Measurement of dissolved organic nitrogen in a drinking water treatment plant: size fraction, fate, and relation to water quality parameters

This paper investigates the characteristics of dissolved organic nitrogen (DON) in raw water from the Huangpu River and also in water undergoing treatment in the full-scale Yangshupu drinking water treatment plant (YDWTP) in Shanghai, China. The average DON concentration of the raw water was 0.34 mg/L, which comprised a relatively small portion (~ 5%) of the mass of total dissolved nitrogen (TDN). The molecular weight (MW) distribution of dissolved organic matter (DOM) was divided into five groups: > 30, 10-30, 3-10, 1-3 and < 1 kDa using a series of ultrafiltration membranes. Dissolved organic carbon (DOC), UV absorbance at wavelength of 254 nm (UV254) and DON of each MW fraction were analyzed. DON showed a similar fraction distribution as DOC and UV254. The < 1 kDa fraction dominated the composition of DON, DOC and UV254 as well as the major N-nitrosodimethylamine formation potential (NDMAFP) in the raw water. However, this DON fraction cannot be effectively removed in the treatment line at the YDWTP including pre-ozonation, clarification and sand filtration processes. The results from linear regression analysis showed that DON is moderately correlated to DOC, UV254 and trihalomethane formation potential (FP), and strongly correlated to haloacetic acids FP and NDMAFP. Therefore, DON could serve as a surrogate parameter to evaluate the reactivity of DOM and disinfection by-products FP.     

Effluent dissolved organic nitrogen and dissolved phosphorus removal by enhanced coagulation and microfiltration

Plants aiming to achieve very low effluent nutrient levels (<3 mg N/L for N, and <0.1 mg P/L for P) need to consider removal of effluent fractions hitherto not taken into account. Two of these fractions are dissolved organic nitrogen (DON) and dissolved non-reactive phosphorus (DNRP) (mainly composed of organic phosphorus). In this research, enhanced coagulation using alum (at doses commonly employed in tertiary phosphorus removal) followed by microfiltration (using 0.22 μm pore size filters) was investigated for simultaneous effluent DON and dissolved phosphorus (DP) fractions removal. At an approximate dose of 3.2 mg Al(III)/L, corresponding to 1.5 Al(III)/initial DON-N and 3.8 Al(III)/initial DP-P molar ratios, maximum simultaneous removal of DON and DP was achieved (69% for DON and 72% for DP). At this dose, residual DON and DP concentrations were found to be 0.3 mg N/L and 0.25 mg P/L, respectively. Analysis of the trends of removal revealed that the DNRP removal pattern was similar to that commonly reported for dissolved reactive phosphorus. Since this study involved intensive analytical work, a secondary objective was to develop a simple and accurate measurement protocol for determining dissolved N and P species at very low levels in wastewater effluents. The protocol developed in this study, involving simultaneous digestion for DON and DNRP species, was found to be very reliable and accurate based on the results.     

Fate of dissolved organic nitrogen in two stage trickling filter process

Dissolved organic nitrogen (DON) represents a significant portion of nitrogen in the final effluent of wastewater treatment plants (WWTPs). Biodegradable portion of DON (BDON) can support algal growth and/or consume dissolved oxygen in the receiving waters. The fate of DON and BDON has not been studied for trickling filter WWTPs. DON and BDON data were collected along the treatment train of a WWTP with a two-stage trickling filter process. DON concentrations in the influent and effluent were 27% and 14% of total dissolved nitrogen (TDN). The plant removed about 62% and 72% of the influent DON and BDON mainly by the trickling filters. The final effluent BDON values averaged 1.8 mg/L. BDON was found to be between 51% and 69% of the DON in raw wastewater and after various treatment units. The fate of DON and BDON through the two-stage trickling filter treatment plant was modeled. The BioWin v3.1 model was successfully applied to simulate ammonia, nitrite, nitrate, TDN, DON and BDON concentrations along the treatment train. The maximum growth rates for ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria, and AOB half saturation constant influenced ammonia and nitrate output results. Hydrolysis and ammonification rates influenced all of the nitrogen species in the model output, including BDON.     

