Article pubs.acs.org/est
A Novel Membrane Inlet Mass Spectrometer Method to Measure 15 NH4+ for Isotope-Enrichment Experiments in Aquatic Ecosystems Guoyu Yin,† Lijun Hou,*,† Min Liu,‡ Zhanfei Liu,§ and Wayne S. Gardner*,§ †
State Key Laboratory of Estuarine and Coastal Research and ‡Department of Geography, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China § The University of Texas at Austin Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, United States S Supporting Information *
ABSTRACT: Nitrogen (N) pollution in aquatic ecosystems has attracted much attention over the past decades, but the dynamics of this bioreactive element are difficult to measure in aquatic oxygentransition environments. Nitrogen-transformation experiments often require measurement of 15N-ammonium (15NH4+) ratios in small-volume 15 N-enriched samples. Published methods to determine N isotope ratios of dissolved ammonium require large samples and/or costly equipment and effort. We present a novel (“OX/MIMS”) method to determine N isotope ratios for 15NH4+ in experimental waters previously enriched with 15 N compounds. Dissolved reduced 15N (dominated by 15NH4+) is oxidized with hypobromite iodine to nitrogen gas (29N2 and/or 30N2) and analyzed by membrane inlet mass spectrometry (MIMS) to quantify 15 NH4+ concentrations. The N isotope ratios, obtained by comparing the 15 NH4+ to total ammonium (via autoanalyzer) concentrations, are compared to the ratios of prepared standards. The OX/MIMS method requires only small sample volumes of water (ca. 12 mL) or sediment slurries and is rapid, convenient, accurate, and precise (R2 = 0.9994, p < 0.0001) over a range of salinities and 15N/14N ratios. It can provide data needed to quantify rates of ammonium regeneration, potential ammonium uptake, and dissimilatory nitrate reduction to ammonium (DNRA). Isotope ratio results agreed closely (R = 0.998, P = 0.001) with those determined independently by isotope ratio mass spectrometry for DNRA measurements or by ammonium isotope retention time shift liquid chromatography for water-column N-cycling experiments. Application of OX/MIMS should simplify experimental approaches and improve understanding of N-cycling rates and fate in a variety of freshwater and marine environments.
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INTRODUCTION Nitrogen (N) pollution in aquatic ecosystems is an urgent environmental issue around the world.1,2 Increasing N inputs from fertilizers and other sources over the past few decades have caused multiple environmental problems in coastal and other natural waters, including coastal eutrophication, seasonal hypoxia, and harmful algae blooms.3−7 The transformation processes among different N forms must be determined to understand the ecology and biogeochemistry of aquatic ecosystems. Enrichment of water or sediments with 15N-labeled compounds (e.g., 15NH4+ or 15NO3−) can help track N dynamics in benthic and pelagic freshwater and marine ecosystems.8 However, the need to measure N isotopic ratios of ammonium in aqueous solution often limits such studies when appropriate equipment is not available.9,10 Several conventional methods to measure 15N in dissolved ammonium and/or nitrate9−16 require long pretreatment times, specialized equipment, complicated procedures, or large volumes of sample to provide sufficient sensitivity. Some recently developed methods17,18 decrease pretreatment times, but require additional conversion steps, and are not easy to implement. A rapid, © 2014 American Chemical Society
simple, precise, and economical method is needed to measure low concentrations of 15N in ammonium and other compounds in aqueous samples. In this paper, we present a novel method to measure the 15N content of dissolved ammonium for 15N-enrichment experiments. In brief, the ammonium is oxidized to N2 with hypobromite iodine solution, and the concentration of the generated N2 is measured with membrane inlet mass spectrometry (MIMS), which is available in many laboratories involved with N-dynamics studies. MIMS is a versatile tool for simultaneous measurements of volatile compounds and requires only a small water sample for analysis. MIMS has been used increasingly to measure dissolved gases over the past several years19−25 and quantify the generated N isotopes and isotope ratios of 28N2, 29N2, and 30N2 in experiments enriched with 15N compounds (e.g., 15NH4+, 15NO3−, or 15N-labled Received: Revised: Accepted: Published: 9555
March 17, 2014 June 20, 2014 July 14, 2014 July 14, 2014 dx.doi.org/10.1021/es501261s | Environ. Sci. Technol. 2014, 48, 9555−9562
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organic compounds26−28). The high accuracy and precision, rapid sample throughput, affordability, and cost-effectiveness of MIMS make this approach attractive for current and future investigations of N dynamics in regions affected by N pollution.27,29,30 A combination of the ammonium oxidation technique and MIMS analysis (here called “OX/MIMS”) provides a powerful and promising approach to decipher N-transformation processes in sediment oxygen-transition zones and water columns of important microbe-dominated biogeochemical ecosystems. Of the N-transformation processes, dissimilatory nitrate reduction to ammonium (DNRA) is a primary nitrate reduction process, as are denitrification and anaerobic ammonium oxidation (anammox).31,32 DNRA is of management interest because it does not remove N directly from aquatic ecosystems in the form of dinitrogen gas as do denitrification and anammox33,34 but instead converts nitrate to ammonium, an inorganic N form that is often a more biologically available and less mobile than nitrate.17,35 DNRA has attracted increasing attention as a pathway for nitrate removal over the past few decades. However, published methods for measuring DNRA rates are time-consuming, making the occurrence and extent of DNRA difficult to investigate.36 The OX/MIMS method provides a convenient way to measure DNRA rates, including the 15NH4+ associated with sediments. Field trials were conducted in Chinese salt marshes to demonstrate its use and provide preliminary information about potential DNRA rates in this region.
