A new method for collection of nitrate from fresh water and the analysis of nitrogen and oxygen isotope ratios

Journal of Hydrology 228 (2000) 22–36 www.elsevier.com/locate/jhydrol

A new method for collection of nitrate from fresh water and the analysis of nitrogen and oxygen isotope ratios S.R. Silva a,*, C. Kendall a, D.H. Wilkison b, A.C. Ziegler c, C.C.Y. Chang a, R.J. Avanzino a a

US Geological Survey, 345 Middlefield Road, Menlo Park, CA, 94025 USA US Geological Survey, 301 West Lexington, Independence, MO 64050 USA c US Geological Survey, 4821 Quail Crest Place, Lawrence, KS 66046 USA

b

Received 10 November 1998; received in revised form 1 September 1999; accepted 19 November 1999

Abstract A new method for concentrating nitrate from fresh waters for d 15N and d 18O analysis has been developed and field-tested for four years. The benefits of the method are: (1) elimination of the need to transport large volumes of water to the laboratory for processing; (2) elimination of the need for hazardous preservatives; and (3) the ability to concentrate nitrate from fresh waters. Nitrate is collected by, passing the water-sample through pre-filled, disposable, anion exchanging resin columns in the field. The columns are subsequently transported to the laboratory where the nitrate is extracted, converted to AgNO3 and analyzed for its isotope composition. Nitrate is eluted from the anion exchange columns with 15 ml of 3 M HCl. The nitrate-bearing acid eluant is neutralized with Ag2O, filtered to remove the AgCl precipitate, then freeze-dried to obtain solid AgNO3, which is then combusted to N2 in sealed quartz tubes for d 15N analysis. For d 18O analysis, aliquots of the neutralized eluant are processed further to remove non-nitrate oxygen-bearing anions and dissolved organic matter. Barium chloride is added to precipitate sulfate and phosphate; the solution is then filtered, passed through a cation exchange column to remove excess Ba 21, re-neutralized with Ag2O, filtered, agitated with activated carbon to remove dissolved organic matter and freeze-dried. The resulting AgNO3 is combusted with graphite in a closed tube to produce CO2, which is cryogenically purified and analyzed for its oxygen isotope composition. The 1s analytical precisions for d 15N and d 18O are ^0.05‰ and ^0.5‰, respectively, for solutions of KNO3 standard processed through the entire column procedure. High concentrations of anions in solution can interfere with nitrate adsorption on the anion exchange resins, which may result in isotope fractionation of nitrogen and oxygen (fractionation experiments were conducted for nitrogen only; however, fractionation for oxygen is expected). Chloride, sulfate, and potassium biphthalate, an organic acid proxy for dissolved organic material, added to KNO3 standard solutions caused no significant nitrogen fractionation for chloride concentrations below about 200 mg/l (5.6 meq/l) for 1000 ml samples, sulfate concentrations up to 2000 mg/l (41.7 meq/l) in 100 ml samples, and Potassium biphthalate for concentrations up to 200 mg/l carbon in 100 ml samples. Samples archived on the columns for up to two years show minimal nitrogen isotope fractionation. Published by Elsevier Science B.V. Keywords: Nitrates; Stable isotopes; Ion exchange; Geochemical methods

1. Introduction Problems associated with anthropogenic nitrogen * Corresponding author. 0022-1694/00/$ - see front matter. Published by Elsevier Science B.V. PII: S0022-169 4(99)00205-X

contributions to the biosphere and hydrosphere are increasingly being recognized. In natural environments, the availability of nitrogen (N) is often limited

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by the rate of N-fixation. Nitrogen availability is one of the main controls on productivity and is an important factor regulating biodiversity. Presently, N input from human sources, chiefly N-fixing crops, synthetic fertilizers, and burning of fossil fuels, approximately equals the input from natural nitrogen fixation (Vitousek et al., 1997). The increased N input from anthropogenic sources causes eutrophication of lakes, streams, and coastal waterways, acidification of susceptible environments, and degradation of drinking water quality (Vitousek et al., 1997). Longer term and largely unpredictable responses in plant and animal communities are due to a shift from N limitation to N saturation. Nitrogen in terrestrial and aquatic ecosystems cycles among oxidized, reduced, organic, and inorganic species, of which nitrate is relatively abundant and mobile. Nitrogen and oxygen isotope ratios of nitrate provide a powerful tool to investigate nitrate sources and cycling mechanisms. The analysis of nitrate for both d 15N and d 18O allows improved discrimination among potential sources and reaction mechanisms (Amberger and Schmidt, 1987; Boettcher et al., 1990). Various methods are currently in use for collection and preparation of nitrate for isotope analysis. The simplest method for preparing nitrate from natural waters for d 15N analysis involves evaporation or freeze-drying of filtered samples. The resulting solids, which include nitrate and nitrite 1, are combusted in the presence of Cu, CuO, and CaO to produce pure N2 (Kendall and Grim, 1990). A more widely used method converts nitrate to ammonium by a Kjeldahl reaction (Bremner, 1965; Bremner and Edwards, 1965). The ammonium is then converted to N2 gas by one of several methods: (1) direct combustion of the dried ammonium salt (Kendall and Grim, 1990); (2) steam distillation of ammonium followed by oxidation with a hypobromite solution, and purification of N2 in a Cu/ CuO furnace (Bremner, 1965; Bremner and Edwards, 1965); or (3) distillation followed by collection of ammonium on an ammonium-specific zeolite, and combustion to N2 (Velinsky et al., 1989). As an alter1

Nitrite generally occurs in very low abundance in natural waters. For simplicity, nitrate plus nitrite will henceforth be referred to as nitrate (NO32).

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native to steam distillation, ammonium may be slowly diffused into an acid solution or onto acidified filter paper, and combusted or reacted as above to form N2 (MacKown et al., 1987; Sigman et al., 1997). These techniques have several drawbacks. They require that the water samples be preserved by chilling, acidifying, or poisoning, and transported back from the field for processing. Waters with low nitrate concentrations are impractical to process because of the large volumes of sample required. The distillation process is time consuming and is subject to isotope fractionation and cross contamination (Mulvaney, 1986). Freeze-drying of large water samples requires considerable time and when these samples are combusted in quartz or Vycor 2 tubes, the tubes often fail because of reaction of alkali metals, particularly Na, with the glass. As an alternative, anion exchange resins have been used for collecting nitrate (e.g. Hoering, 1957; Morrissey, 1989; Garten, 1992; Downs et al, 1999). Amberger and Schmidt (1987) developed the first reliable method for d 18O analysis using HgCN as a carbon source and nitrate in the form of KNO3. The drawbacks of this method are the toxicity of HgCN and the low yields of CO2. A recently developed alternative method combusts nitrate in the form of KNO3 with graphite as a carbon source, and mathematically corrects for the fractionation caused by lower yields of CO2 (Revesz et al., 1997). Our objectives were to develop methods for concentrating dissolved nitrate and to prepare it for nitrogen and oxygen isotope analysis while avoiding or reducing the drawbacks associated with the previously described techniques. This paper describes in detail a new method for nitrate concentration in the field using commercially available, pre-packed, disposable, anion exchange columns, and for analysis of the column eluant for both nitrogen and oxygen isotope ratios. The new method is convenient, economic, and has excellent precision for d 15N. A new preparation procedure for d 18O of nitrate, using graphite as a carbon source with nitrate in the form of AgNO3, produces higher yields of CO2 and eliminates

2

Use of trade names and trademarks in this publication is for descriptive purposes only and does not constitute endorsement by the US Geological Survey.