Organic nitrogen transformations in a 4-stage Bardenpho nitrogen removal plant and bioavailability/biodegradability of effluent DON

Nitrogen species, specifically, the fate and occurrence of organic nitrogen (ON) within a 4-stage Bardenpho process bioreactor producing low total nitrogen (TN) effluents were investigated in this study. The results showed release of ON in primary anoxic zone and no ON release in the first aerobic zone of the process. The research included investigation of biodegradability/bioavailability of wastewater-derived effluent dissolved ON (DON). The final-effluent DON utilization was evaluated by two different bioassay protocols in the presence and absence of nitrate. About 28–57% of the effluent DON was bioavailable/biodegradable. Bioavailable (to algae and bacteria) DON (ABDON) and biodegradable (to bacteria) DON (BDON) results did not show significant differences in terms of quantity, but DON utilization rates by ABDON (0.13 day⁻¹) protocol were higher than that of the BDON (0.04 day⁻¹) protocol in the nitrate-removal samples. As a result, ABDON requires a shorter time to exert the bioavailable fraction due to symbiotic relationship between algae and bacteria. In the nitrate-containing samples, it appears that nitrate competes with labile DON as a nitrogen source to microorganisms in both ABDON and BDON protocols. The first order decay rate of DON in the presence of nitrate was 0.11 day⁻¹ and 0.02 day⁻¹ for ABDON and BDON, respectively.     

Composition of a protein-like fluorophore of dissolved organic matter in coastal wetland and estuarine ecosystems

This study demonstrates the compositional heterogeneity of a protein-like fluorescence emission signal (T-peak; excitation/emission maximum at 280/325 nm) of dissolved organic matter (DOM) samples collected from subtropical river and estuarine environments. Natural water samples were collected from the Florida Coastal Everglades ecosystem. The samples were ultrafiltered and excitation-emission fluorescence matrices were obtained. The T-peak intensity correlated positively with N concentration of the ultrafiltered DOM solution (UDON), although, the low correlation coefficient (r(2)=0.140, p<0.05) suggested the coexistence of proteins with other classes of compounds in the T-peak. As such, the T-peak was unbundled on size exclusion chromatography. The elution curves showed that the T-peak was composed of two compounds with distinct molecular weights (MW) with nominal MWs of about >5 x 10(4) (T(1)) and approximately 7.6 x 10(3) (T(2)) and with varying relative abundance among samples. The T(1)-peak intensity correlated strongly with [UDON] (r(2)=0.516, p<0.001), while T(2)-peak did not, which suggested that the T-peak is composed of a mixture of compounds with different chemical structures and ecological roles, namely proteinaceous materials and presumably phenolic moieties in humic-like substances. Natural source of the latter may include polyphenols leached from senescent plant materials, which are important precursors of humic substances. This idea is supported by the fact that polyphenols, such as gallic acid, an important constituent of hydrolysable tannins, and condensed tannins extracted from red mangrove (Rhizophora mangle) leaves exhibited the fluorescence peak in the close vicinity of the T-peak (260/346 and 275/313 nm, respectively). Based on this study the application of the T-peak as a proxy for [DON] in natural waters may have limitations in coastal zones with significant terrestrial DOM input.     