was derived from the linear relationship between the known concentrations of 15NH4 + and the measured signal intensities of the total produced 29N2 plus 30N2. Step 3: Water sample 15NH4+ analysis. The test samples were transferred into 12 mL gastight vials without gas entrapment and sealed to prevent solution and/or gas leakage. Hypobromite iodine solution (0.2 mL) was injected into each sample vial to oxidize 15NH4+ to 29N2 and/or 30N2. After oxidation, the produced gases were analyzed with MIMS. A long syringe needle was inserted into the bottom of the samples before the samples were connected to the MIMS instrument to avoid exposure to air, and helium gas was replenished through another short syringe needle to eliminate negative pressure. The concentrations of 15NH4+ in these samples were calculated from a standard OX/MIMS calibration curve, prepared with the samples for each analytical run. Salinity Effects. Water samples with different salinities were tested to determine whether OX/MIMS results were comparable in freshwater and other aquatic systems with different salinities. Respective artificial-seawater samples were prepared with salinities of 0, 15, and 30.39 A concentration gradient of 15NH4Cl, including 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, and 200 μmol L−1, was prepared at each salinity. Four replicate samples for each concentration were transferred into respective 12 mL gastight vials, sealed, and analyzed with OX/MIMS as described above. Influence of 15N/14N ratios. A 15N/14N ratio experiment was conducted to determine if the initial 15N/14N ratios in samples would affect OX/MIMS accuracy. The total concentration of NH4Cl was fixed at 100 μmol L−1, and isotope ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 were prepared with pure 14NH4Cl and 15NH4Cl standard solutions. Four replicates, prepared for each isotope ratio, were transferred into respective 12 mL gastight vials and sealed for OX/MIMS analysis of 29N2 and 30N2. Total ammonium concentrations were determined via a continuous-flow nutrient autoanalyzer (SAN plus, Skalar Analytical B.V., Breda, The Netherlands) Application of OX/MIMS to Potential DNRA Measurements. Sediment and near-bottom water samples were collected from seven sites located along the China coast in March 2013 (Figure 1). The sediment samples were homogenized in a glovebox under helium, and the water samples were purged with helium for 40 min. The homogenized sediment (30 g) from each site was mixed with
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MATERIALS AND METHODS Protocol for Determining the Presence and Concentration of 15NH4+. Step 1: Oxidant preparation. Hypobromite iodine solution, to oxidize NH4+−N to N2,37 was prepared as follows. A NaOH solution (16 mol L−1) was cooled in ice water to maintain it below 5 °C. Br2 (100 mL) was added dropwise to the NaOH solution (600 mL) with continuous stirring at a temperature maintained below 5 °C. After refrigeration for 1 week to allow NaBr crystals to precipitate, the precipitate was removed by filtration. The supernatant was mixed with an equal volume of KI solution (0.2%, w/v) to maintain stability. A 1 mL amount of this solution can oxide up to 5−6 mg of NH4+ to N2.37,38 Step 2: Standard calibration curve. A standard solution of 15 NH4Cl (15N, 99%, Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) was prepared with a concentration gradient of 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, and 200 μmol N L−1. Respective 12 mL gastight borosilicate vials (Labco Exetainer, Lampeter, UK) were filled completely with the standard solutions and sealed with silicon septa and screw caps to prevent leakage of solution and gas. Four replicate calibration-curve standards were prepared for each concentration. Hypobromite iodine solution (0.2 mL) was added to each vial to oxidize the NH4+ to N2. The excessive oxidant oxidizes the NH4+ completely.37,38 The produced gases were analyzed with a MIMS instrument, based on the technique described by Kana et al.19 The MIMS instrument consists of a vacuum inlet fitted with gas-permeable silicone tubing. The water sample is pumped through the tubing at a constant speed of 2.5 mL min−1 at a fixed temperature of 25 °C. The inlet allows gas to pass into the vacuum system, where it is routed through a cold trap (liquid N2 to remove CO2 and H2O vapors) and into a HAL/3F 301 RC triple filter mass spectrometer with a Faraday/multiplier detector. The standard calibration curve