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S.R. Silva et al. / Journal of Hydrology 228 (2000) 22–36

Fig. 1. Apparatus used to retain nitrate from water samples on anion exchange resins. See the text for description.

the use of HgCN while producing acceptable precision. The anion column method for d 15N analysis described here has been in use since 1993. Since the procedure for d 18O of nitrate was developed a year later, most of the testing of the field method was done with d 15N only. The combined techniques have been successfully used and published in studies from alpine, agricultural, and urban environments (Kendall et al., 1995; Wassenaar, 1995; Ging et al., 1996; Aravena and Robertson, 1999).

2. Materials and methods 2.1. Procedure for preparing and loading anion exchange columns Bio-Rad 3 (Hercules, California) AG1-X8, 200– 400 mesh anion exchange resin in the chloride form was used for our experiments because of its relatively high affinity for nitrate. The 3

Please refer to footnote 2.

exchange capacity is 1.2 meq/ml. The resin is available in boxes of 50 pre-filled, 12 × 2 cm columns containing 2 ml of resin, 2.4 milliequivalents (for about US$ 160 per box currently). Columns and resin may also be purchased separately. Before use, 2 ml of 1.25 M CaCl2 solution is dripped through the column to insure that the exchange sites are fully occupied by chloride ions; this step is precautionary and may be unnecessary. This is followed by five 2-ml rinses of deionized water (DI) to remove any excess chloride. The last 0.5 ml of deionized water is retained in the column to keep the resin beads fully hydrated. The columns are then tightly capped and stored at room temperature until needed. In the field, NO32 concentrations are measured to determine how much water needs to be processed to retain 100–200 mmol of nitrate on the anion exchange resin. The sample water is then filtered through a 0.45 mm polycarbonate membrane to remove particles that might clog the resin. Nitrate is then sorbed on the anion exchange resin using the apparatus shown in Fig. 1. A flow rate of 500–1000 ml/h is achieved by adjusting the stopcock on the separatory funnel. Multiple apparatuses can be connected in series to a peristaltic vacuum pump for simultaneous processing of samples. After collecting the NO32 the columns are capped and refrigerated until they are transported to the laboratory for analysis. 2.2. Procedure for stripping anion exchange columns In the laboratory, the anion exchange columns are mounted on test tube racks that were modified to allow the simultaneous desorption of nitrate from 25 pre-packed columns. The NO32 is stripped from the columns by gravity dripping 15 ml of 3 M HCl through the column in 3 ml increments. Positive air pressure, supplied by a one-way rubber bulb attached to a #2 stopper, is applied to the columns after each 3 ml increment to remove residual eluant and sometimes is needed to start the subsequent aliquot dripping. Small multiple increments were found to give a consistently high yield with lower variability than that obtained by a single rinse (Section 3.3). The 15 ml of nitrate-bearing eluant is collected in 50 ml glass beakers.

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2.3. Preparation of

NO32-bearing

eluant

Because HNO3 is volatile, it must be neutralized before freeze drying. Silver oxide (Ag2O) is used for neutralization by the reaction HCl 1 HNO3 1 Ag2 O ! AgCl…s† 1 AgNO3 1 H2 O: One benefit of Ag2O as a neutralizing agent is that the resulting silver chloride precipitate (AgCl) can be removed by filtration before the nitrate is freezedried to anhydrous AgNO3. Beakers containing the column eluant are placed in a cold water bath and a total of about 6.5 g Ag2O is added in successive increments of about one gram each to allow the heat of reaction to dissipate without producing vapor. Each Ag2O addition is stirred and crushed with the flattened end of a glass stirring rod to break the crust which tends to encapsulate the unreacted reagent. A final pH of about 5.5–6 is verified by pH paper. The AgCl precipitate is removed by filtration through DI-rinsed, Whatman 2 #1 filters and collected in 100-ml tri-cornered, plastic beakers. Additional DI water is used to rinse the sample nitrate through the filter, bringing the sample volumes to about 40 ml each. At this point the sample is split into aliquots for d 15N, d 18O, and a reserve if excess sample was collected. The d 18O, and reserve aliquots are transferred to 30 ml plastic bottles with tight fitting caps and stored in a freezer for further preparation. Because AgNO3 is light-sensitive, care should be taken in all subsequent steps to minimize exposure to light. 2.3.1. Preparation for nitrogen isotope analysis To prevent spattering during freeze-drying, the beakers are covered with Parafilm 2 and a few small holes are punched adjacent to the rim. The solutions are frozen either overnight in a freezer or immediately in liquid nitrogen prior to being freeze-dried. After freeze-drying, the AgNO3 is redissolved by adding 2 ml of DI water to each beaker and swirling it over the bottom and sides. Preparation of this solution for analysis by a combustion tube method and by continuous flow mass spectrometry (CF-MS) using an elemental analyzer (EA) for combustion are discussed below.

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2.3.1.1. Combustion tube method. The solutions are pipetted into pre-combusted 20 cm by 9 mm (OD) quartz tubes. The solutions are then frozen by slowly immersing the quartz tubes in a dry-ice/ alcohol slush such that the freezing level of the sample remains at or above the level of the slush. After freezing, the tops of the tubes are covered by small squares of Kimwipe 2 held in place with rubber bands to prevent losses of dried salt during the subsequent freeze drying procedure. After freeze-drying, the tubes are loaded with combustion reagents (CaO, CuO, and Cu wire) evacuated, and combusted according to the method of Kendall and Grim (1990). During evacuation, the lower halves of the tubes are covered by opaque paper or foil jackets to prevent photodegredation of AgNO3.

