Wastewater-Derived Dissolved Organic Nitrogen

By Pehlivanoglu-Mantas, Elif; Sedlak, David L

Wastewater-derived dissolved organic nitrogen (DON) accounts for up to 80% of dissolved nitrogen in nitrified-denitrified effluent. The sturdiness of DON measurements hindered the characterization of DON, especially in wastewater matrix, leading to an unsatisfying knowledge level. Measurement of DON and DON species is imortant not only as a measure of treatibility of wastewater in treatment plants, but also for the possible algal growth-stimulating effects after their discharge into receiving waters. In addition, wastewater- derived DON compounds may act as precursors to several disinfection by-products during intentional or unintentional reuse of wastewater. A review of DON compounds in wastewater and wastewater effluents is given.

KEY WORDS: bioavailability, Kjeldahl, NDMA, proteins

The discharge of municipal wastewater effluent and stormwater runoff from urbanized areas are important anthropogenic nitrogen sources. Overall, it has been estimated that wastewater-derived nitrogen accounts for 12 to 33% of the nitrogen pollution in the rivers worldwide, while agriculture and fertilizer runoff accounts for the remainder of the anthropogenic nitrogen released to rivers (Howarth, 2004).

Although the wastewater treatment plants equipped with nitrification and denitrification systems can decrease the concentration of inorganic forms of nitrogen more than 95%, the removal of organic nitrogen is normally much less efficient. Dissolved organic nitrogen (DON) usually accounts for approximately 65% of the dissolved nitrogen in effluents from conventional wastewater treatment plants, and DON can comprise up to 80% of dissolved nitrogen in efficient nitrification-denitrification systems (Qasim, 1999; TMWRF web page).

DON in oceans has been more extensively studied than DON in freshwater systems due to the N-limited nature of most of the saltwater systems. Reviews by Anita et al. (1991) and Berman and Bronk (2003) give details about the structure and bioavailability of DON in natural waters (Anita et al., 1991; Berman and Bronk, 2003). In addition, DON in drinking water has recently been reviewed (Westerhoff and Mash, 2002). The trend in organic nitrogen research is moving toward the anthropogenic effects on the aquatic systems (Seitzinger and Sanders, 1997; Seitzinger et al., 2002), but there is a lack of information about the types of DON and their concentrations in wastewater and wastewater effluents, are important both for cultural eutrophication and as disinfection by-product precursors.

EFFECTS OF WASTEWATER-DERIVED DON

The relatively high contribution of dissolved organic nitrogen to the total nitrogen content of highly treated (i.e., nitrified and denitrified) wastewater effluent is significant for watershed protection plans because most total maximum daily load (TMDL) plans use total nitrogen as the nitrogen parameter and do not consider the possibility that DON and inorganic nitrogen may have different potentials to cause cultural eutrophication. Although both nitrate and ammonium can stimulate primary production, the bioavailability of DON is uncertain (Anita et al., 1991). Chemical oceanographers and limnologists have shown that anywhere between 2 and 70% of the DON in surface waters is bioavailable (Seitzinger and Sanders, 1997; Bushaw-Newton and Moran, 1999; Carlsson et al., 1999; Paerl and Whitall, 1999; Seitzinger et al., 2002; Stepanauskas et al., 2002).

The variability in the bioavailability of DON in natural waters is most likely related to differences in the composition of the DON. For example, free amino acids (Kirchman and Hodson, 1984; Coffin, 1989; Palenik and Morel, 1990; Keil and Kirchman, 1991a, 1999; Suttle et al., 1991; Hammer, 1993; Jorgensen et al., 1993; Pantoja and Lee, 1994; Middelboe et al., 1995; Thomas, 1997; Berman and Bronk, 2003), urea (Jorgensen et al., 1998; Rondell et al., 2000), and nucleic acids (Jorgensen and Jacobsen, 1996; Berman and Bronk, 2003) are taken up readily by heterotrophic bacteria and/or marine and freshwater algae. Other forms of DON, such as humic substances, are less easily used to support growth of algae in N-limited systems (Carlsson and Graneli, 1993; Carlsson et al., 1993, 1995, 1999). Photochemical reactions in natural waters may convert DON into more labile compounds such as primary amines (Bushaw-Newton and Moran, 1999) or ammonia (Bushaw et al., 1996; Vahalato and Zepp, 2005), although photochemical reactions also may affect the bioavailability of DON negatively (Vahalato and Zepp, 2005, and references therein).

