INFLUENCE OF DISSOLVED ORGANIC MATTER ON THE TOXICITY OF COPPER TO CERIODAPHNIA DUBIA: EFFECT OF COMPLEXATION KINETICS

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1 Environmental Toxicology and Chemistry, Vol. 18, No. 11, pp , SETAC Printed in the USA /99 $ INFLUENCE OF DISSOLVED ORGANIC MATTER ON THE TOXICITY OF COPPER TO CERIODAPHNIA DUBIA: EFFECT OF COMPLEXATION KINETICS SANG DON KIM, HUIZHONG MA, HERBERT E. ALLEN, and DANIEL K. CHA* Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware 19716, USA (Received 23 October 1998; Accepted 5 March 1999) Abstract The reaction kinetics of copper interaction with dissolved organic matter (DOM) in water were studied in order to determine the effect of equilibration period on the toxicity of copper to aquatic organisms. The changes in physical and chemical forms of the copper during four reaction times were examined in four completely mixed reactors in series; the bioavailability of the copper as a function of these new forms was then determined with a flow-through bioassay system, using Ceriodaphnia dubia as a test organism. This study showed that the toxicity of copper to C. dubia decreased with increasing copper DOM reaction time, which demonstrated that the copper reaction rate with dissolved organic components in the test water was slow. The toxicity of copper to C. dubia was closely related to the measured free-copper concentration (Cu 2 ) rather than to the total copper concentrations, a fact that supports the free ion activity model. We found that the LC50 of copper for C. dubia increased (i.e., toxicity decreased) linearly with increasing total available binding sites. Although a similar trend was observed in both natural DOM and commercial humic acid, our results indicated that for a given copper organic carbon ratio, copper binds more strongly to humic acid than to the natural DOM. This difference may be attributed to the greater copper binding affinity of humic acid (greater than that of other metal-binding organic fractions present in DOM, i.e., fulvic acid). Keywords Copper Kinetics Toxicity Complexation Dissolved organic matter INTRODUCTION A large body of environmental literature demonstrates that bioavailability or toxicity of trace metals is directly correlated to concentrations of free metal ions rather than to total or complexed metal concentrations [1 4]. Sunda et al. [5] showed that the addition of the synthetic chelator nitrilotriacetic acid reduced the toxicity of cadmium to grass shrimp and that the toxicity was related to the concentration of free cadmium ions present in the test water rather than to the concentration of total metal. Sunda and Guillard [6] demonstrated that the toxicity of copper to phytoplankton in seawater was directly correlated to the concentration of free-copper ions. Allen et al. [7] showed that the toxicity of zinc to a species of algae was also related to the concentration of free metal ions. They used a series of synthetic chelators that had conditional stability constants for zinc that extended over more than six orders of magnitude. Allen and Brisbin [8] showed that the toxicity of copper in natural waters could be predicted based on speciation calculations using stability constants and complexation capacities that had been determined by anodic stripping voltammetry. Recently, Ma et al. [9] used commercial humic acid (HA) to demonstrate a linear relationship between the survival of Ceriodaphnia dubia and Cu 2 concentration. However, most of the described studies have been conducted in the presence of synthetic ligands and commercial HA. A limited number of investigations have been reported with the presence of natural organic matter [10]. More studies examining the interaction of trace metals with aquatic organisms in the presence of natural dissolved organic matter (DOM) are needed so that we can quantitatively correlate the response of aquatic organisms to free-metal ion activity in natural waters. In most bioassay studies, in which trace metal salts are * To whom correspondence may be addressed (cha@ce.udel.edu). added to the test water, there is an implicit assumption that the reaction of the added metal with materials in the water is rapid. Most laboratory aquatic-toxicity test protocols are based on dilutor systems [11 14], in which the toxicant is given