2.3.1.2. Continuous flow mass spectrometry method. Due to the small volume of EA combustion capsules, the sample solutions are further consolidated by pipetting them into plastic centrifuge tubes with pointed bottoms. The solutions are first frozen then freeze-dried with a Kimwipe 2 cover as described above. The resulting precipitate is redissolved in an appropriate volume of DI and pipetted into 4– 5 × 9 mm silver capsules (Elemental Microanalysis 2, Manchester, MA) such that each capsule contains 6– 10 mmol NO32. The more commonly used tin capsules are unsuitable due to reaction with AgNO3, and aluminum capsules tend to react with the quartz combustion tube in the elemental analyzer. The capsules are held in a 0.5 in. thick aluminum block with sample wells drilled to a depth of about 5 mm. The block is partially immersed in liquid N2 until the sample solutions are frozen. The top of each of the capsules is pressed closed such that they may be reopened later. The block is covered in a Kimwipe 2 and freeze-dried. When dry, the capsules are re-opened and 2 mg of sucrose is added. The capsules are then crimped in the usual manner for elemental analysis. We found that EA combustion of pure nitrate salts yielded inconsistent d 15N but the addition of about 2 mg of sucrose (as suggested by Tom Jackson of Elemental Microanalysis) in the same capsules solved this problem (Fig. 2).

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Fig. 2. The d 15N values of AgNO3 run on an Optima continuous flow mass spectrometer. The runs labeled “4 October” and “10 October” were performed by using pure AgNO3 (1 mg samples) in silver capsules. The run labeled “sugar” was conducted using AgNO3 (1 mg samples) plus 2 mg of sugar in silver capsules (see text for discussion). Correct d 15N values (115.95 determined by sealed tube combustion) were obtained by combusting samples with added sugar (see text for discussion).

2.3.2. Preparation for oxygen isotope analysis For accurate d 18O analyses of dissolved nitrate, all O-bearing compounds other than NO32 must be removed from the sample (Amberger and Schmidt, 1987). For this method, 2 ml of a 1 M BaCl2 solution is added to the 30 ml plastic bottles to precipitate SO422 and PO432. Note that this quantity of BaCl2 is approximately a 250% excess necessary for precipitation of BaSO4 from 30 ml of a saturated Ag2SO4 solution (Ag2PO4 is essentially insoluble). After addition of the BaCl2, the bottles are re-capped and stored refrigerated overnight to allow the precipitate to settle. The resulting solids (BaSO4, AgCl, and BaPO4) are filtered from solution through an 0.2 mm nylon filter. The sample is then passed through a column packed with 4 ml of Bio-Rad 100–200 mesh AG 50W X8 cation exchange resin in the H 1 form. The exchange capacity of the cation resin is 1.2 meq/ ml or 4.8 meq total. Excess Ba 12 and the remaining Ag 1 are exchanged for H 1 on the column. The column is blown dry with a one-way rubber bulb, and then rinsed with 5-ml of DI water. The sample is collected in a 50 ml glass beaker and neutralized with an excess (1 g) of Ag2O to achieve a pH of about 6 and the resulting AgCl precipitate and excess Ag2O

is filtered out of solution through a pre-rinsed 0.2 mm nylon filter. To remove DOC, 10 mg of Norit 2 G-60 activated carbon is added per 50 ml solution and shaken in an orbital shaker at 180 rpm for 20 min. The activated carbon is removed with a pre-rinsed, 25 mm diameter, 0.2 mm nylon filter. Because activated carbon can also absorb nitrate, it is important that the ratio of carbon (mg) to solution (ml), or shaking time not be exceeded and that the carbon be removed immediately after shaking. The resulting nitrate solutions are then freeze dried, redissolved in 2 ml of DI, pipetted into quartz tubes, frozen and freeze-dried (as described for d 15N of AgNO3). After freeze-drying, 4–5 mg of finely ground spectrographic graphite is added to the samples and the tubes are evacuated and torch sealed. The samples are combusted in a furnace programmed to heat to 8508C at its maximum rate then shut off. Samples are allowed to cool overnight to room temperature in the oven with the door closed. The resulting CO2 is extracted on a vacuum line, cryogenically purified, gas yields measured by manometer, and transferred to a sample tube for analysis. 2.4. Silver oxide Silver oxide was selected as a neutralizing agent for acid column eluant, because all other reasonable alternatives (hydroxides, oxides, and metals) caused one or more of the following difficulties: (1) reaction of the cations (particularly Na) with the quartz combustion tubes, which causes failure during combustion; (2) formation of a hydrous chloride and/or nitrate salt, which requires an extra drying step; or (3), production of large quantities of fluffy salt (e.g., potassium chloride), which is very cumbersome to handle and leaves insufficient room in the combustion tubes for other reagents. Commercially available Ag2O reagent (Alpha 2, Danvers, MA, cat. #11407) must be treated to remove NO32; different batches used during these experiments had NO32 concentrations ranging from 11–75 ppm. To remove contaminant NO32, 2 l of DI is added to 500 g of Ag2O in a 4 l Erlenmeyer flask and stirred for several hours. The rinse water is decanted off through a Whatman #1 filter. This process is repeated until the

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NO32

level in the rinse water is below detection (0.01 mg-N/l) using the low range HACH 2 kit test. The rinse water is collected and treated with an excess of NaCl to precipitate dissolved Ag as AgCl. The AgCl is filtered out through a 0.45 mm nylon filter before disposal of the rinse water. The rinsed Ag2O is oven dried at 508C and stored in a dark container. One drawback to the use of Ag2O is cost. We are refining a process for regenerating Ag2O from the AgCl waste. The current procedure is to heat at a low boil 500 g AgCl in 2 l of 3 M KOH overnight in a 4 l Erlenmeyer flask with a condenser to recycle the water. Filter out the spent KOH and repeat the process two additional times. The newly-formed Ag2O solid is dissolved in 3 M HNO3 (approx. 1 l). The solution is decanted to separate unconverted AgCl solid. Fresh Ag2O is precipitated from the decanted solution on addition of 3 M KOH (approx. 1 l). The new Ag2O is filtered and rinsed as described above. The AgNO3 solutions are subject to isotope fractionation if processing is delayed. Aqueous AgNO3 samples left in covered glass beakers, and exposed to ambient light for 24 h showed a 0.15‰ enrichment in 15N. A likely cause for fractionation is photo degradation of AgNO3. For this reason we recommend processing samples without delay and restricting light from the samples wherever practical.