Although wastewater-derived DON contributes significantly to anthropogenic nitrogen inputs in a number of receiving waters, there is a lack of information about the bioavailability of wastewater- derived DON. The existing literature data about wastewater-derived organic nitrogen consists mostly of research conducted in the 1970s. The bioavailability of wastewater-derived DON, determined by measuring the uptake of nitrogen by the activated sludge bacteria over a period of 60 days, ranged between 0 and 60% (Parkin and McCarty, 1981a). In another study, wastewater-derived DON (which had been subjected to bacteria for 42 days prior to algal uptake experiments) did not support significant growth of algae (Parkin and McCarty, 1975). However, there are two factors that might have resulted in the underestimation of the bioavailability of the wastewater-derived DON. First, the gravimetric methods used for determination of algal growth might not have been sensitive enough to detect a small growth in algal mass. Second, considering the importance of bacteria in the food web, where bacterial uptake is needed to reincorporate dissolved organic matter from primary production (Tranvik, 1998), and the evidence on the role bacteria play in the availability of DON to algae in natural waters (Carlsson et al., 1999; Stepanauskas et al., 1999a, 1999b), the absence of bacteria in the algal cultures used in these experiments might have underestimated the availability of wastewater-derived DON to algae. The importance of bacteria in the cycling of wastewater-derived organic nitrogen also was indicated by the algal bioavailability experiments conducted in the presence and absence of bacteria (Pehlivanoglu and Sedlak, 2004). Approximately 10% of the wastewater- derived DON was available to algae in the absence of bacteria, whereas the bioavailable fraction increased to 60% in the presence of bacteria. The bioavailability of wastewater-derived DON was mostly due to the low-molecular-weight compounds (i.e., free amino acids and low-molecular-weight amines); although the low-molecular- weight fraction was 30% of the total DON, it accounted for almost all of the algal growth (Pehlivanoglu and Sedlak, 2004).

The presence of dissolved organic nitrogen in wastewater effluent also is important for (intentional or unintentional) indirect potable wastewater reuse because wastewater-derived organic nitrogen may serve as disinfection by-product precursors during wastewater disinfection or when the water undergoes drinking water treatment with chlorine disinfection. Although disinfection of water offers protection against waterborne infectious diseases, it also increases the risk of other diseases such as cancer due to the formation of disinfection by-products (DBPs; Morris et al., 1992). Chlorination of organic matter present in surface water and groundwater can result in the formation of disinfection by-products that pose human health risks (Becher, 1999), and therefore it is likely that chlorination of organic compounds in wastewater effluent could result in the production of similar by-products.

Various different organic compounds, including humic substances, amino acids, and proteins, have been shown to form trihalomethanes (THMs) and dihaloacetic acids (DHAAs) upon chlorination (Hureiki et al., 1994; Plummer and Edzwald, 1998, 2001). For example, chlorination of wastewater effluent resulted in formation of up to 500 g/L THM and approximately 300 g/L HAA (Rebhun et al., 1997).

In addition to acting as a precursor for THMs and DHAAs, which are regulated disinfection by-products (DBPs), wastewater-derived DON may form a variety of DBPs that contain a nitrogen functional group (i.e., haloacetonitriles [HAN] Bieber and Trehy, 1983; Oliver, 1983; Trehy et al., 1986; Ueno et al., 1996; Reckhow, 2003; cyanogen halides [CNCl], Hirose et al., 1988; Pedersen et al., 1999; Zheng et al., 2004a, 2004b, 2004c; and Af-nitrosodimethylamine [NDMA], Mitch and Sedlak, 2002, 2004; Mitch et al., 2003). Furthermore, wastewater- derived DON affects the efficiency of chlorination and dechlorination processes. When chlorine reacts with DON, especially with primary and secondary amines, organic chloramines form (Scully and Bempong, 1982; Fleischacker and Randtke, 1983; Scully et al., 1988a; Rebhun et al., 1997), and many of these species do not have germicidal properties, although standard chlorine residual tests, such as the DPD colorimetric assay (APHA, 1998), do not discriminate between organic and inorganic chloramines and measure them as combined residual chlorine (Jensen and Johnson, 1990). Therefore, the total residual chlorine measurement is not appropriate for assess\ment of disinfection in wastewater that contains relatively high concentrations of DON.

Chlorinated wastewater effluent is often toxic to aquatic organisms due to the presence of residual chlorine; therefore, wastewater effluent usually undergoes dechlorination prior to discharge by addition of SO^sub 2^ gas or sodium bisulfite (Lazarova et al., 1999; Bedner et al., 2004). Some of the N-chloro compounds, such as the chlorination products of free and combined amino acids, react slowly with bisulfite, leading to incomplete dechlorination under the conditions used in wastewater treatment plants (Helz and Nweke, 1995; Jensen and Helz, 1998; MacCrehan et al., 1998; Jameel and Helz, 1999).