only a few minutes to react with the components in the water prior to its introduction into the test chamber. Thus, such a system does not consider the reaction kinetics and may expose organisms to a higher proportion of more toxic forms of metals than would waters that were given a longer time to equilibrate. Using a continuous flow-through bioassay system, Ma et al. [9] showed that longer reaction times of copper with HA solution reduced the toxic effects of this copper on C. dubia. The study clearly demonstrated that laboratory toxicity testing protocols based on dilutor systems may result in a higher concentration of bioavailable toxicant in exposure chambers than would be encountered in receiving waters. As a follow-up to the study of copper interaction with commercial HA, the objective of this study was to investigate both the kinetics of the interaction of copper with natural DOM and its influence on the toxicity of copper to C. dubia. A continuous flow-through bioassay system was used to provide test organisms with a constant free-copper concentration during the exposure period. Because this study used natural DOM, it was designed to quantitatively correlate the response of test organisms to free-metal ion activity in natural water conditions. MATERIALS AND METHODS All reagents were of analytical grade and were used without further purification, except for HNO 3, which was used to preserve samples for atomic absorption analysis of copper; HNO 3 was Optima grade. Humic acid (as the sodium salt) was obtained from Aldrich Chemical (Milwaukee, WI, USA). All the glassware and the polyethylene and polypropylene labware was soaked in 10% HNO 3 (v/v) for at least 48 h before use. 2433

2 2434 Environ. Toxicol. Chem. 18, 1999 S.D. Kim et al. Deionized water from a Barnstead NANOpure ultrapure water system was used throughout the study. Collection and concentration of natural DOM A portable reverse osmosis (RO) system, based on the design of Serkiz and Perdue [15], was used to collect and concentrate natural DOM from Suwannee River (Okefenokee State Park, GA, USA). River water was first pumped through in-line filters (1 and 0.45 m) to remove particulate matter and was then collected in a 40-L sample reservoir. A 1/6 HP submersible pump (Simer Pump, Model 2310, Kansas City, MO, USA) in the sample reservoir pumped the filtered samples through a cation exchanger (Dowex-50 cation exchange resin in the sodium form, Dow Chemical, Midland, MI, USA). The filtered and cation-exchanged sample was then delivered to the RO membrane by a high-pressure pump (Hypro, Model 2230B, New Brighton, MN, USA), which boosted the pressure to 150 to 200 psi. The RO membrane (Filmtec FT30, Dow Chemical) consisted of a 0.2- m thick, highly crosslinked aromatic polyamide skin on a 35- m polysulphone support and was designed for the desalination of tap water. The percent rejection of 0.2% NaCl solution by the Filmtec RO membrane ranged from 99.0 to 99.7%, depending on operating pressure (E.M. Perdue, personal communication). The retentate solution from the RO system was collected into the sample reservoir and mixed with filtered river water. The recycling of the retentate solution to the sample reservoir was continued until the desired enrichment of DOM was achieved. The concentrated sample was desalted with a H -saturated cation exchange resin (Fluka, Milwaukee, WI, USA) in order to remove major ions. The concentration of DOM was reported based on dissolved organic carbon (DOC) content. Test organisms and culture conditions The test organism used in this study, C. dubia, and the food, Selenastrum capricornutum, as well as yeast, trout chow, and Cerophyll mixture were purchased from Aquatic Bio- System, Inc. (Fort Collins, CO, USA). The organisms were cultured and handled according to the procedures outlined in the U.S. Environmental Protection Agency manual [16]. The detailed culturing and toxicity testing conditions are summarized in Ma et al. [9]. We used moderately hard reconstituted water, with a hardness of 85 5 mg/l, an alkalinity of 60 5 mg/l as CaCO 3 mg/l, and a ph of , prepared according to the guidelines given in U.S. Environmental Protection Agency manual [16], as a test water throughout this study. Static toxicity test The effect of DOM on the toxicity of copper was first examined