3. Experimental section 3.1. Experimental procedures, materials, and precision The use of anion exchange resin columns to concentrate NO32 in the field allows the collection of optimal sample sizes for maximum analytical precision, regardless of the nitrate concentration of the water. Approximate nitrate concentrations are determined in the field and enough water is collected and passed through the columns to yield 100–200 mmol of NO32. Two types of test solutions were used to evaluate various aspects of sample collection and preservation: laboratory standard solutions made from Fisher 2 KNO3 standard salt …d 15 N ˆ 13:49‰†; DI water,

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plus other reagents as required, and “natural” samples collected from a USGS field site (Blevins et al., 1994) from which both 15N enriched and natural abundance isotope ratio NO32 samples were collected. Isotope compositions for nitrogen are reported in parts per thousand or per mil (‰) relative to atmospheric air, and relative to VSMOW (Vienna Standard Mean Ocean Water) for oxygen. During the initial development of the method, NO32 yields were measured to assess the efficiency of trial procedures (Section 3.3). After the method for loading and stripping the columns had been established, only isotope ratio measurements were made to test for significant fractionations caused by the adsorption interference and preservation conditions as described below. During sorption and desorption experiments, nitrate concentrations measured in sample solutions before and after passage through the columns were determined using methods described by Fishman and Friedman (1989); the analytical precision is ^1.5%. To determine the accuracy and precision of the anion exchange column method for d 15N analyses, KNO3 solutions were prepared at 25.0 and 0.2 mgN/l from KNO3 standard salt …d15 N ˆ 13:49‰† and deionized water. The average d 15N and 1s standard deviation of dissolved nitrate from the two solutions, after having been sorbed on and stripped from anion columns as described above, was 13:49 ^ 0:04‰ …n ˆ 10†; and 13:48 ^ 0:08‰ …n ˆ 10†: Instrumental precision is ^0.02‰. A laboratory standard for d 18O analyses of NO32 was established by combusting Fisher AgNO3 with graphite (Section 3.7.1). The average yield of CO2 was 98:7 ^ 2:4% …n ˆ 23† and the mean d 18O was 119:7 ^ 0:2‰ …n ˆ 19†: The accuracy and precision of the anion exchange column method for d 18O was assessed with 9.4 mg-N/l solutions made from AgNO3 and DI water. After sorption and desorption of the solution onto and from columns, and preparation of the column eluant for d 18O analysis, as described in the methods section, the average d 18O value was 119:2 ^ 0:04‰ …n ˆ 4†: 3.2. Adsorption efficiency and flow rate Nitrate adsorption efficiency was tested using standard solutions and natural samples by comparing the

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efficiencies were increased by adding the HCl in several increments and blowing the remaining HCl through the columns after each increment. Fifteen ml of 3 M HCl applied in five 3 ml increments consistently removed more than 98% of the NO32. Higher HCl concentrations (up to 6 M) did not improve elution efficiency. 3.4. Nitrogen isotope fractionation by incomplete nitrate elution

Fig. 3. Percentage of nitrate removed from anion exchange columns with increasing amount of eluant.

NO32 content of samples before and after passage through the columns. The test solutions included 77 field samples with NO32 concentrations ranging from 17 to 130 mg-N/l (1.21–9.29 meq/l) and 10 solutions with NO32 concentrations between 25 and 100 mg-N/l (1.79–7.14 meq/l) prepared from KNO3 reagent. The average percentage of NO32 sorbed for the 87 samples was 99:84 ^ 0:16%: There was no correlation …r 2 ˆ 0:13† between the extent of NO32 sorption and flow rate for rates between 90 and 565 ml/h. Nitrate fractionation caused by incomplete adsorption or desorption of nitrate was demonstrated by experiment (Sections 3.4 and 3.6). In later experiments, using KNO3 standard solutions and flow rates of about 1000 ml/h, concentrations were not measured but d 15N analyses showed accuracy and precision equal to that obtained at the lower flow rates, indicating virtually complete sorption and recovery of nitrate. 3.3. Elution of nitrate from the column We tested HCl, KCl, SrCl2, CaCl2, H2SO4, K3C6H5O7· 2H20 (potassium citrate), and Ca3(C6H5O7)2·4H20 (calcium citrate) as possible eluants. Of these, hydrochloric acid was found to be superior both in terms of consistently high nitrate yields and its relative ease of handling. The percent of NO32 recovered with increasing quantities of 3 M HCl is depicted in Fig. 3. Initial experiments indicated that the mean recovery of NO32 with a single 15 ml aliquot of 2.7 M HCl was greater than 95%, but with a large variance. Recovery

To evaluate the effect of incomplete NO32 elution on isotope composition, resin columns were loaded with 100 ml of a standard 25 mg-N/l NO32 (1.8 meq/l) solution …d15 N ˆ 13:49‰† as described above and eluted with three successive 5 ml aliquots of 3 M HCl. Each aliquot eluted was individually measured for NO32 concentration, prepared, and analyzed for d 15N. Dissolved nitrate in the first aliquot was isotopically heavier than the bulk sample; successive aliquots became increasingly depleted in 15N, indicating that 15N was preferentially displaced during elution. The data show that incomplete elution, leaving 3% of the NO32 on the column, caused an increase in d 15N of 0.3‰ with respect to the true 15N value (Fig. 4). Nitrogen isotope fractionation for natural nitrate solutions will likely vary with loading conditions including total sample volume, the concentration of other anions in solution (Section 3.6), and flow rate. 3.5. Sample preservation 3.5.1. Refrigeration vs. HgCl2 treatment plus refrigeration Morrissey (1989) reported that NO32 can be preserved on anion exchange columns at least 35 days when stored moist and refrigerated. In this study, nitrate preservation was tested by comparing nitrate yields of field samples which had been poisoned with HgCl2 before loading on anion exchange columns with sample splits which received no treatment. After loading the columns, both sets of samples were stored moist and refrigerated. No loss of NO32 occurred with either set after storage for 55 days. The mean recovery of NO32 for the HgCl2-treated samples was 100:3% ^ 0:9% …n ˆ 4† and for the refrigerated samples was 99:7% ^ 0:7% …n ˆ 5†:

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3.5.2. Storage of nitrate on columns vs. storage in HCl eluant To determine whether nitrate can be stripped from columns and stored in the HCl eluant for extended periods of time without loss, 10 sample solutions were prepared with 25 mg-N/l NO32 (1.8 meq/l) and the nitrate was sorbed on columns. Five columns were stored refrigerated. Five columns were eluted immediately and the nitrate-bearing eluant was refrigerated in 60 ml glass bottles with Polyseal 2 (conical insert) caps. After one month, both sets of samples were processed and analyzed. Nitrate stored on the columns had an average d 15N of 13:48 ^ 0:04‰; identical to the d 15N of the original KNO3 reagent (13.49‰), whereas nitrate stored in the HCl eluant had an average d 15N of 13:58 ^ 0:04‰: These results suggest a small loss of the lighter nitrogen isotope from the acid solution, possibly due to a loss (evaporation) of minor amounts of HNO3 into the headspace of the bottles used. 3.5.3. Comparison of d 15N values from samples analyzed immediately with duplicates stored on columns for up to two years To evaluate the reproducibility of isotope analysis on nitrate from field samples, nitrate from 18 samples was collected in duplicate on columns. The d 15N compositions ranged from approximately 19– 142‰. One sample from each pair was processed immediately after collection and the isotope