METHODS FOR CHARACTERIZATION OF WASTEWATER-DERIVED DON

Dissolved organic nitrogen in wastewater effluent and surface waters consists of a suite of different compounds of which only a few can be quantified through direct analysis. As a result, the sum of all dissolved organic nitrogen compounds is usually measured as a collective parameter (e.g., Kjeldahl-N). A brief overview of the available methods for quantification and characterization of DON and N-containing compounds that are known to be present in the aquatic environment is summarized in the following paragraphs.

The Kjeldahl-N method has been used for over 120 years for the quantification of DON and total organic nitrogen (Kjeldahl, 1883). In the Kjeldahl-N method, the DON in the N(-III) oxidation state (except azide, azine, azo, hydrazone, nitro, nitroso, oxime, and semicarbozone functional groups) is converted to ammonia, which is distilled and measured by titration, colorimetry, or ion-selective electrode (APHA, 1998). The presence of high concentrations of nitrate (≥10 mg N/L) interferes with the measurement of organic nitrogen (APHA, 1998). Also, high concentrations of ammonia decrease the sensitivity and the precision of DON measurements with the Kjeldahl-N method because ammonia concentration must be subtracted from total nitrogen to estimate DON. This problem can be minimized by distilling away the NH^sub 3^ initially present in the sample prior to converting the DON to NH^sub 3^. The Kjeldahl method requires a relatively large volume of sample to quantify DON at concentrations encountered in the aquatic environment (e.g., the typical detection limit for a 1-L sample is 35 M of N). Furthermore, the Kjeldahl-N digestion step produces a relatively large volume of hazardous waste.

There are several other techniques that have been used to measure DON, most of which have been have been pioneered by marine chemists interested in nitrogen cycling (Solorzano and Sharp, 1980; Koroleff, 1983; Suzuki, 1993; Alvarez-Salgado and Miller, 1998; Bronk et al., 2000; Sharp et al., 2002, 2004). One technique for measuring DON employs persulfate digestion to convert all forms of inorganic and organic N into NO^sup -^^sub 3^ followed by detection by colorimetry, ion-selective electrode, or ion chromatography (APHA, 1998). For a sample volume of 10 ml, the typical detection limit of this method is approximately 10 M of N, depending on the method chosen for nitrate detection. Because the persulfate techniques also require the subtraction of inorganic N from total N, the precision of the measurement can be low in samples that contain high concentrations of inorganic nitrogen relative to DON.

Another method that has been used for measuring DON involves the oxidation of DON to nitrate with ultraviolet (UV) light (e.g., Armstrong et al., 1966). UV oxidation does not always oxidize certain recalcitrant forms of organic nitrogen, and the technique usually yields low DON values compared to the persulfate method (Delia et al., 1977; Cornell et al., 1995; Bronk et al., 2000). Some researchers believe that these incomplete recoveries observed in the UV systems for recalcitrant compounds such as urea may be a result of poor optimization of the UV systems and the use of purified chemicals (Mace and Duce, 2002).

DON also has been measured using high-temperature combustion (HTC) to convert organic nitrogen compounds into NO, which is subsequently detected in the gas phase by chemiluminescence (e.g., Badr et al., 2003). Commercial instruments, such as the Antek 9000N (Shimadzu) and the Apollo 9000 (Tekmar-Dohrmann) have been manufactured to quantify DON by this technique. During interlaboratory studies, the HTC methods have shown good reproducibility among laboratories (Sharp et al., 2004). HTC methods also estimate DON from the difference between the total nitrogen and the inorganic nitrogen forms. Therefore, they are subject to the same loss of precision in samples that contain high concentrations of inorganic nitrogen. Recently, a method using a dialysis pretreatment was developed to decrease the loss of precision in the DON measurements by reducing the dissolved inorganic nitrogen (DIN) concentration (Lee and Westerhoff, 2005). Although the dialysis pretreatment was found useful for drinking and surface water samples with high DIN concentration, the presence of small molecular weight compounds with molecular weights less than the dialysis membrane diameter (e.g., 100 Da) and the membrane fouling due to the high organic matter concentration will probably limit the use of this method in wastewater samples.

Amino acids and proteins are important components of DON and, as a result, numerous researchers have quantified amino acids and proteins along with DON. The most widely used protein assays are the Lowry (Lowry et al., 1951), Bradford (Bradford, 1976), and bicinchoninic acid (BCA; Smith et al., 1985) assays, as well as the ninhydrine method (Moore and Stein, 1948). These assays rely on reagents that undergo a spectral shift when they bind to proteins and amino acids.

