UK) was dissolved in 42 ml of MQ to which 0.068 g sodium nitroprusside was added. The sodium dichloroisocyanurate/sodium hydroxide reagent was made as described in Section 2.2. 42 ml of phenol-3,5-d2/sodium nitroprusside reagent was added to 1 l of 2.5 mmol/ l NH4Cl in MQ and thoroughly mixed. After 5 min, 42 ml of sodium dichloro-isocyanurate/sodium hydroxide reagent was added, the volume was thoroughly mixed and incubated at 18 8C for 24 h for deuterated indophenol development. Deuterated indophenol was collected using 70 ml/10 g C18 SPE cartridges (Kinesis Ltd., UK). Two cartridges were used for the collection of the entire volume of deuterated indophenol. 70 ml/10 g C18 SPE cartridges were first cleaned in a scaled-up version of the protocol described in Section 2.2. Prior to collection, sufficient citric acid was added to the deuterated indophenol solution to turn it from deep blue to red (pH c 5.5). Deuterated indophenol was then collected, and subsequently cleaned with 500 ml MQ per cartridge. The C18 matrix was thoroughly dried under high vacuum for 10 min. Deuterated indophenol was eluted into boiling tubes from the C18 matrices using 20 ml methanol per cartridge. The deuterated indophenol was transferred to a watch glass and placed in an oven at 50 8C in 5 ml batches in order to obtain dry deuterated indophenol powder. 2 mg deuterated indophenol powder was dissolved in 1 l MQ water at pH 10.0. The solution was acidified to pH 6.0. 1 ml deu-
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terated indophenol internal standard was added to seawater samples and standards after they had been acidified by the addition of citric acid and prior to collection by SPE. 2.4. Indophenol analysis by GC/MS Samples and standards were derivitised with Sylon TP (25% trimethylsilylimidazole in pyridine, SigmaAldrich, UK), which was purchased in 1 ml ampoules, each of which was used within 24 h. A volume of between 20 and 100 Al of Sylon TP was added to samples which were incubated at 50 8C for 30 min. Isotopic analysis of bis(trimethylsilyl)-indophenol was performed using a Hewlett-Packard 5890 series II GC coupled to a HP5972 mass selective detector (MSD). Samples with a volume of 0.5–1.0 Al were injected in splitless mode using a HP7973 injection controller onto a HP-5ms column (30 m 0.25 mm internal diameter). The injection port contained a liner packed with glass wool and the capillary column was further protected by a 1-m length of uncoated deactivated pre-column, both of which were replaced at regular intervals during routine maintenance. Injection port and transfer line temperatures were 300 8C. Derivitised indophenol was resolved using temperature programming: GC oven initial temperature of 95 8C; ramped to 210 8C at 45 8C/min; ramped to 310 8C at 20 8C/min; hold for 5 min. For quantitative analysis the MSD was operated using selected ion monitoring (SIM). Ions used for SIM were molecular ions with m/z 345.2 and 346.2 for samples or standards and m/z 349.2 and 350.2 for deuterated internal standard. Bis(trimethylsilyl)-indophenol and deuterated internal standard co-eluted at 6.1 min. Integrated peak area was used for quantitative and isotopic analysis. Capillary column performance was routinely monitored by the use of a test mixture (Grob et al., 1981; Sigma-Aldrich UK).
rinsed with 10 ml methanol and 10 ml MQ, eluted in 5 ml of methanol and blown to dryness. 1 ml of methanol was added to samples which were then quantitatively transferred to micro-centrifuge tubes and centrifuged at 13000 rpm for 20 min. Samples were transferred to GC vials and blown to dryness, ensuring that any particulate material remained undisturbed in the centrifuge tube. 2.6. Blank determination The contribution to sample-N derived from reagents was determined by adding 5, 10 and 15 ml of each reagent (i.e. reagent concentration factors of 1.6, 3.3 and 5 times normal additions respectively) to triplicate 500 ml volumes of 0.22 Am filtered seawater containing 200 nmol/kg 15NH4Cl. A regression between blank concentration (i.e. 14NH4+ derived from seawater and reagents) and reagent concentration factor enabled the contribution to sample-N derived from reagents to be calculated. 2.7. Normalisation of samples for quantification The deuterated internal standard was used to enable the normalisation of all samples and calibration standards thereby removing variance associated with sample manipulation and changes in recovery efficiency or GC/MS performance. The integrated peak area of the deuterated internal standard associated with a calibration standard was used as the benchmark against which all other samples and standards were normalised. In this case a calibration standard at a concentration of 25 nmol/kg was chosen as this was approximately in the middle of the calibration curve concentration range. Peak areas were normalised using the equation below: 349:2 ! d IP Std Peak345:2 PeakNormð345:2Þ ¼ d IP Sample349:2
2.5. Sample cleaning procedure Inappropriate sample storage resulted in SPE matrix degradation and contamination of samples. Contamination derived from the SPE matrix took the form of both particulate material and a chemical component (silane), and necessitated sample re-collection on C18’s and centrifugation in order to remove both contaminating components. Samples were quantitatively transferred from GC vials to test tubes in 3 200 Al aliquots of 3 mol/l NaOH. 5 ml MQ was added and samples were then acidified with 600 Al of 1 mol/l citric acid. Samples were collected on SPE cartridges which had been
where PeakNorm(345.2) was the normalised peak area for m/z 345.2 of a sample or standard, d-IP(Std349.2) was the integrated peak area of the deuterated internal standard at m/z 349.2 associated with the 25 nmol/kg calibration standard benchmark, d-IP(Sample349.2) was the integrated peak area of the deuterated internal standard at m/z 349.2 associated with the sample and Peak345.2 was the peak area for m/z 345.2 of a sample or standard. Noting that m/z 345.2 and 349.2 correspond to 14N of the sample and internal standard, respectively (as represented in the equation), while m/ z 346.2 and 350.2 correspond to 15N of the sample and
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
2.8. Data analysis Regeneration rates (r, nmol N/kg/h) were determined using normalised data according to the Blackburn–Caperon model (Blackburn, 1979; Caperon et al., 1979) described by Laws (1984): Rt=Ro St r ¼ ln So St=So t where So was the pre-incubation ammonium concentration (nmol N/kg) and St was the post-incubation ammonium concentration (nmol N/kg) after t hours of incubation. Ro and Rt were the pre-incubation and postincubation 15N enrichment ratios, respectively. Ro was determined using the equation: Ro ¼
Mo Mn 1 þ ðMo MnÞ
where Mo was the pre-incubation ratio of m/z 346.2/ 345.2, Mn was the natural abundance ratio of m/z 346.2/345.2 derived from calibration standards. Rt was determined by substituting Mo for Mt defined as the post-incubation enrichment ratio of m/z 346.2/345.2 at time t. 2.9. Application of the method to extremely oligotrophic water Samples were taken during a transect of the Atlantic Ocean between the UK and the Falkland Islands on board RRS James Clark Ross during September 2003 as part of the Atlantic Meridian Transect programme (AMT 13). Results for only three stations in the North Atlantic Gyre are presented here. Regeneration was determined on seawater samples collected during a pre-dawn CTD cast from depths corresponding to 55% surface Photosynthetically Active Radiation (sPAR). 2 l was filtered (GF/F) and distributed between three 500 ml samples for the determination of ambient NH4+ concentrations. NH4+ regeneration was measured by adding 0.5–1 nmol/kg 15NH4Cl to 7 l seawater. The seawater was mixed, distributed between two 2.4 l vessels, placed into incubators on deck prior to sunrise and left for 24 h. Irradiance equivalent to the depth from which the samples were taken was simulated using Perspex screens (Joint et al., 2001) and incubation procedures followed JGOFS protocols (IOC, 1994).
The remaining enriched seawater was filtered and distributed between three 500-ml samples for the determination of pre-incubation 15N enrichment. The incubator was maintained at surface sea-temperature by cooling with surface seawater from the ship’s non-toxic supply. 24 h deck incubations were terminated by filtration (GF/F), the filtrate being distributed between three 500-ml replicates for post-incubation enrichment determinations. Indophenol was synthesised, collected and analysed as described in Sections 2.2 and 2.4. 3. Results 3.1. Low ammonium seawater and reagent blank This study was concerned with the measurement of ammonium concentration and isotopic enrichment under extreme oligotrophic conditions. The improved accuracy and precision of the method depended on a number of factors but primarily these were the inclusion of the deuterated indophenol internal standard and the ability to match sample and standard matrices for quantitative analysis. Passing seawater through PTFE tubing immersed in an acid bath (see Section 2.1) was an effective way of removing ammonium from seawater. In order to demonstrate the principle, a 2 l volume of seawater was spiked with 1.2 Amol/kg NH4Cl and the removal of NH4+ from seawater during treatment was measured as a decrease in GC/MS response over time (Fig. 1). Results demonstrated that NH4+ concentration decreased below GC/MS detection limits within 48 h. System performance was subsequently enhanced by increasing the effective PTFE tubing length from 5e+7 4e+7
GC/MS response
internal standard, respectively, appropriate substitutions in the equation above were made in order to normalise 15 N peaks.