Figure 1. Map of coastal regions of China showing the sampling sites. 9556
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200 mL of overlying water, and the slurry was stirred continuously and purged with helium for 20 min. The mixtures were transferred by syringe into 10 12 mL gastight vials as replicates, and each vial was sealed with a silicon septum and screw cap. After a 24 h preincubation, the slurry vials were spiked with 15NO3− (final concentration ca. 100 μmol L−1; final % 15N ca. 90−99%, depending on the background nitrate concentration), and one-half of the replicates were designated as initial samples and preserved with 0.1 mL of saturated HgCl2 solution.40 The remaining slurries were shaken (200 rpm) and incubated for about 8 h at near in situ temperature.40 At the end of the incubation, the remaining final-sample replicates were preserved with HgCl2 as described for initial samples. Slurried samples were stirred and repurged with helium for 30 min to remove any 29 N 2 and/or 30 N 2 generated by denitrification and/or anammox over the incubations. The slurry samples were sealed immediately, and 0.2 mL of hypobromite iodine solution was injected into each Labco Exetainer vial. The samples were analyzed with MIMS after the 15 NH4+ produced from DNRA in the slurry incubations was oxidized. Potential DNRA rates were estimated by the following equation:41 RDNRA =
Figure 2. Measured signal intensity of the total 15N2 (29N2 + 2 × 30N2) at different oxidation times from 0 to 180 min. Vertical bars denote standard error of quadruplicate samples. However, most of the error marks are obscured by the symbols, due to the high precision of OX/ MIMS.
[15 NH4 +]final × V − [15 NH4 +]initial × V W×T
where RDNRA (μmol 15N kg−1 h−1) denotes the total, measured 15 N-based potential DNRA rates, [ 15 NH 4 + ] initial and 15 [ NH4+]final (μmol L−1) are the concentrations of 15NH4+ in the initial and final samples of the slurry experiments, respectively, V (L) is the volume of the incubation vial, W (kg) denotes the dry weight of the sediment, and T (h) is the incubation time.
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RESULTS AND DISCUSSION OX/MIMS Accuracy and Precision. Results from an initial experiment with a constant concentration of 15NH4+ (ca. 100 μmol L−1), to examine optimum oxidation time, confirmed that the ammonium oxidation was almost instantaneous (Figure 2). The signal intensities of measured total 15N2 (29N2 + 2 × 30N2) did not change from 0 to 180 min (one-way ANOVA, p > 0.1). The residual ammonium was measured spectrophotometrically to calculate the oxidation efficiency of ammonium (data not shown). Residual ammonium was below the detection limit of 0.1 μmol L−1, suggesting that the oxidation efficiency of ammonium to N2 was 100%. We did not quantify the oxidation efficiency for labile organic compounds, such as amino acids or proteins but expect similar results.38 Particulate organic compounds were removed by filtration before the oxidation step (see Figure S1, Supporting Information). Dissolved labile organic 15N compounds are not a major issue in natural waters under normal conditions because they occur at concentrations much lower than ammonium, due to rapid bacterial uptake of any labile N-rich compounds.42,43 The OX/MIMS calibration curves were prepared for aqueous samples containing different atom fractions of 15NH4+ at the same salinity as the samples. The Pearson’s correlation between the signal intensities of total 15N2 and the concentrations of 15 NH4+, representing the precision of measured 15NH4+, was linear (R2 = 0.9994, p < 0.0001; Figure 3). However, the signal intensities of 29N2 were low and did not change significantly among standard solution samples with different concentrations
Figure 3. Relationships of the known 15NH4+ concentrations with measured signal intensities of 29N2, 30N2, and total 15N2 (29N2 + 2 × 30 N2). The panel shows the relationships of the low 15NH4+ concentrations (0.2−10 μmol L−1) with measured signal intensities. Vertical bars denote standard error of quadruplicate samples, but some of them are less than the symbol size.