in a static bioassay test, in which we exposed the test organism to 15 ml test water containing 2.5 mg DOC/L of DOM. For one set of bioassay chambers, C. dubia was introduced to the test water immediately after the addition of copper; for the second set, we allowed DOM and copper to equilibrate for 24 h before the addition of the organisms. The third set contained only dilution water without DOM. Each set comprised nine different copper concentrations and a control. Total and free-copper concentrations were determined before and after the 24-h exposure period. Flow-through toxicity test We conducted the flow-through bioassay tests with DOM concentrations of 2.5, 4.5, 7.5, and 10 mg DOC/L and with HA concentrations of 5, 10, 15, and 20 mg/l, using four identical continuous flow-through bioassay systems. For each DOM concentration, we tested seven copper concentrations plus a control. Control experiments without copper were included in order to determine whether the presence of DOM had any detrimental effect on C. dubia. Each system consisted of 4 chemical mixing reactors, 12 flow-through bioassay chambers, and 2 peristaltic pumps. A detailed description and schematic drawing of the experimental setup are provided in our earlier publication [9]. The chemical mixing chambers consisted of four completely mixed reactors in series. The volume of each reactor was selected in order to provide the target hydraulic residence times of 2 min and of 1, 5, and 23 h for copper and DOM reaction. A shaded pole gear motor (Dayton Electric, Niles, IL, USA) and a high-density polyethylene stirrer (Nalgen, Rochester, NY, USA) were used to stir the solution in each chemical mixing reactor (except for the first mixing reactor [V 0.02 L], which used a magnetic stirrer because the water level in it was too low). Two peristaltic pumps delivered DOM and copper solutions into the first chemical mixing reactor at a constant flow rate of 600 ml/h. The DOM and HA solutions were prepared by adding appropriate amounts of concentrated DOM (from the Suwannee River) and commercial HA to the reconstituted dilution water, and the mixture was allowed to equilibrate for at least 7 d at a constant temperature of 25 1 C. A portion of the reactor content from each mixing reactor was continuously delivered to each of three 50-ml flow-through bioassay chambers containing C. dubia neonates. Bioassay chambers with a liquid volume of 50 ml were constructed from a transparent acrylic polymer. The inlets and outlets of the chambers were fitted with mesh/in Nytex screen (McMaster-Carr, New Brunswick, NJ, USA) in order to confine C. dubia and to minimize the hydrodynamic jet effect of influent stream on the test organisms. The flow rate through each of the 12 bioassaychamber effluent flow tubes was adjusted to 50 ml/h using ramp clamps (Cole Parmer, Vernon Hill, IL, USA). The flow rates and hydraulic residence times in each of the bioassay chambers were the same so that we could preclude these variables from affecting the toxicity. Since the hydraulic residence time in the bioassay chamber was 1 h, the overall hydraulic residence time, or chemical reaction time, was equal to the sum of the hydraulic residence time in the mixing reactor and that in the bioassay chamber (or 62 min and 2, 6, and 24 h). Before transferring the neonates of C. dubia into bioassay chambers, the systems were operated for at least 24 h, until all four chemical mixing reactors and 12 bioassay chambers reached a steady-state flow rate. Effluent flow rate was checked hourly to ensure a desired flow rate through each bioassay chamber during the toxicity test. Each bioassay chamber contained seven neonates of C. dubia that were less than 24 h old. The exposure period was 24 h for the toxicity tests. The survival rates were determined to be the numbers of live organisms after 24-h exposure divided by the total number of organisms transferred. The total recoverable copper and free- Cu 2 concentrations from the effluent of each chemical mixing reactor and from the effluents of three bioassay chambers were determined by atomic absorption spectrophotometer and by copper-ion selective electrode, respectively. A preliminary test demonstrated that copper adsorption onto and leaching from the chemical mixing reactors and bioassay chambers were negligible.