Rejection of pharmaceuticals in nanofiltration and reverse osmosis membrane drinking water treatment

This paper investigates the removal of a broad range of pharmaceuticals during nanofiltration (NF) and reverse osmosis (RO) applied in a full-scale drinking water treatment plant (DWTP) using groundwater. Pharmaceutical residues detected in groundwater used as feed water in all five sampling campaigns were analgesics and anti-inflammatory drugs such as ketoprofen, diclofenac, acetaminophen and propyphenazone, beta-blockers sotalol and metoprolol, an antiepileptic drug carbamazepine, the antibiotic sulfamethoxazole, a lipid regulator gemfibrozil and a diuretic hydrochlorothiazide. The highest concentrations in groundwater were recorded for hydrochlorothiazide (58.6-2548ngL(-1)), ketoprofen (85%). Deteriorations in retentions on NF and RO membranes were observed for acetaminophen (44.8-73 %), gemfibrozil (50-70 %) and mefenamic acid (30-50%). Furthermore, since several pharmaceutical residues were detected in the brine stream of NF and RO processes at concentrations of several hundreds nanogram per litre, its disposal to a near-by river can represent a possible risk implication of this type of treatment.     

Optimization of dissolved organic nitrogen (DON) measurements in aqueous samples with high inorganic nitrogen concentrations

Since dissolved organic nitrogen (DON) concentrations in aqueous samples can only be determined by the subtraction of three independently measured concentrations (total dissolved nitrogen (TDN), nitrate and ammonium), analytical errors are compounded in the calculation of DON concentration. Several methods were tested to increase the recovery and precision of the DON determinations in aqueous samples with high inorganic nitrogen concentrations. The first step was the objective selection of the most accurate TDN analysis method. The persulfate oxidation (PO) method had a significantly higher recovery than the high-temperature catalytic oxidation (HTCO) method. The second step involved the lowering of the DIN (dissolved inorganic N)/TDN ratio by applying three sample pretreatment procedures: conventional dialysis, dialysis against a buffered acceptor solution and concentration of the samples combined with the buffered dialysis. Despite the fact that DIN was only partially removed, conventional dialysis pretreatment resulted in higher precision and recovery of the DON determination compared to analysis of untreated samples. Dialysis of the samples against a buffered acceptor solution gave additional improvements in precision, recovery, and reproducibility. The concentration of aqueous samples by lyophilization, however, did not lead to higher recovery rates in the DON measurements.     

Evaluation of the catalytic reduction of nitrate for the determination of dissolved organic nitrogen in natural waters

The catalytic reduction of nitrate ions into nitrogen gas was tested to partly remove dissolved inorganic nitrogen (DIN) before the determination of dissolved organic nitrogen (DON). Experiments were conducted on nitrate solutions enriched with natural organic matter (NOM) isolates previously extracted from surface waters. Three catalysts Pd-In/Al2O3, Pd-Sn/Al2O3 and Pd/SnO2 were tested. Their noble metal (palladium) and promoter metal (indium or tin) contents are 5 and 1.75 wt%, respectively. Preliminary experiments performed on a solution containing 17 amino acids showed that most compounds were removed by less than 15%, probably due to sorption onto the catalysts. Reduction of nitrate in absence of NOM was complete after 20 min of reaction time and the removal of DIN was about 80% (about 19% formation of ammonium). In the presence of NOM (DOC 20 mgC/L, DON 0.67 mg N/L), the kinetic of nitrate reduction was slower and the reduction in DIN content was limited to 15% i.e. selectivity toward ammonium reached 85%. Adsorption tests showed a similar removal of both DOC and DON of about 70% and 30% onto Pd-Sn/Al2O3 and Pd/SnO2 catalysts, respectively, which confirmed that NOM probably compete with nitrate for active catalytic sites. In conclusion, catalytic reduction of nitrate before DON determination cannot be used because of DON sorption and low DIN removal.     