125
3e+7 2e+7 1e+7 0 0
20
40
60
80
100 120 140 160 180
Time (h) Fig. 1. GC-MS response to bis(trimethylsilyl)-indophenol reflecting the change in ammonium concentration during the treatment of seawater with the PTFE–HCl system. 1.2 Amol/kg NH4Cl was added to seawater and passed through 4 1 m lengths of PTFE tubing. Error bars indicate 95% confidence limits about the mean.
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
4 1 m to 8 1 m. The resulting ammonium concentrations in treated seawater derived from oligotrophic oceans (typically 10–100 nmol/kg NH4+) were indistinguishable from MQ water within 12 h of processing. Laboratory reagents are an additional source of ammonium contamination that must be quantified. Table 1 presents isotope dilution data for the quantification of reagent-derived ammonium. A regression of blank (14NH4+ derived from reagents and seawater) against reagent concentration factor indicated that the ammonium concentration that would be added with the reagents was 30 nmol/kg (blank = 30 concentration factor + 253; R 2 = 0.99).
1e+7
A m/z 345
GC/MS response
126
8e+6 6e+6 4e+6 2e+6
m/z 73 m/z 180
0 0
100
8e+6
Concentration 15NH+4 addition factor of (nmol/kg) reagents
At% excess 15 N
1.66 3.33 5.00
39.94 F 0.34 300.8 35.58 F 0.59 362.1 33.22 F 2.71 402.1
200 200 200
Blank (14NH+4 from reagents plus seawater) (nmol/kg)
5, 10 and 15 ml of each reagent were added to triplicate 500 ml volumes of 0.2 Am filtered seawater containing 200 nmol/kg 15 NH4Cl.
400
GC/MS response
deuterated indophenol m/z 349
B
bis(TMS)-indophenol m/z 345
6e+6
4e+6
2e+6
m/z 73 m/z 180
m/z 182
0 0
100
200
300
400
m/z Fig. 2. Electron ionisation mass spectra of (A) bis(trimethylsilyl)indophenol and (B) bis(trimethylsilyl)-indophenol in conjunction with bis(trimethylsilyl)-d4-indophenol internal standard.
2.5e+7
GC/MS response
Table 1 Determination of ammonium concentration in reagents by isotope dilution analysis of 200 nmol/kg 15NH+4 added to a seawater sample containing 253 nmol/kg NH+4
300
m/z
3.2. Indophenol analysis by GC/MS and method optimisation Bis(trimethylsilyl)-indophenol gave a simple electron ionisation (EI) spectra (Fig. 2A) with a prominent molecular ion at m/z 345.2. For quantitative analysis samples were analysed in conjunction with the deuterated internal standard (molecular ion m/z 349.2; Fig. 2B). In order to maximise the yield of indophenol and hence optimise the method for maximum sensitivity, the time of indophenol development was determined (Fig. 3). Reagents were added proportionately (v/v) to a 1 l volume of 400 nmol/kg NH4Cl in 0.22 Am filtered oligotrophic seawater. Sample volume was 50 ml such that GC/MS injections of sample-N were quantitatively comparable to those generated from field samples (100 pmol NH4+–N injected). Results demonstrated that for maximum yield an incubation time in excess of 12 h at 18 8C was required. Further increases in response during GC/MS analysis could be gained by increasing injection port purge timing (Fig. 4), modifying derivatisation volume within the range (20–100 Al) or increasing injection volume within the range 0.5–1.0 Al. With such modifications an
200
2.0e+7 1.5e+7 1.0e+7 5.0e+6 0.0 0
2
4
6
8
10
12
14
16
Time (h) Fig. 3. The development of indophenol over time at 18 8C determined by GC-MS analysis of bis(trimethylsilyl)-indophenol. A concentration of 400 nmol/kg NH4Cl was added to filtered low nutrient seawater allowing for the analysis of small seawater sample volumes (50 ml). Results demonstrate the GC-MS response to an injection of 100 pmol NH+4 –N. Error bars indicate 95% confidence limits about the mean.
7e+6
Original f-ratio
6e+6 5e+6
Dilution factor (x)
Inlet purge timing (min)
Corrected NH+4 uptake rate (nmol/kg/h)
Fig. 4. Influence of injection port purge timing upon the magnitude of the GC-MS response to bis(trimethylsilyl)-indophenol during repeat injections of the same sample at 50 pmol NH4–N per injection. Error bars indicate 95% confidence limits about the mean.
NO 3 assimilation rate (nmol/kg/h)
appropriate level of GC/MS sensitivity could be tailored to seawater samples with a range of ammonium concentrations. However, there are important limits to these modifications as discussed in Section 4.2.
Ammonium regeneration was measured at 3 stations in the oligotrophic North Atlantic (Table 2). NH4+ concentrations were between 10 and 21 nmol/kg—concentrations that have previously presented great problems for the determination of uptake and regeneration rates. The systems developed in this study have enabled accurate and precise determination of regeneration under oligotrophic conditions. These varied between 1.14 (F 0.24) and 1.58 (F0.32) nmol/kg/h, with the higher rate being measured at station A, with the largest NH4+ concentration. 4. Discussion 4.1. Low ammonium blanks and ammonium contamination Studies involving the determination of ammonium concentrations in samples derived from marine and freshwater environments are complicated by the ease with which samples may become contaminated. The significance of these sources is magnified when the ambient concentration diminishes, and may set the lower detection limit in some methods. In an overview of methods for the determination of isotope enrichment of ammonium, Ko¨ster and Jo¨ttner (1999) highlighted the
Table 2 Effect of ammonium regeneration on estimates of nitrogen assimilation and f-ratio
3.3. Ammonium regeneration rates in the North Atlantic Gyre
0.557 0.622 0.552 1.21 1.15 1.13
2.2
1.58 F 0.32a 1.14 F 0.24a 1.18 F 0.31a
2.0
20.9 F 4.8a 10.4 F 1.9a 12.2 F 1.0a
1.8
47.068N; 15.178W 38.108N; 24.418W 21.578N; 20.378W
1.6
A B C
1.4
NH+4 assimilation rate (nmol/kg/h)
1.2
NH+4 regeneration rate (nmol/kg/h)
1.0
NH+4 concentration (nmol/kg)
0.8
Position
0.6
2.84 1.83 2.14
2e+6
0.750b 0.146c 0.167c
3e+6
2.349b 1.589c 1.895c
Regeneration: assimilation ratio (a)
4e+6
Ammonium concentration and regeneration rates were measured at stations A, B and C in 2003. These rates are compared with nitrate and ammonium uptake rates determined at the same stations in 1997 and 1998. a This study. b Data from cruise AMT 5 (September/October 1997). c Data from cruise AMT 6 (May/June 1998).