of 15NH4+ (p > 0.05), mainly because the concentrations of NH4+ in the standard solutions were low. In contrast, the linear relationship between the signal intensities of 30N2 and the concentrations of amended 15NH4+ was excellent (R2 = 0.9998, p < 0.0001). The significant correlation between the signal intensities of total-15N2 and 15NH4+ concentrations illustrates that OX/MIMS can quantify the 15N content of water samples accurately in isotope-enrichment experiments. Effects of Sample Salinity. Some published methods for measuring isotope ratios of 15NH4 are specific to soils or freshwater systems,44,45 which may restrict their application to marine waters or sediments. Therefore, we tested this method for salinity effects, using prepared-sample salinities of 0, 15, and 30 to represent a range of aquatic environments from freshwater to coastal marine systems. The total-15N2 signal 14
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intensity correlated linearly with the concentrations of 15NH4+ at each salinity (Figure 4), indicating that the method is
Figure 5. Relationships of the known 14NH4+/15NH4+ ratios with the measured signal intensities of 28N2, 29N2, 30N2, and total 15N2 (29N2 + 2 × 30N2) at the total NH4Cl concentration of 100 μmol L−1. All curves were fit separately using the 14NH4+/15NH4+ ratios and measured signal intensities. Vertical bars denote standard error of quadruplicate samples.
Figure 4. Relationships of the known 15NH4+ concentrations with measured signal intensities of total 15N2 (29N2 + 2 × 30N2) under different salinity conditions. Vertical bars denote standard error of quadruplicate samples. S represents salinity.
production, as supported by the excellent linear correlation between the total- 15 N 2 signal values and the 15 NH 4 + concentrations (R2 = 0.9967, P < 0.0001). The results confirm that OX/MIMS is appropriate for measuring 15N concentrations in samples with different atom percent 15N ratios. Field Examination of DNRA and Ammonium-Cycling Rates. In this study, measured potential DNRA rates in Chinese salt marshes ranged from 0.21 to 1.54 μmol 15N kg−1 h−1 and accounted for 1.8−13.1% of total dissimilatory reduction of nitrate (Figure 6). In contrast, denitrification was the dominant dissimilatory process in this system with potential rates of 7.90−17.6 μmol 15N kg−1 h−1 and accounted for 83.6−96.1% of nitrate reduction (see Method S1, Supporting Information). Anammox was less important than
effective for aquatic environments of different salinities. Although the calibration curves varied slightly with salinity (R2 = 0.9989, p < 0.0001 for S = 0; R2 = 0.9997, p < 0.0001 for S = 15; R2 = 0.9966, p < 0.0001 for S = 30), the effect was quite small. The respective relative deviations of the measured signal intensities of total 15N2 were below 5% at each salinity value, indicating minimal differences among them. Influence of 15N/14N Ratios in Water Samples. Nitrogen isotopes in field samples are dominated by 14N, whereas 15N is added in isotope-enrichment experiments, causing us to report “potential uptake” results but “actual” regeneration rates.31 However, recent additional calculations for the same data allows estimation of both actual and potential uptake rates.32 The generated 29N2 and 30N2 consist of the existing 14N and spiked 15N, which depend on the atom percent of 15NH4+ in the water samples.26 We hypothesized that measurements of total-15N2 signal intensities are influenced by the N isotopic ratios. An experiment was conducted (1) to examine whether different 15N/14N ratios in samples influence the signal intensities of total 15N2 and (2) to demonstrate the application of this method in a field trial. Although the signal intensities of 29N2 and 30N2 were not linear with 15NH4+ concentrations (Figure 5), the total-15N2 signal intensities increased linearly with the concentrations of 15 NH4+ (R2 = 0.9967, p < 0.0001). Depending on random pairing of 14N and 15N originating from the 14NH4+ and 15 NH4+,46−48 the signal intensities of 29N2 correlated parabolically with the changes of 15NH4+ concentrations and peaked when the atom percent of 15N reached 50%. The production of 28 N2 decreased to the background signal intensity of MIMS, as concentrations of 14NH4+ were reduced from 100 to 0 μmol L−1, whereas the production of 30N2 increased from the background to the peak value as the concentrations of 15NH4+ rose from 0 to 100 μmol L−1 (Figure 5). Both patterns also showed parabolical relationships of the 14NH4+/15NH4+ ratios with the measured signal intensities of 28N2 and 30N2. However, the 15N/14N ratios in samples did not influence the total-15N2
Figure 6. Rates of the dissimilatory nitrogen transformation processes (DNRA, denitrification, and anammox) at seven sites located along the coastal areas of China. QD, LYG, NB, ZS, WL, ND, and SZ are site abbreviations for Qingdao, Lianyungang, Ningbo, Zhoushan, Wenling, Ningde, and Shenzhen, respectively. Vertical bars denote standard error of quadruplicate samples. 9558
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denitrification or DNRA; it ranged from 0.11−0.34 μmol 15N kg−1 h−1 and contributed 0.7−3.3% to the total nitrate loss (see Method S1, Supporting Information). This comparison shows that DNRA is an important, but not dominant, process controlling the nitrogen fate in these estuarine and coastal regions. These measured rates of potential DNRA in coastal salt marshes of eastern China also resemble results from other estuarine and coastal sediments across the world (Table 1).