Characteristics and fate of organic nitrogen in municipal biological nutrient removal wastewater treatment plants

The aim of this study was to investigate the occurrence and fate of colloidal and dissolved organic nitrogen (CON and DON) across biological nutrient removal (BNR) activated sludge bioreactors. Primary and secondary effluent total nitrogen (TN) measurements and component fractionation, CON and DON concentration profiles across BNR bioreactors, and laboratory batch experiments with the process mixed liquor were carried out at several full-scale BNR plants in northern Poland. The organic nitrogen (ON) components were divided into high CON, low CON, and DON based on sequential filtration through 1.2, 0.45 and 0.1 μm pore-size filters. The average influent DON_0.1μm (<0.1 μm) concentrations ranged from 1.1 g N/m³ to 3.9 g N/m³ and accounted for only 4-13% of total organic nitrogen. In the effluents, however, this contribution increased to 12-45% (the DON_0.1μm concentrations varied in a narrow range of 0.5-1.3 g N/m³). Conversions of ON inside the bioreactors were investigated in more detail in two largest plants, i.e. Gdansk (565,000 PE) and Gdynia (516,000 PE). Inside the two studied bioreactors, the largest reductions of the colloidal fraction were found to occur in the anaerobic and anoxic compartments, whereas an increase of DON_0.1μm concentrations was observed under aerobic conditions in the last compartment. Batch experiments with the process mixed liquor confirmed that DON_0.1μm was explicitly produced in the aerobic phase and significant amounts of ON were converted in the anoxic phase of the experiments.     

Sorption of phenols onto sandy aquifer material: the effect of dissolved organic matter (DOM)

The influence of dissolved organic matter (DOM) on the sorption of four phenols, 2,4,6-trichlorophenol (2,4,6-TCP), pentachlorophenol (PCP), 2,4-dinitrophenol (2,4-DNP) and 2-methyl-4,6-dinitrophenol (2-M-4,6-DNP), onto sandy aquifer material at different pH values was investigated using flow through column experiments. The pH-dependent sorption of the chlorinated phenols 2,4,6-TCP and PCP was not significantly affected by DOM (measured as dissolved organic carbon, DOC), whereas in the case of nitrophenols a significant lower retardation was found, depending on the DOC concentration and pH value of the aqueous solution. Sorption decreases with increasing DOC concentration, which indicates a binding of these compounds by DOM. The degree of sorption reduction depends on the pH value and increases with increasing fraction of neutral species. The different behaviour of nitrophenols in comparison to the chlorophenols is assumed to be a result of specific charge-transfer interactions. A combined sorption and complex formation model was used to describe the effect of pH and DOC concentration on the sorption of nitrophenols onto aquifer material and to estimate binding coefficients of neutral nitrophenols on DOM.     

Mechanistic and kinetic evaluation of organic disinfection by-product and assimilable organic carbon (AOC) formation during the ozonation of drinking water

Ozonation of drinking water results in the formation of low molecular weight (LMW) organic by-products. These compounds are easily utilisable by microorganisms and can result in biological instability of the water. In this study, we have combined a novel bioassay for assessment of assimilable organic carbon (AOC) with the detection of selected organic acids, aldehydes and ketones to study organic by-product formation during ozonation. We have investigated the kinetic evolution of LMW compounds as a function of ozone exposure. A substantial fraction of the organic compounds formed immediately upon exposure to ozone and organic acids comprised 60-80% of the newly formed AOC. Based on experiments performed with and without hydroxyl radical scavengers, we concluded that direct ozone reactions were mainly responsible for the formation of small organic compounds. It was also demonstrated that the laboratory-scale experiments are adequate models to describe the formation of LMW organic compounds during ozonation in full-scale treatment of surface water. Thus, the kinetic and mechanistic information gained during the laboratory-scale experiments can be utilised for upscaling to full-scale water treatment plants.     