The exact mechanism of the reactions and the relationship between protein type and light absorption in the colorimetric protein and amino acid measurement techniques is not well understood. For example, the Bradford method appears to require the presence of proteins with a molecular mass above 2 kD and may underestimate the protein concentration in wastewater effluents (Confer et al., 1995). The Lowry method and the BCA method yielded protein concentrations that were consistently about 20% higher than the results obtained with the Bradford assay in studies of the protein content of a marine diatom (Berges et al., 1993). More importantly, the Lowry method has been shown to yield protein concentrations that are four to seven times higher than those obtained with the Bradford method in wastewater effluents (Raunkjaer et al., 1994). These differences could be related to the chemical reaction responsible for the color development in Lowry method: The Lowry method uses a reaction with phenol, and phenolic substances in wastewater organic matter may lead to overestimates of protein content (Dewalle and Chian, 1974). For this reason, some researcher have concluded that the Bradford assay is less susceptible to artifacts from nonprotein compounds than the Lowry method (Berges et al., 1993).

Free amino acids and proteins have been quantified directly by numerous researchers by high-performance liquid chromatography (HPLC) after derivatization of the amino acid with a fluorescent reagent such as ortho-phthaldialdehyde (OPA), dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), and 6- aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; Lindroth and Mopper, 1979; Delmas et al., 1990; Gardner and Stjohn, 1991; Keil and Kirchman, 1991b; Confer et al., 1995; Jorgensen and Jensen, 1997; Sacher et al., 1997; Dignac et al., 2001). The selection of the fluorescent reagent usually depends on its stability and the presence of substances other than proteins and polypeptides that can react with the reagent. The HPLC methods discriminate between free amino acids and combined amino acids, which are measured after liquid- or vapor-phase acid hydrolysis (Keil and Kirchman, 1991b; Jorgensen and Jensen, 1997). Combined amino acids include proteins, polypeptides, and amino acids that are bound or adsorbed onto humic substances (Hubberten et al., 1994). However, not all of the proteins and polypeptides, that are associated with humic substances can be liberated by acid hydrolysis (Thurman and Malcolm, 1989).

Fractionation by affinity for hydrophobic resins are common techniques that are used to gain insight into the structure of DON. These techniques are frequently employed in conjunction with the DON and protein quantification techniques described above using C-18 or C-8 resins. The standard fractionation technique used by the International Humic Substances Society, which employs a XAD-8 resin in conjunction with acid and base precipitation steps to isolate humic substances (Leenheer, 1981, 2001; Leenheer et al., 2000), also can be used to quantify humic-associated DON. The molecular weight distribution of the DON also can be estimated by ultrafiltration or by gel-permeation chromatography (GPC), which is also known as high- performance size-exclusion chromatography (HPSEC). During ultrafiltration, solutes above the molecular weight cutoff (MWCO) of the ultrafilter membrane are retained and those below the MWCO permeate through the membrane. In gel-permeation chromatography, molecules larger than the gel pores move rapidly through the column while the smaller molecules penetrate into the pores of the gel; different effective travel path lengths lead to separation of compounds with different sizes. The molecular weight (MW) distribution determined by either technique may be affected by the pH of the sample and ionic strength (which will alter the organic matter structure and its interaction with the GPC resin or UF membrane through electrostatics), water temperature, interaction of the organic matter with the GPC resin, and, in the case of ultrafiltration, the cell pressure. In addition, size exclusion technique requires c\alibration of the column with appropriate standards (e.g., L-phenylalanine, streptomycin, and poly-DL- alanine; Keller et al., 1978) for the estimation of the MW distribution of nitrogen-containing organic compounds. As a result, molecular weight measurements are inexact and should be interpreted by considering the conditions used for the measurement.

DON COMPOUNDS IN MUNICIPAL WASTEWATER

During the 1970s, researchers studying biological wastewater treatment systems dedicated considerable effort into identifying the forms and fate of organic compounds in wastewater due to their possible effect on dissolved oxygen levels, the toxicity of certain organic compounds to aquatic organisms, and the possibility that nitrogen and phosphorus in organic matter could contribute to cultural eutrophication (Bender et al., 1970; Rebhun and Manka, 1971; Manka et al., 1974). Because DON is an important constituent of organic matter in wastewater and can serve as a source of nitrogen, numerous researchers studied DON in wastewater (Hanson and Lee, 1971; Chudoba and Dalesick, 1973; Randtke and McCarty, 1977, 1979; Keller et al., 1978; Parkin and McCarty, 1981a, 1981b, 1981c; Pehlivanoglu and Sedlak, 2004). Some of the relevant findings of those studies are summarized in the following paragraphs.