OX/MIMS is convenient and time-efficient. For example, DNRA experiments required no elution, extraction, or analytical-enrichment steps. Samples obtained in laboratory experiments were analyzed within a few minutes after pretreatment. However, we speculate that more remote field samples may need preservation to inhibit microbial activities, for example, with ZnCl2 or HgCl2, to maintain high data quality at the time of analysis. Precise OX/MIMS isotopic measurements of dissolved ammonium are cost-effective, relative to analysis by IRMS or high-performance liquid chromatography (HPLC). OX/MIMS is especially useful to measure DNRA in laboratories where MIMS systems are already available to measure denitrification, respiration,29 or other gas transformations, but we expect that future applications may include a broader range of environmental studies. A potential limitation for DNRA measurements by some methods53,60 is that DNRA rates may be underestimated by measuring the 15NH4+ concentration in water above the sediment−water interface, as the 15NH4+ produced via DNRA may exchange with 14NH4+ absorbed on sediments and (or) be assimilated by microorganisms.40 Underestimation of DNRA rates are minimized with OX/MIMS because the oxidant converts the inorganic ammonium, simultaneously with organic N compounds in sediment slurry samples, to N2.61 However, overestimation of DNRA rates could potentially occur within a N-limiting experimental system, because some of the injected tracer 15NO3− may be incorporated directly by microorganisms. That regeneration effect may be small in most dissimilatory processes as they use nitrates more for energy than growth.62 However, filtration or centrifugation of samples may be required to remove particulate organic N produced from microbial assimilation of 15NO3− before samples are oxidized. We conducted an additional experiment on ammonium regeneration and uptake in overlying water from a coastal river near Site Zhoushan to (1) test the efficiency of filtering water samples to reduce the potential interference from organic N and (2) compare results on identical samples by OX/MIMS and ammonium isotope retention time shift (AIRTS)/HPLC63 (see Method S4, Supporting Information). Nearly identical rates were obtained from the two independent analyses techniques (see Figure S3, Supporting Information). The excellent agreement indicates that micropore filtration effectively removed the potential effect of particulate organic N produced from microbial assimilation on the analysis of dissolved 15NH4+. Although the newly produced particulate organic N may be remineralized eventually, the absence of such decomposition can be assumed during the time scale of the incubation experiments.29 Interference from dissolved labile organic N after 15NH4+ oxidation should be minimized, assuming that particulate organic N accumulating is negligible during incubations.42,43 The results also indicate that the developed OX/MIMS method can be applied to measuring rates of N transformations accurately in the water column. OX/MIMS Advantages and Possible Future Applications. OX/MIMS is efficient and accurate for measuring DNRA and ammonium-cycling rates and does not require extensive additional effort or equipment, beyond the MIMS instrument, to yield accurate results. Oxidation of NH4+−N to N 2 is “instantaneous”, and the samples are measured immediately after the oxidation with MIMS, without other pretreatment. Less than 1 h is required to measure a set of 10 samples by OX/MIMS, compared to (1) previous stable
Table 1. Rates of DNRA and Its Contribution to Total Nitrogen Loss in the Study Area and Other Estuarine and Coastal Ecosystems sample type marine sediments estuarine sediments coastal sediments estuarine sediments estuarine sediments estuarine sediments marine sediments estuarine sediments coastal sediments
DNRA rates (μmol 15N m−2 h−1)
percentage of DNRA in total N loss (%)
ref
10−240
14−40
27
3.9−307
29−51
33
0−80
9.0−20.5
35
0−2.9
10
49
10−330
30−40
50
2.1−234
70
51
20−80