Estimation of the hydrophobic fraction of dissolved organic matter in water samples using UV photometry

In this study, we tested a simple and rapid method for the estimation of carbon in the hydrophobic fraction of dissolved organic matter (DOM) of different origin (spruce, pine, and beech litter) in soil water. The method is based on the fact that the hydrophobic fraction of DOM contains almost entirely the aromatic moieties of DOM. Thus, it showed a clearly distinct light absorption at 260 nm compared to the hydrophilic fraction. This light absorption was directly proportional to the concentration of the hydrophobic fraction. Moreover, it was independent of the concentration of the hydrophilic fraction. We compared the concentrations of hydrophobic DOM estimated by the UV method with those of the conventional fractionation using chromatographic columns of XAD-8 macroporous resin and found an excellent agreement between the two methods for both solutions from laboratory sorption experiments and field samples of forest floor leachates and subsoil porewaters. In addition, the absorption at 260 nm of hydrophobic DOM proved to be independent of pH values ranging from 2.0 to 6.5. Compared to the conventional chromatographic fractionation, the method using the UV absorption at 260 nm is less time consuming, needs a much smaller sample volume, and showed a better reproducibility. However, its use is restricted to water samples of low nitrate (<25 mg L⁻¹) and Fe (<5 mg L⁻¹) concentrations and, probably, with the hydrophobic fraction dominated by aromatic compounds deriving from degradation of lignin.     

Formation of assimilable organic carbon (AOC) and specific natural organic matter (NOM) fractions during ozonation of phytoplankton

Ozonation of natural surface water increases the concentration of oxygen-containing low molecular weight compounds. Many of these compounds support microbiological growth and as such are termed assimilable organic carbon (AOC). Phytoplankton can contribute substantially to the organic carbon load when surface water is used as source for drinking water treatment. We have investigated dissolved organic carbon (DOC) formation from the ozonation of a pure culture of Scenedesmus vacuolatus under defined laboratory conditions, using a combination of DOC fractionation, analysis of selected organic acids, aldehydes and ketones, and an AOC bioassay. Ozonation of algae caused a substantial increase in the concentration of DOC and AOC, notably nearly instantaneously upon exposure to ozone. As a result of ozone exposure the algal cells shrunk, without disintegrating entirely, suggesting that DOC from the cell cytoplasm leaked through compromised cell membranes. We have further illustrated that the specific composition of newly formed AOC (as concentration of organic acids, aldehydes and ketones) in ozonated lake water differed in the presence and absence of additional algal biomass. It is therefore conceivable that strategies for the removal of phytoplankton before pre-ozonation should be considered during the design of drinking water treatment installations, particularly when surface water is used.     

Effects of mass retention of dissolved organic matter and membrane pore size on membrane fouling and flux decline

Ultrafiltration (UF) fouling has been attributed to concentration polarization, gel layer formation as well as outer and inner membrane pore clogging. It is believed that mass of humic materials either retained on membrane surface or associated with membrane inner pore surface is the primary cause for permeate flux decline and filtration resistance build-up in water supply industries. While biofilm/biofouling and inorganic matter could also be contributing factors for permeability decline in wastewater treatment practices. The present study relates UF fouling to mass of dissolved organic matter (DOM) retained on membrane and quantifies the effect of retained DOM mass on filtration flux decline. The results demonstrate that larger pore membranes exhibit significant flux decline in comparison with the smaller ones. During a 24-h period, dissolved organic carbon mass retained in 10 kDa membranes was about 1.0 g m⁻² and that in 100 kDa membranes was more than 3 times higher (3.6 g m⁻²). The accumulation of retained DOM mass significantly affects permeate flux. It is highly likely that some DOMs bind or aggregate together to form surface gel layer in the smaller 10 kDa UF system; those DOMs largely present in inner pore and serving as pore blockage on a loose membrane (100 kDa) are responsible for severe flux decline.     

