Environ. Sci. Technol. 2005, 39, 879-884
Dissolved Organic Nitrogen Measurement Using Dialysis Pretreatment WONTAE LEE* AND PAUL WESTERHOFF Department of Civil and Environmental Engineering, Arizona State University, Tempe, Arizona 85287-5306
pollution through wastewater discharges, agricultural runoff, and NOx deposition have increased total dissolved nitrogen (TDN) levels in many surface waters (13-15). Consequently, DON represents a lower percentage of TDN in humanimpacted water (e.g., nitrate inputs). Methods capable of quantifying low DON concentrations in waters with elevated DIN tend to be inaccurate; pretreatment methodologies have the potential to improve this accuracy. DON quantification involves subtracting DIN concentrations from the TDN concentration:
DON ) TDN - NO3- - NO2- - NH3/NH4+
Dissolved organic nitrogen (DON) is important for ecological and engineering researches. Quantification of low DON concentrations in waters with elevated dissolved inorganic nitrogen (DIN) using existing methods is inaccurate. In this study, a dialysis-based pretreatment technique was optimized and adopted to reduce the interference from DIN to the quantification of DON in natural water. A cellulose ester dialysis tube (nominal molecular weight cutoff ) 100 Da) was used in batch and continuousflow dialysis steps with model compounds, natural organic matter isolates, and bulk waters to develop a dialysis pretreatment approach that selectively reduces DIN from solutions containing DON. By reducing DIN concentrations, propagation of analytical variance in total dissolved nitrogen (TDN) and DIN species concentrations allows more accurate determination of DON (DON ) TDN - NO3- - NO2- - NH3/NH4+). Dialysis for 24 h against continuously flowing distilled water reduced DIN species by 70%. With dialysis pretreatment, DON recoveries of more than 95% were obtained for surface water and finished drinking water, but wastewater experienced a slight loss (∼10%) of DON possibly due to the adsorption of organics onto the dialysis membrane, permeation of low molecular weight fractions, or biodegradation. Dialysis experiments using surface water spiked with different DIN/TDN ratios concluded that dialysis pretreatment leads to more accurate DON determination than no dialysis when DIN/TDN ratios exceed 0.6 mg of N/mg of N.
Introduction Elemental composition analysis indicates that lyophilized aquatic natural organic matter (NOM) contains nitrogen (15%), carbon (40-60%), oxygen (∼40%), hydrogen (∼5%), sulfur (99% nitrate removal. Therefore, the ratio was set at 1: 640 and the flow rate at 100 mL/min. Except for experiments to evaluate the effect of dialysis duration on the remaining DIN and DON, samples were dialyzed for 24 h. Each sample was dialyzed in triplicate unless otherwise stated. Optimization of the Dialysis System. Various nitrogencontaining compounds and waters from Arizona, including Arizona Canal water, effluent from a nitrifying-denitrifying (NDN) wastewater treatment plant (WWTP) (Mesa, AZ), and effluent from a trickling filter (TF) WWTP (Tucson, AZ), were used to optimize the dialysis system. Water samples were filtered with pre-ashed (500 °C) glass fiber filters (GF/F; Millipore, Maidstone, UK). Table 1 lists the characteristics of the water sources. Reagent grade nitrogen-containing compounds were dissolved in deionized water (Nanopure Infinity, Barnstead, IA). Solutions containing NOM were prepared by dissolving fractionated NOM isolates in deionized water: (a) a hydrophobic isolate (C/N ratio ) 28) obtained from water from Saguaro Lake, AZ (27), and (b) a colloidal (3500 Da-1 µm) isolate (C/N ratio ) 6.9) obtained from Naintre´ WWTP effluent, France (28). The effect of dialysis duration on remaining DON and DIN was assessed over 5 d with an acceptor flow rate of 100 mL/min. The effect of sample conductance on sample volume change and solute permeability during dialysis was assessed using glutamic acid and tripeptide (Glu-Cys-Gly) in various concentrations of NaCl and Na2SO3 solutions (1-50 mM) and was verified with bulk water samples. Sample volume change was measured by gravimetric volume determination before and after dialysis. Concentration was adjusted for volume changes.