8e+6
Station
GC/MS response
9e+6
0.209 (13.7%) 0.074 (12.1%) 0.072 (10.7%)
1e+7
127
0.242 0.084 0.081
Corrected f-ratio (% decrease)
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
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D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
advances made in constraining the significance of ammonium contamination from contributions N 500 nmol N/kg (Selmer and So¨rensson, 1986) to 150 nmol N/kg (Preston et al., 1996) and more recently 12.75 nmol N/ kg (Ko¨ster and Jo¨ttner, 1999). Reagents have been reported to be a significant source of contamination in methods which utilise the Berthelot or phenol/hypochlorite reaction. In the present study, while the reagent concentrations were the same as those used in Preston et al. (1996), the reagent volume to sample volume ratio was over three fold lower, hence minimising reagent contamination. Isotope dilution analysis of reagent-derived blanks in this study found that reagents contributed 30 nmol N/kg. While this is towards the lower end of the range reported by Ko¨ster and Jo¨ttner (1999), further work is needed to further decrease the significance of this source of contamination. An important principle in any analytical procedure is to ensure that standard and sample matrices are identical, so that reaction chemistry and yield are not adversely affected. This means that MQ water should not be used to generate standards for ammonium quantification in seawater samples. However, it has previously been difficult to obtain seawater with zero ammonium concentration and methods have relied on the use of dcorrection factorsT to allow for salt effects. In this paper we developed a PTFE–HCl system that consistently generated seawater with extremely low ammonium concentrations. Now ammonium determination in seawater can be done using the same seawater for standard curves as is used to determine ambient concentrations. Regression analysis demonstrated that treated seawater used for the generation of calibration standards in this instance contained a background ammonium concentration of 1 nmol/kg. Contributions to this contamination included glassware and sample manipulation, laboratory air and the possibility of residual ammonium not removed by the PTFE–HCl system. The most significant source of contamination to samples and standards was from reagents. 4.2. Sample preparation and analysis The use of SPE cartridges for the collection of samples in the field greatly increased the number of samples which could be generated by a single investigator, since the time-consuming processing could be done subsequently in a shore-based laboratory. The treatment of SPE cartridges prior to storage was crucial in order to ensure recovery efficiencies greater than 95%. Inadequate cleaning and storage of damp cartridges adversely affected the SPE matrix, resulting in
matrix degradation and the elution of particulates with samples. The presence of siloxane in GC/MS chromatographs was further evidence of matrix degradation. Consequently, ensuring that SPE cartridges were thoroughly rinsed with pH neutral MQ and thoroughly dried prior to storage was absolutely essential in order to ensure reproducibility and to avoid any additional sample clean-up prior to analysis. We have recently found that centrifugation (4000 rpm, 1 h) of SPE cartridges prior to storage is a more rigorous means of ensuring that the SPE matrices are moisture free. The method can be tailored to a range of concentrations by modifying derivatisation volume, injection volume or injection port purge timing. However, it is important to monitor chromatographic performance and sensitivity, especially when making any modifications which resulted in a higher sample loading oncolumn. A balance should be obtained to ensure sufficient sensitivity while introducing the minimum sample volume, thus maximising sample through-put between routine maintenance operations. The routine use of a test mixture identified changes in GC/MS performance, thus highlighting the need for maintenance. For example, after approximately 50 sample injections, a small but significant build-up of silane was evident during the analysis of test mixtures, which coincided with changes in chromatographic performance—specifically, a decrease in the resolution of methyl undecanoic acid and dicyclohexylamine test mixture components. Silane was removed and performance was re-established after the GC oven, injection port and transfer line were maintained at 325 8C for 4 h. While filament failure was not an issue during this analysis, decreases in sensitivity due to silica build-up on the source required close monitoring. The most obvious indicators of silica build-up was a decrease in absolute abundance and poor peak shape at m/z 502 for perfluorotributylamine (PFTBA) which the HP5972 MSD uses for electron impact tuning. Changes in performance were accounted for within and between runs by the use of the internal standard. The synthesis of a deuterated version of the sample molecule ensured that the characteristics of the internal standard were as closely matched to the sample as possible in terms of derivatisation efficiency, chromatography and MSD performance. Consequently, steps were taken to ensure that as far as possible, the sample to internal standard ratio was conserved. 4.3. Application of the method to the oligotrophic ocean In the present investigation ammonium regeneration was estimated from the magnitude of the dilution of 15N
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tracer in conjunction with changes in NH4+ concentration. Tracer experiments involve the addition of 15N substrates and it is standard practice to ensure that an upper limit of 10% of the ambient N concentration is not exceeded. However, in the oligotrophic oceans there is the danger that even such low additions might stimulate microbial activity. In this study, average enrichments prior to incubations were 3.6 F 0.45% of ambient ammonium concentration, and reflected the sensitivity of the method. Such additions represented less of a perturbation by comparison to other studies. The Blackburn-Caperon model (Blackburn, 1979; Caperon et al., 1979) was applied in the calculation of regeneration rates, as recommended by Laws (1984). Model assumptions were that regeneration was constant during the incubation and that there was no regeneration of 15N. The validity of making these assumptions can not be tested with this data set, although Laws et al. (1985) suggested that regeneration of 15N ammonium was negligible in 24 h open ocean experiments. Results presented in Table 2 suggest the potential for significant turnover of the ammonium pool during 24 h incubations. However, this type of analysis does not consider the finite time associated with processes such as assimilation, transformations, catabolism and excretion of Ncontaining intermediates by the various components of the microbial community in order to regenerate enriched NH4+. Nor does this type of analysis consider the sequential dilution of 15N within the various Npools prior to NH4+ regeneration. However, if 15NH4+ were regenerated this would result in an underestimation of regeneration rates. Consequently, the impact of not satisfying the assumptions recommended by Laws (1984) is likely to be minimised with lower regeneration rates, lower 15N tracer additions and shorter incubation times. On these grounds the data compared favourably with other studies in which the Blackburn–Caperon model has been applied. Given the sensitivity of the method, future studies should employ shorter incubation times in order to avoid the potential for the regeneration of enriched ammonium. The ammonium regeneration rates determined for three stations in the NE Atlantic are comparable to those reported for other regions. Raimbault et al. (1999) reported rates of 0.5–4.0 nmol N/kg/h in the Equatorial Pacific, Diaz and Raimbault (2000) reported rates of 2–9 nmol N/kg/h in the NW Mediterranean and Maguer et al. (1999) reported rates of 0.6–27 nmol N/ kg/h in the Western English Channel. What effect might these regeneration rates have on estimates of new production? In the absence of direct measurements, the ratio of regeneration to assimilation
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has generally been assumed to be in the range of 1–2 (Kanda et al., 1987). We cannot assess this ratio directly because we did not make measurements of nitrate or ammonium uptake on this cruise. However, data are available from other studies of this meridional transect which allow us to determine the possible magnitude of the effect. Nitrate and ammonium assimilation were measured in September–October 1997 and May–June 1998 (A.P. Rees, Plymouth Marine Laboratory, Personal Communication). Samples taken from depths equivalent to 55% sPAR were incubated for 6 h on-deck. Ambient ammonium concentrations in the three regions were comparable to those measured in the present study (26, 23 and 10 nmol/kg for stations A, B and C), so we have used the assimilation rates to get an indication of the likely scale of effect of isotope dilution by NH4+ regeneration processes during an incubation period. In 1997 and 1998, nitrate uptake rates were 0.15– 0.75 nmol/kg/h and ammonium uptake ranged between 1.6 and 2.3 nmol/kg/h (Table 2). These rates are consistent with other studies in oligotrophic oceans (Harrison et al., 1996). The equations of Kanda et al. (1987) were used to calculate the isotope dilution factor (x) defined as the ratio of actual to apparent NH4+ uptake rate. From these data, the likely ratio (a) of ammonium assimilation rate (corrected for isotope dilution) to regeneration rate was also determined (Table 2). Values of (a) b 1 imply an imbalance between ammonium regeneration and assimilation processes at all stations, with ammonium regeneration rates potentially limiting assimilation rates. Including ammonium regeneration estimates, the correction for isotope dilution also has an impact on f-ratio. In these regions that are dominated by regenerative productivity, the influence of isotope dilution only enhanced the importance of ammonium regeneration by decreasing the f-ratio by 11–14%. This is greater than the 5% overestimation observed in the NW Mediterranean by Diaz and Raimbault (2000). Given that oligotrophic oceans represent a major proportion of the global ocean, an overestimation of f-ratios of the order of 10% will have a significant impact on model predictions of primary productivity and C-export. 5. Conclusions The specificity of the phenol/hypochlorite reaction, in conjunction with GC/MS analysis, provided sensitivity gains over IRMS and allowed the analysis of dissolved inorganic nitrogen concentrations typical of oligotrophic regions. The protocol, which is readily applied at sea, enabled replicated samples to be gener-
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ated with only modest volumes of seawater, and avoided complications associated with distillation/diffusion/solvent extraction and associated fractionation/ efficiency issues. GC/MS provides a robust method to quantify sample-derived NH4+ concentration and isotopic enrichment. The impact of ammonium regeneration on NH4+ assimilation rates is non-trivial and should be considered in any study of new production. Acknowledgements This study was supported by the U.K. Natural Environment Research Council through grant number NER/A/S/2001/00636. We thank the steering committee of the Atlantic Meridional Transect consortium for the opportunity to participate in an AMT cruise. This is contribution number 98 of the AMT programme. The authors would like to thank Dr. Andrew Rees for providing N-assimilation data. References Blackburn, T.H., 1979. Method for measuring rates of NH+4 turnover in anoxic marine sediments, using a 15N–NH+4 dilution technique. Appl. Environ. Microbiol. 37, 760 – 765. Bremner, J.M., Keeney, D.R., 1965. Steam distillation methods for the determination of ammonium, nitrate and nitrite. Anal. Chim. Acta 32, 485 – 495. Caperon, J., Schell, D., Hirota, J., Laws, E., 1979. Ammonium excretion rates in Kaneohe Bay, Hawaii, measured by a 15N isotope dilution technique. Mar. Biol. 54, 33 – 40. Diaz, F., Raimbault, P., 2000. Nitrogen regeneration and dissolved organic nitrogen release during spring in a NW Mediterranean coastal zone (Gulf of Lions): implication for the estimation of new production. Mar. Ecol., Prog. Ser. 197, 51 – 65. Dugdale, R.C., Goering, J.J., 1967. Uptake of new and regenerated forms of nitrogen in primary production. Limnol. Oceanogr. 12, 199 – 206. Eppley, R.W., Peterson, B.J., 1979. Particulate organic matter flux and planktonic new production in the deep ocean. Nature 282, 677 – 680. Glibert, P.M., 1982. Regional studies of daily, seasonal and size fraction variability in ammonium remineralization. Mar. Biol. 70, 209 – 222. Grob, K., Grob, G., Grob Jr., K., 1981. Testing capillary gas chromatography columns. J. Chromatogr. 219, 13 – 20. Haesgawa, T., Koike, I., Mukai, H., 2000. Dissolved organic nitrogen dynamics in coastal waters and the effect of copepods. J. Exp. Mar. Biol. Ecol. 244, 219–238. Harrison, W.G., Harris, L.R., 1986. Isotope-dilution and its effects on measurements of nitrogen and phosphorus uptake by oceanic microplankton. Mar. Ecol., Prog. Ser. 27, 253 – 261.