Bio-diatomite dynamic membrane reactor for micro-polluted surface water treatment

This work investigated the feasibility of treating micro-polluted surface water for drinking water production with a bio-diatomite dynamic membrane reactor (BDDMR) at lab-scale in continuous-flow mode. Results indicate that the BDDMR was effective in removing COD_Mn, DOC, UV₂₅₄, NH₃-N and trihalomethanes' formation potential (THMFP) at a hydraulic retention time (HRT) of 3.5 h due to its high concentrations of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS). The removal of pollutants was mainly ascribed to microbial degradation in BDDMR because the dynamic membrane alone was much less effective in pollutant removal. Though the diatomite particles (5-20 μm) were much smaller in size than the aperture of the stainless steel support mesh (74 μm), microorganisms and their extracellular polymer substances could bind these particles tightly to form bio-diatomite particles which were completely retained by the support mesh. The analysis of molecular weight (MW) distribution by gel permeation chromatography (GPC) shows that the BDDMR could effectively remove the hydrophilic fraction of dissolved organic materials present in the raw water.     

Characterization of dissolved organic matter in drinking water sources impacted by multiple tributaries

The characterization of dissolved organic matter (DOM) in drinking water sources is important as this material contributes to the formation of disinfection by-products (DBPs) and affects how water treatment unit operations are optimized. Drinking water utilities often draw water from sources impacted by multiple tributaries, with possible shifts in DOM concentrations and reactivity over time, depending on specific environmental conditions. In this study, results are presented on the characterization of DOM under varying ambient conditions from the four main tributaries of Lake Mead, a large reservoir in the southwest United States. The tributaries include the Las Vegas Wash (LVW), Muddy River (MR), Virgin River (VR) and the upper Colorado River (UCR). One additional sample was collected at the outflow of the reservoir (lower Colorado River (LCR)). The DOM was characterized by both bulk parameters (specific ultraviolet absorbance (SUVA)) and specific physicochemical properties, i.e. size, polarity and fluorescence. The analyses were performed emphasizing limited changes in its natural configuration by eliminating analytical preparation steps, excluding sample filtration (0.45 microm filter). Results indicate that each tributary had a different molecular weight distribution, as well as fluorescence properties, which helped in the identification of the relative source of DOM (allochthonous versus autochthonous). The largest apparent molecular weight distribution was observed for DOM samples collected at the MR site, which is fed mostly by groundwater seepage. The smallest apparent molecular weight was observed for DOM collected at the LCR site, suggesting that retention in the reservoir resulted in a decrease in molecular weight as a probable result of photo oxidation and microbial processes. Fluorescence analysis aided the differentiation of DOM by clearly identifying waters that were affected by microbial activity (LVW, UCR, and LCR), either by wastewater influence or by autochthonous processes, versus limited microbial influence (MR and VR). Polarity analysis revealed clear differences in the hydrophobic/hydrophilic nature between waters, including temporal differences within individual waters at a particular site. The DOM from the LVW and VR sites had higher hydrophobic character, as measured by retention onto non-polar sorbents. Additionally, the DOM collected at the LCR had the least hydrophobic character. This type of analysis would be beneficial to utilities who want to better understand and manage their source waters, especially in the evaluation of temporal variation within a watershed.     

UV-induced photochemical heterogeneity of dissolved and attached organic matter associated with cyanobacterial blooms in a eutrophic freshwater lake

Cyanobacterial blooms represent a significant ecological and human health problem worldwide. In aquatic environments, cyanobacterial blooms are actually surrounded by dissolved organic matter (DOM) and attached organic matter (AOM) that bind with algal cells. In this study, DOM and AOM fractionated from blooming cyanobacteria in a eutrophic freshwater lake (Lake Taihu, China) were irradiated with a polychromatic UV lamp, and the photochemical heterogeneity was investigated using fluorescence excitation–emission matrix (EEM)-parallel factor (PARAFAC) analysis and synchronous fluorescence (SF)-two dimensional correlation spectroscopy (2DCOS). It was shown that a 6-day UV irradiation caused more pronounced mineralization for DOM than AOM (59.7% vs. 41.9%). The EEM-PARAFAC analysis identified one tyrosine-, one humic-, and two tryptophan-like components in both DOM and AOM, and high component photodegradation rates were observed for DOM versus AOM (k > 0.554 vs. <0.519). Moreover, SF-2DCOS found that the photodegradation of organic matters followed the sequence of tyrosine-like > humic-like > tryptophan-like substances. Humic-like substances promoted the indirect photochemical reactions, and were responsible for the higher photochemical rate for DOM. The lower photodegradation of AOM benefited the integrality of cells in cyanobacterial blooms against the negative impact of UV irradiation. Therefore, the photochemical behavior of organic matter was related to the adaptation of enhanced-duration cyanobacterial blooms in aquatic environments.     