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composition was measured; the other nitrate sample was stored refrigerated on the column for 3 to 24 months prior to preparation. In general, the d 15N values of nitrate stored on anion exchange columns showed good agreement with those of the nitrate, which was analyzed immediately. Excluding two outliers, the average difference in d 15N values was 0:21 ^ 0:19‰: The two outliers, with deviations of 22.25‰ and 14.10‰, were both spiked samples (135.55 and 1138.22‰, respectively), stored for 3 mon. The relatively short storage time and the opposite signs of deviations from the initial d 15N values of the outliers suggest that these differences are unrelated to storage time. In fact, for all samples, there was no correlation between the magnitude of deviations from the original d 15N measurements and preservation time …r2 ˆ 0:05†: Although only five of the 18 replicate samples showed a positive difference in d 15N between the original and stored samples, a Wilcoxon’s signed-rank test of the data indicates that there is no significant tendency for the differences to be in a positive or negative direction. 3.6. Adsorption interferences The ability of the anion exchange resin to adsorb and retain nitrate is a function of the selectivity (affinity) of the anion for the resin, the exchange capacity of the resin, and the competition for sites among other anions in solution. If the presence of other

Fig. 4. Variations in d 15N values resulting from incomplete desorption of nitrate from columns. The HCl eluant preferentially elutes 15NO3 causing an isotopic enrichment in the recovered sample if nitrate recovery is not about 100%. Each line represents the incremental desorption of one column. Samples were eluted with three 5 ml increments of 3 M HCl. The width of the symbols corresponds to a 1s analytical precision of 0.05‰.

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Table 1 Relative selectivity of counterions for Bio-Rad AG1-X8 resin (modified from Bio-Rad, Guide to Ion Exchange, 1993) Counterion

Relative selectivity

Salicylate (C7H5O3) Citrate (C6H5O7) Bisulfate (HSO32) Nitrate (NO32) Bromide (Br 2) Nitrite (NO22) Chloride (Cl 2) Bicarbonate (HCO32)

450 220 85 65 50 24 22 6

anions cause NO32 to be incompletely sorbed during loading or incompletely eluted from the column during stripping, isotope fractionation could result. The potential interference by chloride, sulfate, and dissolved organic carbon was tested, as described below. The most abundant anions in natural waters are Cl 2, SO42, and HCO32 (Hem, 1992). The AG 1-X8 resin exhibits selectivity in the order 2 2 . Cl . HCO (Table 1). Although the selecNO2 3 3 tivity of SO422 has not been determined, its higher valence suggests a stronger selectivity than singlecharged species such as NO32 (personal communication, D. Hardy, Bio-Rad Corp., 1994). Therefore, Cl 2 and SO422 probably have the highest potential for inhibiting adsorption of NO32 on anion exchange resin columns. Bicarbonate was considered less likely to cause interference because the relative selectivity of HCO32 is approximately 1/6 that of Cl 2. Because DOC may contain as much as 2% N, and its behavior on anion exchange resin columns relative to NO32 is unknown, there was concern that DOC might interfere with adsorption or be eluted with nitrate and influence d 15N analyses of the sample (see Sections 3.7.3 and 4.2 for discussion of d 18O and DOC). 3.6.1. Chloride interference The effect of incomplete NO32 adsorption on the measured d 15N values caused by Cl 2 in sample solutions was measured by passing Cl-amended NO32 solutions through anion exchange columns, eluting the nitrate, and analyzing the nitrate-bearing eluant for d 15N as described above. Three parameters varied: volume of solution passed through the columns (100– 1000 ml), nitrate concentrations (2.5–25 mg-N/l), and

chloride concentrations (200–1000 mg/l). Solutions were prepared with a KNO3 standard …d15 N ˆ 13:49‰† and CaCl2. Since the AG1-X8 resin has a higher affinity for NO32 than Cl 2 and is in the chloride form, incomplete adsorption of NO32 can only be caused by displacement of the NO32 and not by exceeding the exchange capacity of the resin with Cl 2. Fig. 5A–C illustrate the results from experiments with 100, 500, and 1000 ml solutions, respectively. For each experiment, five samples were prepared in duplicate and in increasing Cl 2 concentrations from 200 to 1000 mg/l (5.6–28.2 meq/l). Experiments with 500 and 1000 ml solutions were performed with two different NO32 concentrations. In general, the extent of nitrogen isotope fractionation increased with increasing Cl 2 concentration and sample volume, and with decreasing NO32 concentration. 3.6.2. Sulfate interference Sulfate was expected to displace both NO32 and Cl 2 due to its divalent nature and potentially higher selectivity for adsorption onto the resin. During sorption, one SO422 ion should displace two single-charged species (personal communication, D. Hardy, BioRad Corp.). Therefore, SO422 was expected to cause fractionation more readily than Cl 2. To test this assumption, KNO3 standard solutions …d15 N ˆ 13:49‰† with 25 mg-N/l (1.8 meq) were amended with Na2SO4 to yield 15 solutions with SO422 concentrations ranging from 20 and 2000 mg/l (0.4– 41.7 meq/l). Five columns were loaded with 100 ml each of solutions with SO422 concentrations between 20 and 100 mg/l (0.4–2.1 meq/l), and 20 columns were loaded with 100 ml each of solutions with SO422 concentrations between 200 and 2000 mg/l (4.2–41.7 meq/l). The resin exchange capacity of the columns (2.4 meq per column) was exceeded by the combined masses of NO32 and SO422 for 10 columns loaded with SO422 solutions having concentrations above 1000 mg/l (20.8 meq/l). All columns were immediately eluted, prepared, and analyzed for their isotope composition. Contrary to expectations, sorption of sulfate caused no nitrogen isotope fractionation even when the combined concentrations of NO32 and SO422 exceeded the column capacity; the mean d 15N value for all 25 samples was 13:50 ^ 0:04‰: Nitrate yields for the 10 samples which exceeded the column exchange

S.R. Silva et al. / Journal of Hydrology 228 (2000) 22–36

NO32

capacity indicated losses ranging between 1.3 and 10.7%. If all nitrate had been excluded from adsorption after the column capacity had been reached due to the presence of sulfate, NO32 losses would have ranged from 10.4 to 44.8% for the 1200–2000 mg/l SO422 samples. 3.6.3. DOC interference In order to test the potential interference caused by DOC on NO32 adsorption on anion exchange resin columns, potassium biphthalate, an organic acid

31

typically used to prepare concentration standards for analyses of dissolved carbon, was used as a proxy for DOC. It bonds to the resin by both ion exchange and hydrophobic interaction, resulting in a much stronger bond than that of NO32 (personal commun., 1993, D. Hardy, Bio-Rad Corp.) Potassium biphthalate was added to KNO3 standard solutions (25 mg-N/l NO32) as described above to produce solutions with five DOC concentrations ranging from 10 to 200 mg/l C. Two columns each were loaded with 100 ml of each of the five solutions. The dissolved nitrate was

Fig. 5. Effects of Cl 2 interference on d 15N values for different solution volumes and NO32 concentrations: (A) a 100-ml solution containing 25 mg-N/l NO32; (B) two 500-ml solutions containing 6.25 and 12.5 mg-N/l NO32; and (C) two 1000-ml solutions containing 2.5 and 6.25 mg-N/l NO32. Each symbol represents the results from a single column; curves connect the average values. The d 15N values of the KNO3 standard used to prepare the solutions is 13.49‰ (open rectangle on X-axis). The width of the symbols corresponds to a 1s analytical precision of 0.05‰.