Batch Leaching and Adsorption Experiments. Two types of batch experiments were conducted in 40 mL amber glass bottles filled with samples and cut-sections of the dialysis membrane. Unless stated, all experiments were conducted at room temperature. First, membrane dissolution was evaluated by placing cleaned membrane (80 cm2) in deionized water (40 mL) adjusted to pH 2-10. Samples were agitated for 24 h. Second, adsorption onto the membrane was evaluated by agitating cleaned membrane in solutions (40 mL) containing adsorbates for 24 h. The solutions were adjusted to pH 7 with 1 mM phosphate buffer, and the ratios of membrane surface area to sample volume were 0.25-6.0 cm2/cm3. Adsorbates evaluated include Arizona Canal water, effluents from NDN WWTP and TF WWTP, NOM isolates (hydrophobic and colloidal), amino acids (glutamic acid (acidic), arginine (basic), glycine (neutral), tryptophan (aromatic)), aniline, and tributylamine. In all cases control samples without membrane added were conducted. Application and Validation of the Dialysis Pretreatment. To compare DON measurements with and without the dialysis pretreatment, 30 surface water samples and 30 finished drinking water samples were collected from 14 U.S. states (Arkansas, Arizona, California, Illinois, Indiana, Kentucky, Massachusetts, Michigan, Missouri, Nevada, Pennsylvania, Texas, Virginia, Wisconsin). Table 1 lists the characteristics of the water sources. All samples were filtered with pre-ashed (500 °C) GF/F and stored at 4 °C until analysis. DON concentrations were measured after the dialysis pretreatment and compared to the calculated concentrations without pretreatment. To validate the accuracy of DON determination using the dialysis pretreatment, Arizona Canal water was spiked with both nitrate and ammonia in the same proportion as the original Arizona Canal water (different DIN/ TDN ratios ) 0.41-0.98 mg of N/mg of N; nitrate-to-ammonia ratio ) 6 mg of N/mg of N) and DON values at the DIN/TDN ratios were compared during 24 h dialysis. Analytical Methods. DOC and TDN were measured using a Shimadzu TOC-VCSH analyzer (high-temperature combustion at 720 °C; nondispersive infrared detection) with TNM-1 TN unit (chemiluminescence detection) (Shimadzu Corp., Japan). MDLs were calculated for several model compounds (29). Briefly, the MDL was determined by multiplying the standard deviation between the replicates for each species by the t-value at 99% confidence and n - 1 df. MDLs for TDN ranged from 0.009 to 0.019 mg of N/L depending on the chemicals (see Supporting Information). The instrument was checked for nitrogen recovery with various nitrogencontaining compounds (1 mg of N/L), and the recoveries were 97-103% depending on the compounds (see Supporting Information). Nitrate (MDL ) 0.005 mg of N/L) and nitrite (MDL ) 0.005 mg of N/L) were measured using a Dionex DX-120 ion chromatography system (Dionex Corporation, CA). Ammonia (MDL ) 0.005 mg of N/L) was measured by the automated phenate method (SM 4500-NH3 G) (18) using a TRAACS 800 autoanalyzer (Bran-Luebbe, Germany). A VWR conductivity meter (model 2052) and a Beckman pH meter (model 511201) were calibrated prior to each use. Independent t-test was used based on normality to compare two groups of data. In cases in which the data were not normally distributed and did not have equal variances, the Wilcoxon signed-rank test was used. Statistical analysis of experimental data was performed using SPSS V. 11.0 (SPSS Inc., IL).