In most municipal wastewater treatment plants, approximately 90% of the dissolved organic nitrogen is removed during secondary biological treatment (Qasim, 1999). DON concentration in secondary treated effluent typically ranges from 70 to 350 M of N (Parkin and McCarty, 1975, 1981a; Keller et al., 1978; Randtke and McCarty, 1979; Isaac and Morris, 1980; Scully et al., 1988b; Qasim, 1999; Dignac et al., 2000; Pehlivanoglu and Sedlak, in preparation). DON in wastewater effluent includes inert DON in the raw sewage that is resistant to removal during biological treatment, biodegradable DON that was not completely removed during treatment, and DON produced during the treatment process (Parkin and McCarty, 1981b). For example, at the Palo Alto, CA, wastewater treatment plant the DON produced during activated sludge treatment accounted for approximately 35 16% of the DON in the effluent, with the remainder consisting mainly of inert DON originally present in the sewage (Parkin and McCarty, 1981c).

The first specific compounds studied in the dissolved organic nitrogen pool of wastewater are urea and free amino acids (Kahn and Wayman, 1964; Hanson and Lee, 1971). In raw sewage, 80% of organic nitrogen consists of urea that normally is converted to ammonia through hydrolysis in the wastewater collection system. Wastewater originating in small communities (i.e., treatment plants serving fewer than 1000 people) often has a higher urea concentration due to the short travel times in the wastewater collection system (Hanson and Lee, 1971).

Prior to the use of HPLC methods, protein content of the wastewater effluent had been measured by colorimetry (Manka et al., 1974; Manka and Rebhun, 1982; Hejzlar and Chudoba, 1986a, 1986b). Using the Lowry method and the ninhydrine method, proteins were estimated to account for 14 to 25% of the soluble organic compounds (measured as COD; Manka et al., 1974; Manka and Rebhun, 1982).

TABLE 1. Total Amino Acid Concentrations in the Secondary Treated Wastewater Effluents (Scully et al., 1988b; Confer et al., 1995; Grohmann et al., 1998; Pehlivanoglu and Sedlak, in preparation)

The total amino acid concentration in the secondary wastewater effluent is approximately 2 M of N (Table 1), and approximately 0.4 M of N is accounted by the free amino acids (Table 2). Assuming a DON of 2 mg N/L (i.e., 143 M of N) in the wastewater effluent, the concentration of free amino acids accounts for between 0.05 and 3% of the DON, while the combined amino acids account for approximately 0.6 to 13% of the DON (Parkin and McCarty, 1981 a; Confer et al., 1995; Dignac et al., 2000b; Pehlivanoglu and Sedlak, in preparation). The studies indicating that the DCAA accounts for a higher percentage of the DON (Confer et al., 1995; Pehlivanoglu and Sedlak, in preparation) used a vapor phase hydrolysis method which has been shown to result in better digestions and higher recoveries than the liquid phase hydrolysis method (Keil and Kirchman, 1991b).

The concentration of free and combined amino acids decreases during secondary biological treatment (Confer et al., 1995; Dignac et al., 2000b; Pehlivanoglu and Sedlak, in preparation). For example, Confer et al. (1995) observed more than 98% decrease in the concentration of DFAA and 90% decrease in DCAA in an activated sludge system, and 96% decrease in DFAA and 76% decrease in the DCAA concentration in a trickling filter. The larger decrease in DFAA concentration may be due to the need to hydrolyze polypeptides prior to uptake of amino acids. In despite of the decrease in the concentration of DCAA, the percentage of DCAA increased in the DON pool after secondary treatment whereas the DFAA fraction did not change (Pehlivanoglu and Sedlak, in preparation). An analogous observation was made by Parkin and McCarthy (1981a), who observed a decrease in the low-molecular-weight fraction (<1.8 kD) of the wastewater DON during activated sludge treatment, whereas the DON in the high-molecular-weight fraction (>1.8 kD) increased slightly. The preferential removal of low-molecular-weight DON probably corresponds to the uptake of free amino acids or small polypeptides.

TABLE 2. Free Amino Acid Concentrations in Untreated Waste-water and Secondary Treated Wastewater Effluents (Hunter and Faust, 1971; Burleson et al., 1980; Confer et al., 1995; Grohmann et al., 1998; Pehlivanoglu and Sedlak, in preparation)

Low-molecular-weight aliphatic amines such as methylamine, dimethylamine, piperidine, and pyrroline may be formed during the oxidation of complex organic nitrogen compounds (Sacher et al., 1997). Although these compounds are likely to be important components of DON in wastewater, less information is available on their occurrence and fate because they are more difficult to measure than proteins and amino acids. Liquid-liquid and solid-phase extractions are not suitable for many of the polar short chain aliphatic amines. However, the compounds can be analyzed directly by HPLC or can be analyzed by GC after an aqueous phase derivatization step (Sacher et al, 1997).