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S.R. Silva et al. / Journal of Hydrology 228 (2000) 22–36

Table 2 Effects of graphite mixing and quantity on CO2 yield after thermal decomposition of AgNO3 Preparation

Preparation as described in text Graphite and AgNO3 not mixed Graphite excess ˆ 150% Graphite excess ˆ 300% Graphite excess ˆ 500%

Yield (%)

Std. Dev.

Replicates

98.7 96.8 98.9 99.6 97.1

2.4 1.1 1.2 2.3 2.9

23 4 3 3 3

immediately eluted, prepared, and analyzed for its isotope composition. The d 15N for all 10 samples was 13:48 ^ 0:08‰; indicating no measurable nitrogen isotope fractionation. 3.7. Experiments related to d 18O–NO3 3.7.1. Combustion of AgNO3 with graphite Attempts to produce CO2 for d 18O analysis from KNO3 and Ba(NO3)2 by sealed-tube combustion with graphite resulted in yields between 60 and 80%. Only thermal decomposition of AgNO3 with graphite gave consistently high yields of CO2 approaching 100%, (Table 2). Graphite added in stoichiometric excesses of 150, 300, and 500% had little effect on the CO2 yield. A comparison of thoroughly mixed graphite and AgNO3 with nonmixed reagents also showed little difference in yield (Table 2). Gas species produced, in addition to CO2, were measured on a gas chromatograph for one sample each combusted at 800, 850, and 9008C. The results indicated that CO production increased with temperature from 0 to 3% while NO decreased from 2.35 to 0% (Table 3).

Table 3 CO and NO contents of gas generated by thermal decomposition of AgNO3 and graphite at various temperatures Temp. (8C)

CO (%)

NO (%)

800 850 900

0 0.01 3.00

2.35 1.24 0

Table 4 Comparison of d 18O and d 15N values for AgNO3 obtained after nitrate solutions were passed through anion, cation, and both anion and cation columns Column type

d 18O avg.

d 15N avg.

Replicates

Anion Cation Anion 1 Cation

126:43 ^ 0:46 126:53 ^ 0:23 126:05 ^ 0:08

13:52 ^ 0:06 13:56 ^ 0:07 13:48 ^ 0:05

3 3 3

3.7.2. Ion exchange columns and d 18O–NO3 As described above, the neutralized NO32 solution is passed through cation columns (after the addition of BaCl2 and subsequent filtration to remove sulfate) (Section 2.3.2). Nitrate is then converted from HNO3 (after passage through the cation exchange column) back to AgNO3 by reaction with Ag2O. To test for oxygen isotope fractionation after both of these steps, nine 100 ml aliquots of 100 mmol nitrate were prepared from standard KNO3 and DI water. Although the d 18O value of the KNO3 was not previously known, the d 15N value was well known. Since d 15N analyses are currently more precise, nitrogen isotope ratios were used as an indicator of fractionation while d 18O was assessed for precision among the three preparations. Nitrate in three aliquots was loaded onto anion exchange columns, stripped, converted to AgNO3, and combusted to CO2. Three other aliquots were passed through cation exchange columns, and the nitrate was converted to AgNO3 and combusted to CO2 as described above; the remaining three aliquots were passed through both anion and cation exchange columns as would natural samples. All the nine samples were analyzed for both d 15N and d 18O. The average isotope composition of nitrate for the three sets of samples agreed within 0.08‰ for d 15N and the mean value agreed within 0.03‰ of the accepted d 15N value for the standard indicating no significant isotope fractionation of nitrate. The average d 18O values for the three sets of samples agreed within 0.5‰ (Table 4). 3.7.3. Removal of dissolved organic carbon DOC contains roughly 30–50% oxygen and therefore must be removed from samples prior to combustion for the purpose of d 18O-NO3 analysis. Common methods for the removal of DOC include the use of XAD-type resins, activated charcoal, and

S.R. Silva et al. / Journal of Hydrology 228 (2000) 22–36

ultrafiltration. No method known to us removes all organics while leaving nitrate intact. Activated carbon was chosen for DOC removal because it is reasonably effective, inexpensive, and easy to use. Much of the DOC in the sample was eliminated through collection and preparation while a small fraction was added from the resins and plastic ware; therefore, activated carbon was added to the sample at the last step of wet preparation to eliminate as much organic material as possible from all sources. A series of experiments were performed to assess the fate of natural DOC during the entire column procedure (Chang et al., 1999). These experiments used 5 ml of anion exchange resin in larger columns, however, the results are generally applicable here because the resin-type and procedures were otherwise identical. The findings demonstrated that DOC collected from various natural sources behaves quite differently on the columns. In general, an average of about 50% DOC was retained on the anion exchange resin, a considerable fraction permanently. The mass of DOC remaining in the final solution before treatment with activated carbon was consistently less than 0.8 mg (about 20 mg/l in 40 ml of solution) regardless of the mass of DOC exposed to the columns (10–40 mg) or the mass retained by the columns, suggesting that most of DOC released from the resin during stripping was subsequently eliminated by adsorption, precipitation, or volatilization. Two sets of blanks were prepared to determine the quantity of carbon (measured as CO2) that was derived from the reagents and equipment. One set of blanks consisted of graphite combusted in quartz tubes and the other consisted of DI water processed through the entire column procedure exclusive of activated carbon. CO2 derived from the two sets of blanks averaged 1.4 and 2.8 mmol each, indicating that about half of the blank originated from the graphite. Activated carbon has a lower affinity for NO32 than for larger organic molecules but can adsorb significant quantities of NO32 if applied in too high concentrations. It was found that a ratio of 20 mg Norit G60 activated carbon to 100 ml of solution and a shaking time of 20 min was optimal. Under these conditions, Norit G-60 activated carbon reduced DOC concentrations to about 1 mg/l in the final solution.