Harrison, W.G., Harris, L.R., Irwin, B.D., 1996. The kinetics of nitrogen utilisation in the oceanic mixed layer: nitrate and ammonium interactions at the nanomolar concentrations. Limnol. Oceanogr. 41, 16 – 32. Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B., Peterson, B.J., 1998. Measuring 15N–NH+4 in marine, estuarine and fresh waters: an adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar. Chem. 60, 235 – 243. IOC, Paris (France), 1994. Protocols for the Joint Global Flux Study (JGOFS) core measurements. Manuals Guides 29:126 pp. Joint, I., Rees, A.P., Woodward, E.M.S., 2001. Primary production and nutrient assimilation in the Iberian upwelling in August 1998. Prog. Oceanogr. 51, 303 – 320. Jones, R.D., 1991. An improved fluorescence method for the determination of nanomolar concentrations of ammonium in natural waters. Limnol. Oceanogr. 36 (4), 814 – 819. Kanda, J., Laws, E.A., Saino, T., Hattori, A., 1987. An evaluation of isotope dilution effect from conventional data sets of 15N uptake experiments. J. Plankton Res. 19, 79 – 90. Ko¨ster, O., Jo¨ttner, F., 1999. NH+4 utilisation and regeneration rates in freshwater lakes determined by GC-MS of derivatised dihydroindophenol. J. Microbiol. Methods 37, 65 – 76. Laws, E., 1984. Isotope dilution models and the mystery of the vanishing 15N. Limnol. Oceanogr. 29, 379 – 386. Laws, E.A., Harrison, W.G., DiTullio, G.R., 1985. A comparison for nitrogen assimilation rates based on 15N uptake and autotrophic protein synthesis. Deep-Sea Res. 32, 85 – 95. Maguer, J., L’Helguen, S., Madec, C., Corre, P., 1999. Seasonal patterns of ammonium regeneration from size-fractioned microheterotrophs. Cont. Shelf Res. 19, 1755 – 1770. Preston, T., Bury, S., Presing, M., Moncoiffe, G., Slater, C., 1996. Isotope dilution analysis of combined nitrogen in natural waters: 1. Ammonium. Rapid Commun. Mass Spectrom. 10, 959 – 964. Raimbault, P., Slawyk, G., Boudjella, B., Coatanon, C., Conan, P., Coste, B., Garcia, N., Moutin, T., Pujo-Pay, M., 1999. Carbon and nitrogen uptake and export in the equatorial Pacific at 150 8W: evidence for an efficient regenerated production cycle. J. Geophys. Res. 104, 3341 – 3356. Selmer, J.S., So¨rensson, F., 1986. New procedure for extraction of ammonium from seawater for 15N isotopic ratio determinations. Appl. Environ. Microbiol. 52, 577 – 579. Sigman, D.M., Altabet, M.A., Michener, R., McCorkle, D.C., Fry, B., Holmes, R.M., 1997. Natural abundance level measurement of nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonium diffusion method. Mar. Chem. 57, 227 – 242. Slawyk, G., Raimbault, P., 1995. Simple procedure for simultaneous recovery of dissolved inorganic and organic nitrogen in 15N-tracer experiments and improving the isotopic mass balance. Mar. Ecol., Prog. Ser. 124, 289 – 299. Tupas, L.M., Koike, I., Karl, D.M., Holm-Hansen, O., 1994. Nitrogen metabolism by heterotrophic bacterial assemblages in Antarctic coastal waters. Polar Biol. 14, 195 – 204. Ward, B.B., Bronk, D.A., 2001. Net nitrogen uptake and DON release in surface waters: importance of trophic interactions implied from size fractionation experiments. Mar. Ecol., Prog. Ser. 219, 11 – 24.
Determination of ammonium regeneration rates in the oligotrophic ocean by gas chromatography/mass spectrometry Darren R. Clark *, Tim W. Fileman, Ian Joint Plymouth Marine Laboratory, Prospect Place, Plymouth, Devon, PL1 3DH, United Kingdom Received 14 December 2004; received in revised form 31 May 2005; accepted 19 August 2005 Available online 12 October 2005
Abstract A method has been developed for the determination of ammonium concentration and isotopic enrichment in seawater samples at the low nanomolar range (10–100 nmol/kg). It is based on the reaction of phenol/hypochlorite with ammonium to form indophenol, with subsequent solid phase extraction, derivatisation and analysis by Gas Chromatography Mass Spectrometry. The precision of the method was maximised by incorporating a deuterated indophenol internal standard. A system was developed which generated seawater with extremely low ammonium concentrations thus matching sample and standard matrices for quantitative analysis. Data are presented from a study of ammonium regeneration rates at three stations in the oligotrophic North–East Atlantic where ambient ammonium concentrations were b 21 nmol/kg. Results suggested that ammonium availability for phytoplankton was limited by the rate of ammonium regeneration. Efficient ammonium assimilation contributed to the very low ambient ammonium concentrations measured at these stations. The study highlights the need for the accurate determination of ammonium regeneration rates in studies of new production, particularly in extreme oligotrophic conditions. If not corrected for isotope dilution, f-ratio estimates may be overestimated by 10.7–13.7%. D 2005 Elsevier B.V. All rights reserved. Keywords: Ammonium; Regeneration;
15
N; GC/MS; Indophenol; f-ratio; North–East Atlantic
1. Introduction Ammonium regeneration is an important component of N-cycling, often providing the dominant form of nitrogen for N-assimilation by marine phytoplankton in many regions of the global ocean. Estimates of phytoplankton inorganic N demand (primarily as NH4+ and NO3) are usually determined by 15N tracer techniques that generate assimilation data. These rates are used to estimate the f-ratio (Dugdale and Goering,
* Corresponding author. Tel.: +44 1752 633100; fax: +44 1752 633101. E-mail address: [email protected] (D.R. Clark). 0304-4203/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2005.08.006
1967; Eppley and Peterson, 1979) and hence, to determine global-ocean carbon flux. However, many estimates of new production may be in error because most studies have not taken into account the regeneration of unlabeled 14NH4+ in tracer experiments (Glibert, 1982; Harrison and Harris, 1986). This omission creates uncertainties concerning the extent of isotope dilution and consequently may lead to underestimation of NH4+ assimilation rates. The majority of ammonium regeneration studies have been limited to marine provinces with relatively high NH4+ concentrations, usually in the low Amol/kg range, where the recovery of sufficient N for Emission Spectrometry or Isotope Ratio Mass Spectrometry (IRMS) analysis has rarely been an issue. Previous
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approaches have included the diffusion (Slawyk and Raimbault, 1995; Sigman et al., 1997) or distillation methods (Bremner and Keeney, 1965; Tupas et al., 1994; Haesgawa et al., 2000; Ward and Bronk, 2001), with detection limits generally in the order of 2 Amol/kg of ambient NH4+. In an adaptation of the diffusion method, Holmes et al. (1998) improved the limit of detection to 0.5 Amol/kg of ambient NH4+ (1 Amol of sample-N for IRMS), partly by increasing sample volume (and hence total sample N) and also by correcting for isotopic fractionation associated with the diffusion method. For the analysis of samples with yet lower ammonium concentrations, previous methods have perturbed samples by the addition of unlabelled N dcarrierT in order to provide sufficient sample N for IRMS analysis (Slawyk and Raimbault, 1995; Diaz and Raimbault, 2000). These methods have proven to be relatively labour intensive and time consuming, limiting the scope and replication of samples generated. In addition, the detection limit of IRMS makes studies of N flux in the oligotrophic oceans extremely challenging, because NH4+ concentrations are typically in the range of 10– 100 nmol/kg. In a departure from the IRMS approach, Preston et al. (1996) and Ko¨ster and Jo¨ttner (1999) presented methods based on the use of Gas Chromatography Mass Spectrometry (GC/MS) for marine and freshwater environments respectively. These methods employed either the phenol/hypochlorite or Berthelot reactions forming indophenol from dissolved NH4+, with subsequent collection and isotopic analysis of volatile derivatives. With injections of sample N for GC/MS analysis typically in the order of 200 pmol, the method of Preston et al. (1996) demonstrated the sensitivity that could be gained by the development of GC/MS methods, with measurements at ambient concentrations as low as 190 nmol/kg. The method presented here is a development of that of Preston et al. (1996) and combines the advantages of ammonium-specific derivatisation and pre-analysis concentration of sample-N by solid phase extraction, with the sensitivity of GC/MS analysis. A system is presented which removes ammonium and provides seawater with very low ammonium concentrations (~ 1 nmol/l) suitable for the generation of calibration standards. Greatly improved precision over the Preston et al. (1996) method was obtained by incorporating a deuterated indophenol internal standard, which minimised cumulative error during the preparation and analysis of samples. The procedures were tested in the oligotrophic waters of the North Atlantic Gyre. Regeneration rate data are presented from three stations in
which the ambient ammonium concentrations were in the range of approximately 10–20 nmol/kg. The impact of the measured NH4+ regeneration rates upon estimations of N-assimilation rates and f-ratio values is discussed. 2. Materials and methods 2.1. Low-ammonium seawater generation for standards—the PTFE–HCl system Studies of ammonium cycling in the sea are often frustrated by the problem of obtaining seawater with sufficiently low NH4+ concentrations for the preparation of standards. On this study, we developed a method to obtain seawater with extremely low NH4+ concentrations. The basis of the method is the diffusion of ammonium from seawater through a PTFE membrane into acid, building on the principles of Jones (1991). A 2 l volume of low nutrient seawater, collected from oligotrophic regions, typically containing 10–100 nmol/kg NH4+, was filtered through a 0.2Am membrane filter and placed in an acid-cleaned 2 l borosilicate reservoir vessel containing a magnetic stirrer bar. The vessel was sealed with a gas tight cap fitted with a 3-valve adaptor. Water was pumped at a flow rate of 10 ml/min from this reservoir through 8 1 m lengths of PTFE tubing (2 Am porosity, 1 mm internal diameter, 0.4 mm thick, W.L. Gore and Associates U.K.), immersed in 10% HCl. The treated seawater was returned to the 2-l vessel, completing a continuous loop. Seawater was recirculated through this system for N 24 h to obtain essentially ammonium-free seawater. It was noted that the pH of seawater produced with new PTFE tubing decreased, typically in the order of 0.1–0.5 pH units. However, this effect became less significant with successive seawater batches without affecting system performance, i.e. the PTFE tubing in the system required dagingT. Seawater which had been processed with the PTFE– HCl system was used for the generation of calibration standards. 2 l of processed seawater was split into 4 500 ml volumes and placed in 550 ml amber glass bottles. A spike of NH4Cl was added to three of the standards to give a final concentration within the range of 5–50 nmol/kg. No addition was made to the fourth standard which served as a zero. During the application of the method at sea calibration standards were generated throughout the duration of the fieldwork. A minimum of 3 measurments was made for each calibration concentration.