The short chain aliphatic amines have been studied mostly due to their contribution to odor problems associated with wastewater treatment plants (Hwang et al., 1995). The concentration of aliphatic amines in wastewater effluent samples ranged between less than 0.01 and 5 M of N (Table 3). Among the amines reported, dimethylamine (DMA) has the highest concentration in the wastewater effluent, and the removal efficiency of DMA in the WWTP ranged between O and 95% (Hwang et al, 1995; Abalos et al, 1999; Mitch and Sedlak, 2004). It is possible that the data from Hwang et al. (1995, i.e., 0% removal) represent an analytical artifact associated with collection of samples on different dates. Disregarding those data, the DMA removal efficiency ranged between 30 and 95%. Other volatile amines such as isoamylamine, isobutylamine, and 2-methylbutylamine also have been detected in the wastewater effluents, but their concentrations in the secondary effluents were below approximately 0.15 M of N (Scully et al, 1988b), suggesting that their contribution to the DON pool of 143 M of N would be less than 0.1%.

TABLE 3. Concentration of Amines in Wastewater Effluents (M N)

In rivers that do not receive significant discharges of wastewater effluent, the concentration of volatile amines are usually below 0.07 M of N (Sacher et al., 1997). Conversely, in rivers that receive considerable discharges of wastewater effluents, the concentration of volatile amines may be comparable to those detected in wastewater (Table 3; Abalos et al., 1999).

In addition to proteins, polypeptides, and aliphatic amines, nitrogen-containing synthetic chelating agents are present in wastewater effluent. The synthetic chelating agent ethylenediamine tetraacetic acid (EDTA) is one of the most widely used and most studied synthetic chelating agents in wastewater effluents. The concentration of EDTA in wastewater effluents typically ranges between 0.1 and 1 M of N; however, concentrations up to 3.6 M of N have been reported (Alder et al., 1990; Kari and Giger, 1996; Nowack et al., 1996; Bedsworth and Sedlak, 1999; Pehlivanoglu and Sedlak, in preparation). Nitrilotriacetic acid (NTA), another chelating agent, has been detected at concentrations between 0.1 and 0.5 M of N in wastewater effluent samples (Bucheli-Witschel and Egli, 2001).

A variety of other N-containing organic compounds have been detected in wastewater effluent at trace concentrations. For example, benzothiazole has been detected in wastewater effluent at concentrations between 0.02 and 0.42 M of N O(Ellis and Soper, 1954; Legube et al., 1987; Clark et al., 1991; Gulyas et al., 1994; Grohmann et al., 1998). Pharmaceuticals are another class of nitrogen-containing compounds that have been detected in wastewater effluent at trace concentrations. The concentration of N-containing pharmaceuticals is usually less than 0.001 M of N (Hirsch et al., 1996; Ternes, 1998).

Since measurements of the concentration of specific compounds such as free and combined amino acids and amines account for only a small fraction of the DON pool in the wastewater effluent, researchers studied the chemical properties of the unidentified DON as an alternative to identification and quantification of specific nitrogen-containing compounds. Molecular weight (MW) fractionation and fractionation of organics according to their hydrophobicity are two important ways of characterizing wastewater-derived DON (Henze, 1992).

Solid-phase extraction (SPE) results indicated that the unidentified DON was relatively hydrophilic (Pe\hlivanoglu and Sedlak, in preparation). This finding is consistent with our understanding of the physical mechanisms through which organic compounds are removed in wastewater treatment plants: Hydrophobic compounds would be much more easily removed by adsorption onto the surfaces of activated sludge and other particles present in secondary treatment systems. Thus, the recalcitrant wastewater- derived DON compounds consist of hydrophilic compounds that have a low affinity for the surfaces of organic particles.

Molecular weight fractionation of wastewater has been used to study the behavior of soluble microbial products (SMP) produced during biological wastewater treatment. Because SMP contributes most of the biochemical and chemical oxygen demand (BOD and COD) in the wastewater effluent, its molecular weight distribution and bioavailability have been studied extensively and in some cases, DON has been measured as part of these studies (Barker and Stuckey, 1999). The molecular weight distribution of organic carbon in wastewater effluent typically exhibits a bimodal distribution, with the majority of the organic carbon in forms with molecular weights greater than 10 kD or less than 1 kD (Barker and Stuckey, 1999). On the other hand, DON in secondary effluent consists mostly of low (< 10 kD) molecular weight compounds and the <1.8-kD or <1-kD fraction accounts for approximately the half of wastewater-derived DON (Keller et al., 1978; Parkin and McCarty, 1981a; Pehlivanoglu and Sedlak, in preparation).

Another parameter for the measurement of organic compounds in the wastewater is the soluble chemical oxygen demand (SCOD). All the oxidizable organic compounds are included in the SCOD parameter, whereas DON is a measurement of only nitrogen-containing organic compounds. The measurement of MW distribution with both DON and SCOD parameters indicated that the 165-800 Da range was the most nitrogen- rich fraction among the different molecular weight ranges, whereas the >1800 Da fraction was the most carbon-rich fraction (Keller et al., 1978), probably due to the presence of free amino acids and small polypeptides in the 165-800 Da MW fraction and humic substances in the >1800 fraction.