3 Effect of copper DOM complexation kinetics on toxicity Environ. Toxicol. Chem. 18, Table 1. Elemental analyses of Suwannee River dissolved organic matter samples; the percentages of C, H, N, and O S are on a dry, ash-free basis Collection date C H N O S Ash Reference June 1997 April 1987 October This study [15] [15] Table 2. Metal elements in the Suwannee River dissolved organic matter sample Element Na K Ca Mg Fe Before cation-exchange resin ( g/l) 55, ,030 After cation-exchange resin ( g/l) ,280 Laboratory procedures The DOC content of the DOM solution was determined using a Dohrmann DC-190 TOC analyzer. A Perkin-Elmer Model 5000 atomic absorption spectrophotometer (Norwalk, CT, USA) with a graphite furnace accessory was used for the analysis of total copper concentrations. The free-cu 2 activity was determined by a cupric ion-selective electrode (Cu-ISE, model 94-29, Orion Research, Boston, MA, USA) and a double-junction Ag/AgCl reference electrode (Model 90-02, Orion Research). Detailed descriptions of Cu-ISE apparatus and calibration procedure were presented in Ma et al. [9]. Before each use, the Cu-ISE was polished with polishing paper, soaked in 0.01 M H 2 SO 4 solution, and rinsed with deionized water to restore the electrode to good operating condition. The reference electrode was also refilled with filling solutions before each use. We calibrated the Cu-ISE and reference electrode using copper-ethylenediamine buffer for pcu range of 6.7 to 12.5 and Cu(NO 3 ) 2 solution without ethylenediamine buffer for pcu range of 5 to 7. A preliminary test showed that triplicate measurements of Cu 2 in the presence of DOM were within 5%. Elemental analyses of the DOM sample for C, H, N, O S, and ash were obtained from a commercial laboratory (Atlantic Microlab, Norcross, GA, USA). We also analyzed for major ions, Cu, Na, K, Ca, Mg, and Fe using inductively coupled plasma (Spectro Analytical Instrument, Kleve, Germany) before and after the desalting process (through the H -saturate cation-exchange resin). copper. Figure 1 shows that the addition of 2.5 mg DOC/L of DOM to the test water (a mixture of dilution water and copper solution) shifted the copper toxicity curve to considerably higher copper concentrations. Furthermore, the equilibration of 2.5 mg/l of DOM with the test water for 24 h prior to adding the organisms further increased the survivability of C. dubia in the DOM copper solution, thereby indicating that the reaction of DOM with added copper ions is not rapid. The results demonstrate that the kinetics of copper interactions with DOM is a significant factor and that the normal testing protocols, which give no more than a few minutes for the reaction of the toxicant with the components in the water prior to its introduction to the test chamber, may expose organisms to a higher proportion of copper in more toxic forms than would be present if the water was given a longer time to equilibrate. The LC50 value of copper for C. dubia was increased from g/l in dilution water without DOM to g/l in the unequilibrated DOM solution. The LC50 value was further increased to g/l when copper ions were allowed to equilibrate with DOM for 24 h. Flow-through toxicity test with DOM and HA In order to determine the changes in the toxicity of copper to C. dubia as a function of equilibration time with DOM, we ran flow-through toxicity tests with four copper DOM and copper HA reaction times: 62 min and 2, 6, and 24 h. Figure 2 shows a set of four toxicity curves corresponding to the four RESULTS AND DISCUSSION Characteristics of DOM sample The DOM concentration of the raw water at our sampling site was about 47 mg DOC/L. We achieved at least a 25-fold concentration of DOM during the sampling. The probability of chemical alteration during the DOM-concentration process is low because the RO system does not expose the DOM to extremes of temperature or to chemical reagents [15]. As a result, we believe that the DOM samples collected with the RO process closely resembled the DOM in the Suwannee River. The elemental composition of our DOM sample is presented in Table 1. The characteristics of our sample were comparable to those of the DOM samples obtained from the same location [15]. Major ion contents of our concentrated DOM samples before and after the desalting process are presented in Table 2. The H -saturated cation-exchange resin successfully reduced all of the major ions present in the DOM sample. Effect of DOM on toxicity We studied the effect of DOM on the toxicity of copper ions by examining the survival rate of C. dubia after a 24-h exposure period in static bioassay chambers (Fig. 1). It is evident that the presence of DOM decreased the toxicity of Fig. 1. Effect of dissolved organic matter (DOM) on the survival of Ceriodaphnia dubia in static test. The DOM concentration is 2.5 mg/ L. Equilibrated DOM was prestabilized with reconstituted dilution water for 24 h. The data points were fitted to sigmoidal function in order to assist with visualizing the trend.