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The exact quantity and isotope composition of nonnitrate oxygen derived from sample and laboratory materials is difficult to assess; therefore, we recommend the preparation of relatively large samples ($100 mmol CO2) for d 18O analysis to minimize contaminant effects. 3.7.4. The d 18O values for KNO3 standards USGS-32 and N3 Five replicates of 100 mmol each of KNO3 standards USGS-32 and N3 were analyzed for their oxygen isotope composition by dissolving them in DI water and passing them through a cation exchange column. The columns eluants were prepared is as described above (Section 2.3.2). The d 18O values of USGS-32 and N3 were 23:1 ^ 0:1‰; and 22:9 ^ 0:1‰; respectively, in good agreement with values reported by Revesz et al. (1997).

4. Discussion 4.1. The anion exchange column technique and preparation for d 15N-NO32 The resin column technique for concentrating and preparing nitrate for isotope analysis appears well suited for waters of moderate nitrate concentrations and low to moderate concentrations of common anions and DOC. The preservation experiments indicate that samples can be archived on columns for up to two years and that no additional preservatives such as HgCl2 are required if samples are passed through ion exchange resins shortly after collection. This alleviates the need to dispose of sample waters as hazardous waste. The sequential desorption experiment indicated that 15N is preferentially lost during competition for exchange sites. This phenomenon accounts for the 15N depletion of nitrate noted during the Cl 2 interference experiments and indicates that the d 15N value of a sample which experienced partial desorption on loading, will be d 15N-depleted. Complete sorption and recovery of nitrate onto and from anion exchange columns ensures that no nitrogen and oxygen fractionation occurs. The nitrate loss and isotope fractionation observed during the Cl 2 interference experiments was

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principally a function of Cl concentration and volume of sample passed through the columns, while nitrate concentrations had relatively little control on fractionation. The data indicate that 1000 ml of solution with chloride concentrations of up to 200 mg/l can be passed through a column without causing significant isotope fractionation of the NO32; higher chloride concentrations can be tolerated in smaller-volume samples. Although flow rate may also have an effect on nitrate loss, it was not specifically tested in these experiments. In practice, the drip rate tends to slow somewhat with resin packing and accumulation of adsorbed organic matter and therefore varies with loading time and from sample to sample. We strongly recommend that representative samples be tested for column breakthrough before beginning full-scale collection. The SO422 interference experiments produced no measurable nitrogen isotope fractionation even when the column capacity was exceeded by the combined mass of NO32 and SO422. In addition, NO32 losses were much less than predicted for samples that should have exceeded the column capacity. This is surprising because SO422 is thought to have a higher affinity for the anion exchange resin than NO32 due to its double charge. Besides out-competing NO32 for exchange sites, each SO422 ion should liberate two singlycharged anions which are then available to interfere with NO32 adsorption; hence, SO422 should have at least double the interference potential of an equal molar concentration of Cl 2. By this reasoning, the 2000 mg/l SO422 solution should have produced isotope fractionation effects at least equivalent to a 1480 mg/l (41.7 meq/l) Cl 2 solution, however, this was not the case. One possible explanation for the unexpected results is that SO422 may actually have a lower affinity than NO32 at any individual site. In this case, newly arriving SO422 ions will not displace NO32 but will bond at the “exchange front”, which progressively travels down the column during loading, leaving the previously sorbed NO32 protected and above the rain of Cl 2 ions liberated from the resin by SO422 adsorption. After the column capacity is reached by NO32 plus SO422, additional NO32 applied to the column may pass through in bulk causing no fractionation. The K-biphthalate experiments suggested that DOC in concentrations up to 200 mg/l C does not cause

measurable nitrogen isotope fractionation for nitrate on the column. This conclusion is valid only to the extent that the behavior of K-phthalate accurately reflects that of DOC, which is composed of a spectrum of species, mostly organic acids. Another concern is that DOC may contribute some amount of N to the sample. The content of N in humic and fulvic acids, which make up most of DOC, ranges between about 0.4 and 2% (Drever, 1988); therefore, a 1 l sample with 2 mg/l DOC could contain as much as 3 mmol N. However, a variable but significant fraction of DOC is lost during the column procedure. Since the average DOC concentration in groundwater is less than 1 mg/l (Thurman, 1985), DOC should generally contribute only a very small fraction of the total N analyzed. In cases of high DOC concentrations, it may be possible to remove much of it from the samples solution by adsorption with activated carbon, XAD-resins, or through ultrafiltration before loading of the sample on the anion exchange columns. For moderate to low DOC concentrations, most is eliminated by a combination of passage through the column on loading, permanent adhesion to the resin, and either adsorption, precipitation, or volatilization through the preparation process (Chang et al., 1999). In samples with low NO32 concentrations, requiring large volumes of water to be passed through the columns, accumulated DOC tends to clog the resins. Possible solutions in these cases include: (1) loading of sample solutions on multiple columns to be recombined in the lab; (2) using a larger capacity resin column with a bigger cross-sectional area, a coarser resin (lower mesh size) to increase flow rate, and a cation column in line ahead of the anion column to adsorb DOC or neutralize anionic DOC (Chang et al., 1999). 4.2. Preparation for d 18O–NO32 analysis Nitrate collected on anion exchange columns may be converted to AgNO3 and subsequently to CO2 for d 18O analysis with a precision of 0.5‰. Thermal decomposition of AgNO3 with graphite produces a nearly complete conversion of nitrate oxygen to CO2. Gas chromatographic analyses indicated that non-CO2 oxygen-bearing species were mostly or entirely CO and NO. Refinement of the combustion conditions and possibly the use of activated carbon in

S.R. Silva et al. / Journal of Hydrology 228 (2000) 22–36

place of graphite may ensure a more complete conversion of AgNO3 to CO2 (Revesz et al., 1997). The greatest obstacle to d 18O-NO3 analysis is the presence of other oxygen-bearing substances, particularly DOC, in the water samples. These constitute not only a potential oxygen contaminant but add considerably to the time and complexity of the preparation procedure. Measurements of DOC contents throughout the collection and preparation process indicated that the majority is eliminated before the application of activated carbon. The activated carbon step at the end of the preparation procedure lowers the DOC concentration to approximately 1 mg/l. Two unexplored, and possibly more effective alternatives to activated carbon are ultrafiltration where the final solution is centrifuged through a filter to remove the residual DOC and osmotic membrane filtration (Feuerstein et al., 1997) where small ions such as NO3 are allowed to pass through a membrane while large species including organics are retained.