Adsorption to activated carbon, ion exchange, and chemical precipitation have been used to characterize the wastewater-derived DON (Keller et al., 1978; Randtke and McCarty, 1979; Parkin and McCarty, 1981a) and to provide insight into the efficacy of treatment processes that might be used to reduce DON concentrations (i.e., whether the use of more advanced techniques such as nanofiltration or reverse osmosis is needed). More than half of the DON was removed by contact with 15-50 g/L of activated carbon, suggesting that wastewater-derived DON is relatively hydrophilic (Table 4). Comparison of the removal of DON and SCOD indicated that the nitrogen-containing organic compounds measured as DON are slightly more hydrophilic than the other forms of organic matter (i.e., nonnitrogenous organics) measured as SCOD in the wastewater.

TABLE 4. Change in Organics Concentrations in Secondary Treated Wastewater Effluents, Measured as DON and SCOD, During Characterization with Activated Carbon Treatment

TABLE 5. Ion Exchange Characteristics of Secondary Wastewater Effluent Measured as DON and SCOD Removal

To illustrate the ion exchange characteristics of wastewater- derived DON, strong anion and cation exchangers were used and the compounds that were removed by these exchangers were measured both as DON and as SCOD (Table 5). Cation exchange resins remove more DON than SCOD, indicating that the nitrogenous organic compounds tend to be more positively charged than the rest of the organic matter represented by SCOD. Similarly, anion exchange resins remove more SCOD than DON, signifying that the organics measured as DON and SCOD have different characteristics. The increase in the DON removal during cation exchange with the decrease of pH from 7 to 2 suggests the presence of compounds such as amino acids, amines, nucleic bases, and so on, which will protonate at low pH values.

Chemical precipitation with ferric chloride, lime, and alum is less effective than activated carbon, but more effective than ion exchange at neutral pH (Tables 5 and 6). Ferric chloride and lime removed significantly more DON than SCOD.

TABLE 6. Removal Efficiencies for Several Treatment Technologies for DON and SCOD in Wastewater Effluent (Randtke and McCarty, 1979)

Chemical oxidation with potassium permanganate, hydrogen peroxide, and chlorine works better for DON removal than SCOD, but ozone removes more SCOD than DON (Table 6). The authors found that if ozonation precedes chlorination, then there is an increase in the removal of DON, but no increase in SCOD, suggesting that ozonation makes the nitrogen moiety, which could not be chlorinated prior to the ozonation, available for chlorination (Randtke and McCarty, 1979).

The data presented here suggest that the two parameters widely used to characterize wastewater organics, DON and SCOD, are not always interchangeable due to the differences in the characteristics of the organic compounds such as molecular weight, C:N ratio, and charge. The differences are especially pronounced when ion exchange, oxidation, and chemical precipitation are used to characterize the wastewater organics (Keller et al., 1978; Randtke and McCarty, 1979; Parkin and McCarty, 1981a, 1981b, 1981c). Therefore, in organic nitrogen research, it is important to use DON consistently and not to assume that the parameters SCOD and/or DOC will behave similarly to DON and are therefore acceptable substitutes for DON parameter.

Characterization and quantification efforts through spectrophotometric analysis of wastewater effluent have not been very successful, due to the matrix effects and the complexity of the wastewater. Other spectroscopic methods such as NMR (nuclear magnetic resonance) and FTIR (Fourier transform infrared) also have been used to study the structure of wastewater-derived organic matter. In addition to ^sup 13^C, recent developments in the instruments allow ^sup 15^N spectra to be observed in nonenriched samples (Dignac et al., 2000b, 2001; Knicker et al., 2002). Although obtaining a quantitative analysis of the compounds in the wastewater would be hard if not completely impossible, a comparison between untreated and treated wastewater could explain which compounds undergo treatment and which ones do not. The ^sup 15^N-NMR spectra of two wastewater effluents indicate that the DON composition does not change too much during the biological treatment and most of the DON consists of amide/peptide groups (Dignac et al., 2000a). The persistence of such high concentrations of proteinaceous matter can be explained by their protection in "highly cross-linked, complex" structures.

N-Acetylamino sugars, which are the main compounds of bacterial cell walls (McCarthy et al., 1997), are observed during pyrolysis of both untreated and treated wastewater effluent (Dignac et al., 2000b), and their presence is confirmed with solid-state cross- polarization magic-angle spinning (CPMAS) ^sup 13^C-NMR spectra (Dignac et al., 2001).