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Indophenol was developed, collected and analysed as described in Sections 2.2 and 2.4. After normalisation (see Section 2.7), a calibration curve was constructed by plotting integrated peak area (m/z 345.2 plus m/z 346.2 corresponding to 14N and 15N, respectively) from a calibration standard against the standard concentration. 2.2. Indophenol development All chemicals were supplied by Sigma-Aldrich Co. Ltd., unless otherwise stated. Acid-cleaned borosilicate glasswear was used exclusively. The use of plastics was avoided and all experimental work was undertaken while wearing nitrile gloves. Reagents were made fresh daily and stored in gas-tight borosilicate bottles at 4 8C until use. The first reagent was made by dissolving 2.35 g phenol in 100 ml of milli-Qk water (MQ) followed by 0.16 g sodium nitroprusside. The second reagent was made by dissolving 0.6 g sodium dichloro-isocyanurate in 100 ml of MQ followed by 1.4 g sodium hydroxide. Standard and sample volumes (500 ml) were placed in 550 ml amber glass bottles. Indophenol synthesis was initiated by adding 3 ml of phenol/sodium nitroprusside reagent to the sample or standard, which was thoroughly mixed and left for 5 min. 3 ml of sodium dichloro-isocyanurate/sodium hydroxide reagent was added to the sample or standard which was again thoroughly mixed. Samples and standards were placed in the dark at 18 8C. Indophenol development was complete after 12 h. Indophenol generated in samples and standards was collected by solid phase extraction (SPE) using 6 ml/ 500 mg C18 cartridges (Kinesis Ltd., UK) that required a preparatory cleaning procedure. It was important to ensure that the SPE matrix did not dry between the conditioning and sample collection stages. After soaking the SPE in methanol for 5 min, it was rinsed sequentially with 10 ml methanol, 10 ml MQ and 10 ml 1% NaCl in MQ. Samples and standards were acidified by the addition of 700 Al of 1 mol/l citric acid, resulting in a pH of approximately 5.5. A deuterated indophenol internal standard (see Section 2.3) was added prior to the collection of samples and standards by SPE under low vacuum (120 mm Hg). The SPE cartridges were then rinsed with 50 ml of MQ and allowed to run dry. The SPE matrix was then rigorously dried under high vacuum (360 mm Hg) for 10 min. At this stage, indophenol could be eluted from the C18 as described below, or the cartridge could be capped and stored at 20 8C. It was found to be imperative that the
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SPE matrix was pH-neutral and completely dry prior to storage. If this was not the case, matrix degradation would occur resulting in analytical complications. Indophenol was eluted from the SPE cartridges by first allowing 7 ml of methanol to soak into the SPE matrix for 5 min, before drawing the solvent through the cartridge under low vacuum for collection in a 10ml test tube. Sample volume was decreased to approximately 200 Al using a Zymark Turbo-vap (high capacity evaporating unit using oxygen-free nitrogen). Samples were quantitatively transferred to GC vials with 300-Al inserts (Chromacol Ltd., UK) and blown to dryness. Sample tubes were rinsed with a further 200 Al methanol which was also transferred to GC vials and blown to dryness. Once dry, samples could be analysed as described in Section 2.4, or capped and refrigerated until analysis. 2.3. Deuterated indophenol internal standard The accuracy and precision of the method was greatly improved by including an internal standard, which in this case was a deuterated form of the sample molecule. 1 g of phenol-3,5-d2 (Qmx Laboratories Ltd., UK) was dissolved in 42 ml of MQ to which 0.068 g sodium nitroprusside was added. The sodium dichloroisocyanurate/sodium hydroxide reagent was made as described in Section 2.2. 42 ml of phenol-3,5-d2/sodium nitroprusside reagent was added to 1 l of 2.5 mmol/ l NH4Cl in MQ and thoroughly mixed. After 5 min, 42 ml of sodium dichloro-isocyanurate/sodium hydroxide reagent was added, the volume was thoroughly mixed and incubated at 18 8C for 24 h for deuterated indophenol development. Deuterated indophenol was collected using 70 ml/10 g C18 SPE cartridges (Kinesis Ltd., UK). Two cartridges were used for the collection of the entire volume of deuterated indophenol. 70 ml/10 g C18 SPE cartridges were first cleaned in a scaled-up version of the protocol described in Section 2.2. Prior to collection, sufficient citric acid was added to the deuterated indophenol solution to turn it from deep blue to red (pH c 5.5). Deuterated indophenol was then collected, and subsequently cleaned with 500 ml MQ per cartridge. The C18 matrix was thoroughly dried under high vacuum for 10 min. Deuterated indophenol was eluted into boiling tubes from the C18 matrices using 20 ml methanol per cartridge. The deuterated indophenol was transferred to a watch glass and placed in an oven at 50 8C in 5 ml batches in order to obtain dry deuterated indophenol powder. 2 mg deuterated indophenol powder was dissolved in 1 l MQ water at pH 10.0. The solution was acidified to pH 6.0. 1 ml deu-
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terated indophenol internal standard was added to seawater samples and standards after they had been acidified by the addition of citric acid and prior to collection by SPE. 2.4. Indophenol analysis by GC/MS Samples and standards were derivitised with Sylon TP (25% trimethylsilylimidazole in pyridine, SigmaAldrich, UK), which was purchased in 1 ml ampoules, each of which was used within 24 h. A volume of between 20 and 100 Al of Sylon TP was added to samples which were incubated at 50 8C for 30 min. Isotopic analysis of bis(trimethylsilyl)-indophenol was performed using a Hewlett-Packard 5890 series II GC coupled to a HP5972 mass selective detector (MSD). Samples with a volume of 0.5–1.0 Al were injected in splitless mode using a HP7973 injection controller onto a HP-5ms column (30 m 0.25 mm internal diameter). The injection port contained a liner packed with glass wool and the capillary column was further protected by a 1-m length of uncoated deactivated pre-column, both of which were replaced at regular intervals during routine maintenance. Injection port and transfer line temperatures were 300 8C. Derivitised indophenol was resolved using temperature programming: GC oven initial temperature of 95 8C; ramped to 210 8C at 45 8C/min; ramped to 310 8C at 20 8C/min; hold for 5 min. For quantitative analysis the MSD was operated using selected ion monitoring (SIM). Ions used for SIM were molecular ions with m/z 345.2 and 346.2 for samples or standards and m/z 349.2 and 350.2 for deuterated internal standard. Bis(trimethylsilyl)-indophenol and deuterated internal standard co-eluted at 6.1 min. Integrated peak area was used for quantitative and isotopic analysis. Capillary column performance was routinely monitored by the use of a test mixture (Grob et al., 1981; Sigma-Aldrich UK).