5. Conclusions The anion exchange resin technique offers an efficient and reliable means of collecting, transporting, and storing, water samples for nitrogen and oxygen isotope analysis of nitrate. Experimental results indicate that the method is most applicable to fresh waters with moderate to high nitrate concentrations, although it may be modified for lower concentrations (Chang et al., 1999). Multiple samples may be sorbed on columns in the field using common and relatively inexpensive equipment. The laboratory preparation of samples for d 15N analysis requires little technician time per sample (about 30 min) compared to the Kjeldahl distillation technique because many samples can be processed simultaneously. Most of the total time required for sample preparation is spent in the freeze-drying and combustion steps. The preparation of samples for d 18O analyses is more time consuming (about 2 h) because of the necessity of eliminating non-nitrate, oxygen-bearing anions and DOC, and cryogenic extraction of CO2. The main strengths of this technique are the high

35

yield of CO2 and the elimination of highly toxic HgCN. Although the anion exchange column method and particularly the d 18O–NO3 preparation portion will benefit from further refinement, it has proven its usefulness providing precise d 15N and d 18O values for nitrate in field studies for over four years to date. Acknowledgements We thank Bernhard Mayer, Len Wassenaar, J.K. Bohlke, and Brian Fry for thorough review and valuable criticism of this paper and John Radyk, Jim Langston, Doug White, Dale Blevins, Bill Evans, Theresa Presser, and Tina Saad for their advice and help with the method development. References Amberger, A., Schmidt, H.-L., 1987. Natu¨rliche Isotopengehalte von Nitrat als Indikatoren fu¨r dessen Herkunft. Geochim. Cosmochim. Acta 51, 2699–2705. Aravena, R., Robertson, W.D., 1999. Use of multiple isotope tracers to evaluate denitrification in groundwater: case study of nitrate from a large-flux septic system plume. Ground Water 36 (6), 975–982. Blevins, D.W., Wilkison, D.H., Silva, S.R., Kelly, B.P., 1994. Use of 15N to trace movement of nitrogen fertilizer at a field plot. J. Environ. Quality 25 (3), 584–593. Boettcher, J., Strebel, O., Voerkelius, S., Schmidt, H.-L., 1990. Using isotope fractionation of nitrate-nitrogen and nitrateoxygen for evaluation of microbial denitrification in a sandy aquifer. J. Hydrol. 114, 413–424. Bremner, J.M., 1965. Isotope-ratio analysis of nitrogen in nitrogen15 tracer investigations. In: Black, C.A. (Ed.), Methods of Soil Analysis. Part 2. Agronomy 9, pp. 1256–1286. Bremner, J.M., Edwards, A.P., 1965. Determination and isotoperatio analysis of different forms of nitrogen in soils: I. Apparatus and procedure for distillation and determination of ammonium. Soil Sci. Soc. Am. Proc. 29, 504–507. Chang, C.C.Y., Langston, J., Riggs, M., Campbell, D.H., Silva, S.R., Kendall, C., 1999. A method for nitrate colleciton for d 15N and d 18O analysis from waters with low nitrate concentrations. Can. J. Fish. Aquatic Sci. 56, pp. 1856–1864. Downs, M.R., Michener, R.H., Fry, B., Nadelhoffer, K.J., 1999. Routine measurement of dissolved inorganic 15N in precipitation and streamwater. Environmental Monitoring and Assessment 55, pp. 211–220. Drever, J.I., 1988. The Geochemistry of Natural Waters. PrenticeHall, Englewood Cliffs, NJ, 437 pp. Feuerstein, T.P., Ostrom, P.H., Ostrom, N.E., 1997. Isotopic

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biogeochemistry of dissolved organic nitrogen: a new technique and application. Org. Geochem. 27 (7/8), 363–370. Fishman, M.J., Friedman, L.C., 1989. Methods for determination of inorganic substances in water and fluvial sediments, in: Techniques of Water-Resources Investigations of the US Geological Survey, Book 5, Chapter A1, 545 pp. Garten Jr, C.T., 1992. Nitrogen isotope composition of ammonium and nitrate in bulk precipitation and forest throughfall. Int. J. Environ. Anal. Chem. 47, 33–45. Ging, P.B., Lee, R.W., Silva, S.R., 1996. Water Chemistry of Shoal Creek and Waller Creek, Austin, Texas, and potential sources of nitrate, USGS, Water Resources Investigations Report 96-4167. Hem, J.D., 1992. Study and interpretation of the chemical characteristics of natural waters, in: US Geological Survey Water Supply Paper 2254, 263 pp. Hoering, T., 1957. The isotopic composition of ammonia and nitrate ion in rain. Geochim. Cosmochim. Acta 12, 97–102. Kendall, C., Campbell, D.H., Burns, D.A., Shanley, J.B., Silva, S.R., Chang, C.C., 1995. Tracing sources of nitrate in snowmelt runoff using the oxygen and nitrogen isotopic compositions of nitrate, in: Biogeochemistry of Seasonally Snow-Covered Catchments (Proceedings of a Boulder Symposium, July 1995) IAHS Publ. no. 228, pp. 339–347. Kendall, C., Grim, E., 1990. Combustion tube method for measurement of nitrogen isotope ratios using calcium oxide for total removal of carbon dioxide and water. Anal. Chem. 62, 526–529. MacKown, C.T., Brooks, P.D., Smith, M.S., 1987. Diffusion of Nitrogen-15 Kjeldahl digests for isotope analysis. Soil Sci. Soc. Am. J. 51, 87–90.

Morrissey, K.M., 1989. Determining the source of nitrate contamination in ground water using 15-N as a tracer: method development. Boston, Massachusetts, University of Maryland, Master’s thesis, 75 pp, unpublished. Mulvaney, R.L., 1986. Comparison of procedures for reducing cross-contamination during steam distillations in nitrogen-15 tracer research. Soil Sci. Am. J. 50, 92–96. Revesz, K., Bohlke, J.K., Yoshinari, T., 1997. Determination of d 18O and d 15N in nitrate. Anal. Chem. 69, 4375–4380. Sigman, D.M., Altabet, M.A., Michener, R., McCorkle, D.C., Fry, B., Holmes, R.M., 1997. Natural abundance-level measurement of the nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonia diffusion method. Marine Chem. 57, 227–242. Thurman, E.M., 1985. Humic substances in groundwater. In: Aiken, G.R., McKnight, D.M., Wershaw, R.L., MacCarthy, P. (Eds.), Humic Substances in Soil, Sediment, and Water. Wiley, New York, pp. 87–103. Velinsky, D.J., Cifuentes, L.A., Pennock, J.R., Sharp, H., Fogel, M.L., 1989. Determination of the isotope composition of NH41-nitrogen at the natural abundance level from estuarine waters. Marine Chem. 26, 351–361. Vitousek, P.M., Aber, J.D., Howarth, R.W., Likens, G.E., Matson, P.A., Schindler, D.W., Schlesinger, W.H., Tilman, D.G., 1997. Human alteration of the global nitrogen cycle: sources and consequences. Ecol. Appl. 7 (3), 737–750. Wassenaar, L.I., 1995. Evaluation of the origin and fate of nitrate in the Abbotsford Aquifer using the isotopes of 15N and 18O in NO32. Appl. Geochem. 10, 391–405.