Results and Discussion Effect of Dialysis Duration. Using various nitrogen containing compounds, an acceptable operation time of the dialysis pretreatment was assessed for both effective DIN removal and less DON loss. As shown in Figure 2, low molecular weight ions (nitrate and ammonia) and neutral solutes (urea) readily pass through the dialysis membrane. Ammonia and nitrate
FIGURE 2. Effect of dialysis duration on percentage DON or DOC retained within dialysis tubes for various nitrogen containing compounds. Initial concentrations were 5 mg of N/L for ammonia, nitrate, glutamic acid, tryptophan, tripeptide (Glu-Cys-Gly), and urea solutions, 1 mg of N/L for a hydrophobic NOM isolate solution, and concentrations listed in Table 1 for Arizona Canal water and NDNWWTP effluent. were reduced by 70-80% in 24 h and eliminated (below the MDL) in 120 h. Ammonia permeated slightly faster than nitrate. The first-order permeation rate constants for ammonia and nitrate were 4.0 × 10-2 and 3.5 × 10-2 h-1, respectively. During 120 h of dialysis, 97% of the urea (MW ) 60 g/mol) was removed; the concentrations of tryptophan (MW ) 204 g/mol) and tripeptide (Glu-Cys-Gly; MW ) 307 g/mol) did not change in the same time (significance level(R) ) 0.05). Since the molecular weight of urea is smaller than the membrane MWCO (100 Da), urea permeation was expected. This indicates the possible loss of low molecular weight materials (e.g., free amino acids, organic amines, and amides less than or around 100 Da) during the dialysis pretreatment. However, most organic materials in natural water generally have molecular weights larger than 100 Da (30). To assess the possible loss of small molecules in natural water during the dialysis pretreatment, NOM isolate solution, Arizona Canal water, and WWTP effluents were dialyzed (Figure 2). There was no difference (R ) 0.05) between the initial concentration and the concentrations over time of a hydrophobic NOM isolate. The Arizona Canal water sample also yielded a good DON recovery, but a slight loss of DON (∼5%) occurred from 72 h. Although this potential loss of small molecules cannot be eliminated, changing the operation time to 24 h could minimize its effect. The NDN WWTP effluent sample lost 10-15% of its DOC during the dialysis pretreatment (Figure 2). Similar results were observed for the TF WWTP effluent sample and a colloidal NOM isolate solution (data not shown). Note that DOC concentration was used as an indirect parameter for the wastewater sample to investigate the loss of organics because the initial DON concentration (TDN minus DIN) of the wastewater sample before dialysis pretreatment had a large variation. Possible explanations for the loss of organics are adsorption of organics onto the membrane, permeation of small molecular weight fractions, and/or biodegradation. Wastewater and NOM colloidal fractions contain bacterial cell fragments (31). These could be adsorbed onto the membrane (see the next section). Free amino acids, organic amines and amides, and small nucleic acid bases less than or around 100 Da may possibly contribute to the DON loss of the treated wastewater during dialysis pretreatment. Parkin and McCarty (32) reported that the percent of 0.6 mg of N/mg of N) require dialysis pretreatment for more accurate DON determination. The cumulative analytical variance is not significant for water samples with DIN/TDN ratios less than 0.6 mg of N/mg of N. The dialysis pretreatment system may be of limited use on wastewater samples due to adsorption of organics onto the dialysis membrane and/or permeation of small molecules present in wastewater. The proposed dialysis pretreatment system provides researchers with a tool for acquiring accurate DON concentrations in surface water and drinking water with high DIN/TDN.
Acknowledgments This research was supported by a grant from the American Water Works Association Research Foundation (project 2900). We thank Drs. Gary Amy and Jean-Philippe Croue´ for their advice and Dr. Mario Esparza-Soto and Baiyang Chen for their assistance. We also thank Dr. Croue´ for providing NOM isolates.
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Supporting Information Available One table showing MDLs and percent recovery of nitrogencontaining compounds on high-temperature combustion. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review July 28, 2004. Revised manuscript received October 26, 2004. Accepted November 1, 2004. ES048818Y