rinsed with 10 ml methanol and 10 ml MQ, eluted in 5 ml of methanol and blown to dryness. 1 ml of methanol was added to samples which were then quantitatively transferred to micro-centrifuge tubes and centrifuged at 13000 rpm for 20 min. Samples were transferred to GC vials and blown to dryness, ensuring that any particulate material remained undisturbed in the centrifuge tube. 2.6. Blank determination The contribution to sample-N derived from reagents was determined by adding 5, 10 and 15 ml of each reagent (i.e. reagent concentration factors of 1.6, 3.3 and 5 times normal additions respectively) to triplicate 500 ml volumes of 0.22 Am filtered seawater containing 200 nmol/kg 15NH4Cl. A regression between blank concentration (i.e. 14NH4+ derived from seawater and reagents) and reagent concentration factor enabled the contribution to sample-N derived from reagents to be calculated. 2.7. Normalisation of samples for quantification The deuterated internal standard was used to enable the normalisation of all samples and calibration standards thereby removing variance associated with sample manipulation and changes in recovery efficiency or GC/MS performance. The integrated peak area of the deuterated internal standard associated with a calibration standard was used as the benchmark against which all other samples and standards were normalised. In this case a calibration standard at a concentration of 25 nmol/kg was chosen as this was approximately in the middle of the calibration curve concentration range. Peak areas were normalised using the equation below: 349:2 ! d IP Std Peak345:2 PeakNormð345:2Þ ¼ d IP Sample349:2
2.5. Sample cleaning procedure Inappropriate sample storage resulted in SPE matrix degradation and contamination of samples. Contamination derived from the SPE matrix took the form of both particulate material and a chemical component (silane), and necessitated sample re-collection on C18’s and centrifugation in order to remove both contaminating components. Samples were quantitatively transferred from GC vials to test tubes in 3 200 Al aliquots of 3 mol/l NaOH. 5 ml MQ was added and samples were then acidified with 600 Al of 1 mol/l citric acid. Samples were collected on SPE cartridges which had been
where PeakNorm(345.2) was the normalised peak area for m/z 345.2 of a sample or standard, d-IP(Std349.2) was the integrated peak area of the deuterated internal standard at m/z 349.2 associated with the 25 nmol/kg calibration standard benchmark, d-IP(Sample349.2) was the integrated peak area of the deuterated internal standard at m/z 349.2 associated with the sample and Peak345.2 was the peak area for m/z 345.2 of a sample or standard. Noting that m/z 345.2 and 349.2 correspond to 14N of the sample and internal standard, respectively (as represented in the equation), while m/ z 346.2 and 350.2 correspond to 15N of the sample and
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2.8. Data analysis Regeneration rates (r, nmol N/kg/h) were determined using normalised data according to the Blackburn–Caperon model (Blackburn, 1979; Caperon et al., 1979) described by Laws (1984): Rt=Ro St r ¼ ln So St=So t where So was the pre-incubation ammonium concentration (nmol N/kg) and St was the post-incubation ammonium concentration (nmol N/kg) after t hours of incubation. Ro and Rt were the pre-incubation and postincubation 15N enrichment ratios, respectively. Ro was determined using the equation: Ro ¼
Mo Mn 1 þ ðMo MnÞ
where Mo was the pre-incubation ratio of m/z 346.2/ 345.2, Mn was the natural abundance ratio of m/z 346.2/345.2 derived from calibration standards. Rt was determined by substituting Mo for Mt defined as the post-incubation enrichment ratio of m/z 346.2/345.2 at time t. 2.9. Application of the method to extremely oligotrophic water Samples were taken during a transect of the Atlantic Ocean between the UK and the Falkland Islands on board RRS James Clark Ross during September 2003 as part of the Atlantic Meridian Transect programme (AMT 13). Results for only three stations in the North Atlantic Gyre are presented here. Regeneration was determined on seawater samples collected during a pre-dawn CTD cast from depths corresponding to 55% surface Photosynthetically Active Radiation (sPAR). 2 l was filtered (GF/F) and distributed between three 500 ml samples for the determination of ambient NH4+ concentrations. NH4+ regeneration was measured by adding 0.5–1 nmol/kg 15NH4Cl to 7 l seawater. The seawater was mixed, distributed between two 2.4 l vessels, placed into incubators on deck prior to sunrise and left for 24 h. Irradiance equivalent to the depth from which the samples were taken was simulated using Perspex screens (Joint et al., 2001) and incubation procedures followed JGOFS protocols (IOC, 1994).
The remaining enriched seawater was filtered and distributed between three 500-ml samples for the determination of pre-incubation 15N enrichment. The incubator was maintained at surface sea-temperature by cooling with surface seawater from the ship’s non-toxic supply. 24 h deck incubations were terminated by filtration (GF/F), the filtrate being distributed between three 500-ml replicates for post-incubation enrichment determinations. Indophenol was synthesised, collected and analysed as described in Sections 2.2 and 2.4. 3. Results 3.1. Low ammonium seawater and reagent blank This study was concerned with the measurement of ammonium concentration and isotopic enrichment under extreme oligotrophic conditions. The improved accuracy and precision of the method depended on a number of factors but primarily these were the inclusion of the deuterated indophenol internal standard and the ability to match sample and standard matrices for quantitative analysis. Passing seawater through PTFE tubing immersed in an acid bath (see Section 2.1) was an effective way of removing ammonium from seawater. In order to demonstrate the principle, a 2 l volume of seawater was spiked with 1.2 Amol/kg NH4Cl and the removal of NH4+ from seawater during treatment was measured as a decrease in GC/MS response over time (Fig. 1). Results demonstrated that NH4+ concentration decreased below GC/MS detection limits within 48 h. System performance was subsequently enhanced by increasing the effective PTFE tubing length from 5e+7 4e+7
GC/MS response
internal standard, respectively, appropriate substitutions in the equation above were made in order to normalise 15 N peaks.
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3e+7 2e+7 1e+7 0 0
20
40
60
80
100 120 140 160 180
Time (h) Fig. 1. GC-MS response to bis(trimethylsilyl)-indophenol reflecting the change in ammonium concentration during the treatment of seawater with the PTFE–HCl system. 1.2 Amol/kg NH4Cl was added to seawater and passed through 4 1 m lengths of PTFE tubing. Error bars indicate 95% confidence limits about the mean.
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
4 1 m to 8 1 m. The resulting ammonium concentrations in treated seawater derived from oligotrophic oceans (typically 10–100 nmol/kg NH4+) were indistinguishable from MQ water within 12 h of processing. Laboratory reagents are an additional source of ammonium contamination that must be quantified. Table 1 presents isotope dilution data for the quantification of reagent-derived ammonium. A regression of blank (14NH4+ derived from reagents and seawater) against reagent concentration factor indicated that the ammonium concentration that would be added with the reagents was 30 nmol/kg (blank = 30 concentration factor + 253; R 2 = 0.99).
1e+7
A m/z 345
GC/MS response
126
8e+6 6e+6 4e+6 2e+6
m/z 73 m/z 180
0 0
100
8e+6
Concentration 15NH+4 addition factor of (nmol/kg) reagents
At% excess 15 N
1.66 3.33 5.00
39.94 F 0.34 300.8 35.58 F 0.59 362.1 33.22 F 2.71 402.1
200 200 200
Blank (14NH+4 from reagents plus seawater) (nmol/kg)
5, 10 and 15 ml of each reagent were added to triplicate 500 ml volumes of 0.2 Am filtered seawater containing 200 nmol/kg 15 NH4Cl.
400
GC/MS response
deuterated indophenol m/z 349
B
bis(TMS)-indophenol m/z 345
6e+6
4e+6
2e+6
m/z 73 m/z 180
m/z 182
0 0
100
200
300
400
m/z Fig. 2. Electron ionisation mass spectra of (A) bis(trimethylsilyl)indophenol and (B) bis(trimethylsilyl)-indophenol in conjunction with bis(trimethylsilyl)-d4-indophenol internal standard.
2.5e+7
GC/MS response
Table 1 Determination of ammonium concentration in reagents by isotope dilution analysis of 200 nmol/kg 15NH+4 added to a seawater sample containing 253 nmol/kg NH+4
300
m/z
3.2. Indophenol analysis by GC/MS and method optimisation Bis(trimethylsilyl)-indophenol gave a simple electron ionisation (EI) spectra (Fig. 2A) with a prominent molecular ion at m/z 345.2. For quantitative analysis samples were analysed in conjunction with the deuterated internal standard (molecular ion m/z 349.2; Fig. 2B). In order to maximise the yield of indophenol and hence optimise the method for maximum sensitivity, the time of indophenol development was determined (Fig. 3). Reagents were added proportionately (v/v) to a 1 l volume of 400 nmol/kg NH4Cl in 0.22 Am filtered oligotrophic seawater. Sample volume was 50 ml such that GC/MS injections of sample-N were quantitatively comparable to those generated from field samples (100 pmol NH4+–N injected). Results demonstrated that for maximum yield an incubation time in excess of 12 h at 18 8C was required. Further increases in response during GC/MS analysis could be gained by increasing injection port purge timing (Fig. 4), modifying derivatisation volume within the range (20–100 Al) or increasing injection volume within the range 0.5–1.0 Al. With such modifications an
200
2.0e+7 1.5e+7 1.0e+7 5.0e+6 0.0 0
2
4
6
8
10
12
14
16
Time (h) Fig. 3. The development of indophenol over time at 18 8C determined by GC-MS analysis of bis(trimethylsilyl)-indophenol. A concentration of 400 nmol/kg NH4Cl was added to filtered low nutrient seawater allowing for the analysis of small seawater sample volumes (50 ml). Results demonstrate the GC-MS response to an injection of 100 pmol NH+4 –N. Error bars indicate 95% confidence limits about the mean.