The identifiable nitrogen-containing organic compounds in wastewater effluent usually account for less than 10% of the DON. Therefore, the majority of the DON likely consists of complex organic compounds derived from natural organic compounds. A subgroup of these complex organic compounds is humic substances, which are defined based on their solubility in acidic solutions and are usually measured by XAD resin extraction. Humic substances in the source water that ultimately becomes municipal wastewater probably contribute to wastewater DON. The concentration of humic substances, in wastewater effluent is estimated to range between 1 and 5 mg C/L (Amy et al., 1993; Barber et al., 2001; Ma et al., 2001). Although it is hard to give a specific formula for humic substances, the average composition of humic acid (C^sub 187^H^sub 186^O^sub 89^N^sub 9^S; Sposito, 1989) can be used to calculate the DON contribution of humic substances in the wastewater effluent. Using this stoichiometry, we estimate that the humic substances that are derived from the drinking water source will contribute between approximately 4 and 20 M of N in the wastewater effluent. Therefore, humic substances do not account for more than a small percentage of the unidentifiable DON.

Some of the unidentified wastewater-derived DON may consist of polymerized organic compounds derived from activated sludge and other organisms, which might include a mixture of amino acids, proteins, nucleic acids, steroids, and humic and fulvic acids (Rittmann et al., 1987). We hypothesize that the unidentified DON is similar to the amide-containing complex organic matter that has been observed in surface waters (McCarthy et al., 1997, 1998) and that polymerized biological compounds account for the unidentified organic nitrogen in the wastewater effluent.

CONCLUSION

The problems associated with the analysis of dissolved organic nitrogen (DON) limited the number of studies about DON in wastewater, although wastewater-derived DON accounts for up to 80% of dissolved nitrogen in nitrified-denitrified effluent. Even though several methods exist to measure DON, they either are subject to interference of inorganic nitrogen concentrations (e.g., Kjeldahl, persulfate digestion, UV oxidation) or require costly equipment (e.g., high-temperature combustion). The concentration of wastewater- derived DON varies between 70 and 350 M of N (1 and 5 mg N/L). Free amino acids comprise 0.05 to 3% of the DON, while the combined amino acids account for approximately 0.6 to 13% of the DON in the wastewater. The concentration of free and combined amino acids depends not only on the specific treatment plant or the type of treatment processes used but also on the method that is employed to \determine the concentration. Other specific nitrogen-containing organic compounds, such as EDTA or pharmaceuticals, which are significant for their effect on metal transportation and on environmental health, respectively, account for approximately 1% of the wastewater-derived DON. The sum of all identified nitrogen- containing compounds comprises only 10% of the DON, leaving the majority of DON unidentified. The unidentified DON most probably consist of polymerized biological compounds.

Due to the problems related to the measurement of DON, several other parameters, such as soluble chemical oxygen demand (SCOD) or dissolved organic carbon (DOC), have been used in organic nitrogen research. Although DON is a subgroup of DOC, the molecular weight distribution of DON is a slightly different than for DOC. In addition, the effect of several treatment processes on the removal efficiencies of the nitrogen-containing organics is not the same as their effect on DOC. Therefore, the behavior of nitrogen-containing organic compounds in wastewater treatment plants and in the environment may be different than the behavior of the total pool of organic compounds.

The presence of DON in wastewater may affect the quality of the receiving water in several ways. One probable negative impact of wastewater-derived DON is its support for bacterial and/or algal growth in the receiving water (i.e., lake, river, or sea). DON may support growth of algal either directly or indirectly through bacterial mineralization. In addition, the photochemical reactions in the receiving water may convert wastewater-derived DON into more labile forms.

Another effect of wastewater-derived DON is its role as a disinfection by-product (DBP) precursor for NDMA, HAA, or THM. Wastewater-derived organic nitrogen may act as DBP precursors during chlorination/chloramination in the wastewater treatment plant. DON may also be converted to DBPs in a downstream water treatment facility in case of an intentional or unintentional water reuse.

To understand the fate of wastewater-derived organic nitrogen in the environment and its relevance to cultural eutrophication and disinfection by-product formation, it is important to understand the nature and properties of the nitrogen-containing compounds in wastewater effluent. The knowledge of what particular fraction of wastewater-derived DON (e.g., low molecular weight vs. high molecular weight, hydrophilic vs. hydrophobic) may lead to eutrophication or may act as NDMA precursors will be very helpful to tailor the treatment scheme accordingly to ensure the removal of this fraction. More studies using new and improved analytical techniques such as LC-MS will be important in determining the constituents of the wastewater-derived DON pool.

ACKNOWLEDGMENT

Dr. Elif Pehlivanoglu-Mantas is grateful to the Higher Education Council of Turkey for the award of a PhD scholarship.

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