Assimilable organic carbon (AOC) and biodegradable dissolved organic carbon (BDOC):: complementary measurements

The objective of this study was to evaluate the necessity of measuring both assimilable organic carbon (AOC) and biodegradable dissolved organic carbon (BDOC) as indicators of bacterial regrowth potential. AOC and BDOC have often been measured separately as indicators of bacterial regrowth, or together as indicators of bacterial regrowth and disinfection by-product formation potential, respectively. However, this study proposes that both AOC and BDOC should be used as complementary measurements of bacterial regrowth potential. In monitoring of full-scale membrane filtration, it was determined that nanofiltration (NF) removed over 90% of the BDOC while allowing the majority of the AOC through. Heterotrophic plate counts (HPC) remained low during the entire period of monitoring due to high additions of disinfectant residual. In a two-year monitoring of a water treatment plant that switched its treatment process from chlorination to chlorination and ozonation, it was observed that the plant effluent AOC increased by 127% while BDOC increased by 49% after the introduction of ozone. Even though AOC is a fraction of BDOC, measuring only one of these parameters can potentially under- or over-estimate the bacterial regrowth potential of the water.     

Characterization of copper complexation with natural dissolved organic matter (DOM)--link to acidic moieties of DOM and competition by Ca and Mg

We investigated Cu complexation by three dissolved organic matters (DOMs) collected by reverse osmosis (RO). Alkalimetric titration, pH-stat Cu and Ca titrations, pH edges of Cu–DOM complexation, and Ca/Mg–Cu exchange experiments were investigated at I=10⁻² M for DOM samples of 10 mg C/L. The proton and Cu binding characteristics indicated similarity for all three DOMs. All Cu titrations employed ion selective electrode measurement and indicated the presence of relatively small amounts of strong Cu-binding sites. Four distinct classes of Cu binding sites are required for FITEQL 4.0 to provide good fits to the entire curves. The estimated total Cu binding site density is 4.55 mmol/g C, much less than the total acidity but very close to the phenolic site content. Cu–DOM complexation increases approximately 10-fold per pH unit, even at relatively high pH (>8). We suggest that sites characterized as phenolic based on alkalimetric titration, not carboxyl sites, account for the majority of Cu complexation under natural water conditions, and Cu–DOM complexation is principally through the replacement of H⁺ by Cu²⁺ at the phenolic binding sites. The Cu–H exchange ratio is 1:1 for the first three sites and about 1:2 for the 4th site. This 4-site model describes well the pH dependency of Cu–DOM complexation and provides good estimates of free Cu concentrations throughout wide total copper (Cu_T) and pH ranges. Comparison between Ca–DOM and Cu–DOM complexation demonstrated that (i) Ca–DOM complexation increases much less than an order of magnitude per pH unit and decreases at higher Ca concentration, different from that of Cu–DOM complexation; and (ii) Cu–DOM complexation is highly non-linear, in contrast to the much reduced extent of non-linearity of Ca–DOM complexation. Ca/Mg–Cu exchange experiments showed small competition effect, less than expected by a simple competition model, and the competition tended to reduce with increasing Ca or Mg concentrations. The extent of the competition by Mg and Ca are essentially comparable. Put all together, it suggests that Ca and Mg are preferably bound by carboxyl sites, especially at relatively high concentrations, resulting in a weakened apparent competition effect.