7e+6
Original f-ratio
6e+6 5e+6
Dilution factor (x)
Inlet purge timing (min)
Corrected NH+4 uptake rate (nmol/kg/h)
Fig. 4. Influence of injection port purge timing upon the magnitude of the GC-MS response to bis(trimethylsilyl)-indophenol during repeat injections of the same sample at 50 pmol NH4–N per injection. Error bars indicate 95% confidence limits about the mean.
NO 3 assimilation rate (nmol/kg/h)
appropriate level of GC/MS sensitivity could be tailored to seawater samples with a range of ammonium concentrations. However, there are important limits to these modifications as discussed in Section 4.2.
Ammonium regeneration was measured at 3 stations in the oligotrophic North Atlantic (Table 2). NH4+ concentrations were between 10 and 21 nmol/kg—concentrations that have previously presented great problems for the determination of uptake and regeneration rates. The systems developed in this study have enabled accurate and precise determination of regeneration under oligotrophic conditions. These varied between 1.14 (F 0.24) and 1.58 (F0.32) nmol/kg/h, with the higher rate being measured at station A, with the largest NH4+ concentration. 4. Discussion 4.1. Low ammonium blanks and ammonium contamination Studies involving the determination of ammonium concentrations in samples derived from marine and freshwater environments are complicated by the ease with which samples may become contaminated. The significance of these sources is magnified when the ambient concentration diminishes, and may set the lower detection limit in some methods. In an overview of methods for the determination of isotope enrichment of ammonium, Ko¨ster and Jo¨ttner (1999) highlighted the
Table 2 Effect of ammonium regeneration on estimates of nitrogen assimilation and f-ratio
3.3. Ammonium regeneration rates in the North Atlantic Gyre
0.557 0.622 0.552 1.21 1.15 1.13
2.2
1.58 F 0.32a 1.14 F 0.24a 1.18 F 0.31a
2.0
20.9 F 4.8a 10.4 F 1.9a 12.2 F 1.0a
1.8
47.068N; 15.178W 38.108N; 24.418W 21.578N; 20.378W
1.6
A B C
1.4
NH+4 assimilation rate (nmol/kg/h)
1.2
NH+4 regeneration rate (nmol/kg/h)
1.0
NH+4 concentration (nmol/kg)
0.8
Position
0.6
2.84 1.83 2.14
2e+6
0.750b 0.146c 0.167c
3e+6
2.349b 1.589c 1.895c
Regeneration: assimilation ratio (a)
4e+6
Ammonium concentration and regeneration rates were measured at stations A, B and C in 2003. These rates are compared with nitrate and ammonium uptake rates determined at the same stations in 1997 and 1998. a This study. b Data from cruise AMT 5 (September/October 1997). c Data from cruise AMT 6 (May/June 1998).
8e+6
Station
GC/MS response
9e+6
0.209 (13.7%) 0.074 (12.1%) 0.072 (10.7%)
1e+7
127
0.242 0.084 0.081
Corrected f-ratio (% decrease)
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
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D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
advances made in constraining the significance of ammonium contamination from contributions N 500 nmol N/kg (Selmer and So¨rensson, 1986) to 150 nmol N/kg (Preston et al., 1996) and more recently 12.75 nmol N/ kg (Ko¨ster and Jo¨ttner, 1999). Reagents have been reported to be a significant source of contamination in methods which utilise the Berthelot or phenol/hypochlorite reaction. In the present study, while the reagent concentrations were the same as those used in Preston et al. (1996), the reagent volume to sample volume ratio was over three fold lower, hence minimising reagent contamination. Isotope dilution analysis of reagent-derived blanks in this study found that reagents contributed 30 nmol N/kg. While this is towards the lower end of the range reported by Ko¨ster and Jo¨ttner (1999), further work is needed to further decrease the significance of this source of contamination. An important principle in any analytical procedure is to ensure that standard and sample matrices are identical, so that reaction chemistry and yield are not adversely affected. This means that MQ water should not be used to generate standards for ammonium quantification in seawater samples. However, it has previously been difficult to obtain seawater with zero ammonium concentration and methods have relied on the use of dcorrection factorsT to allow for salt effects. In this paper we developed a PTFE–HCl system that consistently generated seawater with extremely low ammonium concentrations. Now ammonium determination in seawater can be done using the same seawater for standard curves as is used to determine ambient concentrations. Regression analysis demonstrated that treated seawater used for the generation of calibration standards in this instance contained a background ammonium concentration of 1 nmol/kg. Contributions to this contamination included glassware and sample manipulation, laboratory air and the possibility of residual ammonium not removed by the PTFE–HCl system. The most significant source of contamination to samples and standards was from reagents. 4.2. Sample preparation and analysis The use of SPE cartridges for the collection of samples in the field greatly increased the number of samples which could be generated by a single investigator, since the time-consuming processing could be done subsequently in a shore-based laboratory. The treatment of SPE cartridges prior to storage was crucial in order to ensure recovery efficiencies greater than 95%. Inadequate cleaning and storage of damp cartridges adversely affected the SPE matrix, resulting in
matrix degradation and the elution of particulates with samples. The presence of siloxane in GC/MS chromatographs was further evidence of matrix degradation. Consequently, ensuring that SPE cartridges were thoroughly rinsed with pH neutral MQ and thoroughly dried prior to storage was absolutely essential in order to ensure reproducibility and to avoid any additional sample clean-up prior to analysis. We have recently found that centrifugation (4000 rpm, 1 h) of SPE cartridges prior to storage is a more rigorous means of ensuring that the SPE matrices are moisture free. The method can be tailored to a range of concentrations by modifying derivatisation volume, injection volume or injection port purge timing. However, it is important to monitor chromatographic performance and sensitivity, especially when making any modifications which resulted in a higher sample loading oncolumn. A balance should be obtained to ensure sufficient sensitivity while introducing the minimum sample volume, thus maximising sample through-put between routine maintenance operations. The routine use of a test mixture identified changes in GC/MS performance, thus highlighting the need for maintenance. For example, after approximately 50 sample injections, a small but significant build-up of silane was evident during the analysis of test mixtures, which coincided with changes in chromatographic performance—specifically, a decrease in the resolution of methyl undecanoic acid and dicyclohexylamine test mixture components. Silane was removed and performance was re-established after the GC oven, injection port and transfer line were maintained at 325 8C for 4 h. While filament failure was not an issue during this analysis, decreases in sensitivity due to silica build-up on the source required close monitoring. The most obvious indicators of silica build-up was a decrease in absolute abundance and poor peak shape at m/z 502 for perfluorotributylamine (PFTBA) which the HP5972 MSD uses for electron impact tuning. Changes in performance were accounted for within and between runs by the use of the internal standard. The synthesis of a deuterated version of the sample molecule ensured that the characteristics of the internal standard were as closely matched to the sample as possible in terms of derivatisation efficiency, chromatography and MSD performance. Consequently, steps were taken to ensure that as far as possible, the sample to internal standard ratio was conserved. 4.3. Application of the method to the oligotrophic ocean In the present investigation ammonium regeneration was estimated from the magnitude of the dilution of 15N
D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
tracer in conjunction with changes in NH4+ concentration. Tracer experiments involve the addition of 15N substrates and it is standard practice to ensure that an upper limit of 10% of the ambient N concentration is not exceeded. However, in the oligotrophic oceans there is the danger that even such low additions might stimulate microbial activity. In this study, average enrichments prior to incubations were 3.6 F 0.45% of ambient ammonium concentration, and reflected the sensitivity of the method. Such additions represented less of a perturbation by comparison to other studies. The Blackburn-Caperon model (Blackburn, 1979; Caperon et al., 1979) was applied in the calculation of regeneration rates, as recommended by Laws (1984). Model assumptions were that regeneration was constant during the incubation and that there was no regeneration of 15N. The validity of making these assumptions can not be tested with this data set, although Laws et al. (1985) suggested that regeneration of 15N ammonium was negligible in 24 h open ocean experiments. Results presented in Table 2 suggest the potential for significant turnover of the ammonium pool during 24 h incubations. However, this type of analysis does not consider the finite time associated with processes such as assimilation, transformations, catabolism and excretion of Ncontaining intermediates by the various components of the microbial community in order to regenerate enriched NH4+. Nor does this type of analysis consider the sequential dilution of 15N within the various Npools prior to NH4+ regeneration. However, if 15NH4+ were regenerated this would result in an underestimation of regeneration rates. Consequently, the impact of not satisfying the assumptions recommended by Laws (1984) is likely to be minimised with lower regeneration rates, lower 15N tracer additions and shorter incubation times. On these grounds the data compared favourably with other studies in which the Blackburn–Caperon model has been applied. Given the sensitivity of the method, future studies should employ shorter incubation times in order to avoid the potential for the regeneration of enriched ammonium. The ammonium regeneration rates determined for three stations in the NE Atlantic are comparable to those reported for other regions. Raimbault et al. (1999) reported rates of 0.5–4.0 nmol N/kg/h in the Equatorial Pacific, Diaz and Raimbault (2000) reported rates of 2–9 nmol N/kg/h in the NW Mediterranean and Maguer et al. (1999) reported rates of 0.6–27 nmol N/ kg/h in the Western English Channel. What effect might these regeneration rates have on estimates of new production? In the absence of direct measurements, the ratio of regeneration to assimilation
129
has generally been assumed to be in the range of 1–2 (Kanda et al., 1987). We cannot assess this ratio directly because we did not make measurements of nitrate or ammonium uptake on this cruise. However, data are available from other studies of this meridional transect which allow us to determine the possible magnitude of the effect. Nitrate and ammonium assimilation were measured in September–October 1997 and May–June 1998 (A.P. Rees, Plymouth Marine Laboratory, Personal Communication). Samples taken from depths equivalent to 55% sPAR were incubated for 6 h on-deck. Ambient ammonium concentrations in the three regions were comparable to those measured in the present study (26, 23 and 10 nmol/kg for stations A, B and C), so we have used the assimilation rates to get an indication of the likely scale of effect of isotope dilution by NH4+ regeneration processes during an incubation period. In 1997 and 1998, nitrate uptake rates were 0.15– 0.75 nmol/kg/h and ammonium uptake ranged between 1.6 and 2.3 nmol/kg/h (Table 2). These rates are consistent with other studies in oligotrophic oceans (Harrison et al., 1996). The equations of Kanda et al. (1987) were used to calculate the isotope dilution factor (x) defined as the ratio of actual to apparent NH4+ uptake rate. From these data, the likely ratio (a) of ammonium assimilation rate (corrected for isotope dilution) to regeneration rate was also determined (Table 2). Values of (a) b 1 imply an imbalance between ammonium regeneration and assimilation processes at all stations, with ammonium regeneration rates potentially limiting assimilation rates. Including ammonium regeneration estimates, the correction for isotope dilution also has an impact on f-ratio. In these regions that are dominated by regenerative productivity, the influence of isotope dilution only enhanced the importance of ammonium regeneration by decreasing the f-ratio by 11–14%. This is greater than the 5% overestimation observed in the NW Mediterranean by Diaz and Raimbault (2000). Given that oligotrophic oceans represent a major proportion of the global ocean, an overestimation of f-ratios of the order of 10% will have a significant impact on model predictions of primary productivity and C-export. 5. Conclusions The specificity of the phenol/hypochlorite reaction, in conjunction with GC/MS analysis, provided sensitivity gains over IRMS and allowed the analysis of dissolved inorganic nitrogen concentrations typical of oligotrophic regions. The protocol, which is readily applied at sea, enabled replicated samples to be gener-
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D.R. Clark et al. / Marine Chemistry 98 (2006) 121–130
ated with only modest volumes of seawater, and avoided complications associated with distillation/diffusion/solvent extraction and associated fractionation/ efficiency issues. GC/MS provides a robust method to quantify sample-derived NH4+ concentration and isotopic enrichment. The impact of ammonium regeneration on NH4+ assimilation rates is non-trivial and should be considered in any study of new production. Acknowledgements This study was supported by the U.K. Natural Environment Research Council through grant number NER/A/S/2001/00636. We thank the steering committee of the Atlantic Meridional Transect consortium for the opportunity to participate in an AMT cruise. This is contribution number 98 of the AMT programme. The authors would like to thank Dr. Andrew Rees for providing N-assimilation data. References Blackburn, T.H., 1979. Method for measuring rates of NH+4 turnover in anoxic marine sediments, using a 15N–NH+4 dilution technique. Appl. Environ. Microbiol. 37, 760 – 765. Bremner, J.M., Keeney, D.R., 1965. Steam distillation methods for the determination of ammonium, nitrate and nitrite. Anal. Chim. Acta 32, 485 – 495. Caperon, J., Schell, D., Hirota, J., Laws, E., 1979. Ammonium excretion rates in Kaneohe Bay, Hawaii, measured by a 15N isotope dilution technique. Mar. Biol. 54, 33 – 40. Diaz, F., Raimbault, P., 2000. Nitrogen regeneration and dissolved organic nitrogen release during spring in a NW Mediterranean coastal zone (Gulf of Lions): implication for the estimation of new production. Mar. Ecol., Prog. Ser. 197, 51 – 65. Dugdale, R.C., Goering, J.J., 1967. Uptake of new and regenerated forms of nitrogen in primary production. Limnol. Oceanogr. 12, 199 – 206. Eppley, R.W., Peterson, B.J., 1979. Particulate organic matter flux and planktonic new production in the deep ocean. Nature 282, 677 – 680. Glibert, P.M., 1982. Regional studies of daily, seasonal and size fraction variability in ammonium remineralization. Mar. Biol. 70, 209 – 222. Grob, K., Grob, G., Grob Jr., K., 1981. Testing capillary gas chromatography columns. J. Chromatogr. 219, 13 – 20. Haesgawa, T., Koike, I., Mukai, H., 2000. Dissolved organic nitrogen dynamics in coastal waters and the effect of copepods. J. Exp. Mar. Biol. Ecol. 244, 219–238. Harrison, W.G., Harris, L.R., 1986. Isotope-dilution and its effects on measurements of nitrogen and phosphorus uptake by oceanic microplankton. Mar. Ecol., Prog. Ser. 27, 253 – 261.
Harrison, W.G., Harris, L.R., Irwin, B.D., 1996. The kinetics of nitrogen utilisation in the oceanic mixed layer: nitrate and ammonium interactions at the nanomolar concentrations. Limnol. Oceanogr. 41, 16 – 32. Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B., Peterson, B.J., 1998. Measuring 15N–NH+4 in marine, estuarine and fresh waters: an adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar. Chem. 60, 235 – 243. IOC, Paris (France), 1994. Protocols for the Joint Global Flux Study (JGOFS) core measurements. Manuals Guides 29:126 pp. Joint, I., Rees, A.P., Woodward, E.M.S., 2001. Primary production and nutrient assimilation in the Iberian upwelling in August 1998. Prog. Oceanogr. 51, 303 – 320. Jones, R.D., 1991. An improved fluorescence method for the determination of nanomolar concentrations of ammonium in natural waters. Limnol. Oceanogr. 36 (4), 814 – 819. Kanda, J., Laws, E.A., Saino, T., Hattori, A., 1987. An evaluation of isotope dilution effect from conventional data sets of 15N uptake experiments. J. Plankton Res. 19, 79 – 90. Ko¨ster, O., Jo¨ttner, F., 1999. NH+4 utilisation and regeneration rates in freshwater lakes determined by GC-MS of derivatised dihydroindophenol. J. Microbiol. Methods 37, 65 – 76. Laws, E., 1984. Isotope dilution models and the mystery of the vanishing 15N. Limnol. Oceanogr. 29, 379 – 386. Laws, E.A., Harrison, W.G., DiTullio, G.R., 1985. A comparison for nitrogen assimilation rates based on 15N uptake and autotrophic protein synthesis. Deep-Sea Res. 32, 85 – 95. Maguer, J., L’Helguen, S., Madec, C., Corre, P., 1999. Seasonal patterns of ammonium regeneration from size-fractioned microheterotrophs. Cont. Shelf Res. 19, 1755 – 1770. Preston, T., Bury, S., Presing, M., Moncoiffe, G., Slater, C., 1996. Isotope dilution analysis of combined nitrogen in natural waters: 1. Ammonium. Rapid Commun. Mass Spectrom. 10, 959 – 964. Raimbault, P., Slawyk, G., Boudjella, B., Coatanon, C., Conan, P., Coste, B., Garcia, N., Moutin, T., Pujo-Pay, M., 1999. Carbon and nitrogen uptake and export in the equatorial Pacific at 150 8W: evidence for an efficient regenerated production cycle. J. Geophys. Res. 104, 3341 – 3356. Selmer, J.S., So¨rensson, F., 1986. New procedure for extraction of ammonium from seawater for 15N isotopic ratio determinations. Appl. Environ. Microbiol. 52, 577 – 579. Sigman, D.M., Altabet, M.A., Michener, R., McCorkle, D.C., Fry, B., Holmes, R.M., 1997. Natural abundance level measurement of nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonium diffusion method. Mar. Chem. 57, 227 – 242. Slawyk, G., Raimbault, P., 1995. Simple procedure for simultaneous recovery of dissolved inorganic and organic nitrogen in 15N-tracer experiments and improving the isotopic mass balance. Mar. Ecol., Prog. Ser. 124, 289 – 299. Tupas, L.M., Koike, I., Karl, D.M., Holm-Hansen, O., 1994. Nitrogen metabolism by heterotrophic bacterial assemblages in Antarctic coastal waters. Polar Biol. 14, 195 – 204. Ward, B.B., Bronk, D.A., 2001. Net nitrogen uptake and DON release in surface waters: importance of trophic interactions implied from size fractionation experiments. Mar. Ecol., Prog. Ser. 219, 11 – 24.