4 2436 Environ. Toxicol. Chem. 18, 1999 S.D. Kim et al. Fig. 2. Comparison of toxicity curves corresponding to the four copper dissolved organic matter (DOM) reaction times with different DOM concentrations (HRT hydraulic retention time). HRT 1h; HRT 2h; HRT 6h; HRT 24 h; 2.5 mg/l DOM; 4.5 mg/l DOM; 7.5 mg/l DOM; and 10 mg/l DOM. copper DOM reaction times for each DOM concentration. The result clearly demonstrates that the toxicity of copper decreases with an increase in the copper DOM reaction time. For example, the addition of 57 g/l Cu to 2.5 mg/l of DOM solution resulted in a 10% survival of test organisms with a 1- h reaction time, whereas 100% survival was observed with a 24-h reaction time. With higher DOM concentrations, a higher concentration of total copper was required in order to achieve the same mortality. In order to reach 50% of mortality for 24- h copper DOM reaction time, we needed to add 75, 130, 190, and 270 g/l of total copper for 2.5, 4.5, 7.5, and 10 mg/l of DOM, respectively (Fig. 3). For each DOM control experiment without copper addition, 100% survival was observed in all bioassay chambers, which indicates that the presence of DOM did not have any detrimental effect on the test organisms. Fig. 3. Relationships between 24-h LC50 and dissolved organic matter (DOM) concentration at different hydraulic retention times. Fig. 4. Effect of free ions in %survival of Ceriodaphnia dubia in bioassay chambers. Similarly, the addition of commercial HA decreased the toxicity of copper [9]. Copper toxicity curves were displaced to considerably higher total copper concentration with higher concentrations of commercial HA for the same copper HA reaction time and with increased reaction time for the same HA concentration. Relationship of free-copper ion and toxicity In order to normalize the bioassay results by the free-copper ion concentrations in the chamber rather than total copper concentrations, we measured the Cu 2 concentrations in effluent samples collected from each chemical reactor using an ISE. Since the Cu-ISE took 30 min to reach a stable reading, the ISE measurements represent Cu 2 concentrations at reaction times corresponding to the hydraulic residence times of the chemical mixing reactors plus one-half the hydraulic residence times of the bioassay chambers. The normalization of the bioassay results of both DOM and HA flow-through toxicity tests by the free-copper ion concentrations resulted in a single toxicity curve (Fig. 4). From the linear regression analysis, we obtained the relationship between survival percentage and Cu 2 concentrations: survival (%) [Cu 2 ] with a correlation coefficient of (n 232). The results demonstrate that the bioavailability or toxicity of copper is directly correlated to the free-copper ion activity in the presence of both natural DOM and commercial HA. Copper-binding capacity and toxicity The concentrations of total available binding sites in the bioassay chambers were calculated by multiplying the total site density ( M Cu 2 binding sites/g organic C) and the measured total organic carbon concentrations. Using the procedure described in detail by Ma et al. [9], copper titrations of HA and DOM resulted in total site density values of 3.60 and 3.91 mmol Cu/g C for HA and DOM, respectively. We found that the LC50 of copper for C. dubia increased (i.e., toxicity decreased) linearly with increasing total available binding sites (Fig. 5). Figure 5 also showed that the influence of increasing binding sites on toxicity was greater with HA than with DOM. This indicates that for a given copper organic carbon ratio,

5 Effect of copper DOM complexation kinetics on toxicity Environ. Toxicol. Chem. 18, copper binds more strongly to HA than to the natural DOM. A statistical test (F-test) revealed that this difference in the linear relationship correlating LC50 of copper to total available binding sites in HA and DOM was significant ( 0.05) when the results from the same reaction times were compared. This difference may be attributed to the greater copper-binding affinity of HA compared to the binding ability of other metalbinding organic fractions present in DOM (i.e., fulvic acid). Shuman and Cromer [17] reported that the stability constant for copper HA complex (K ) was about two times greater than the value for copper fulvic acid complex (K ). Humic acids represent a minor fraction of natural DOM; the typical HA contents of natural DOM are about 10% [18]. However, our results suggest that even though HA contents of natural DOM are relatively small compared to other fractions, its contribution to the amelioration of copper toxicity in natural waters may be significant because of its strong binding affinity. In most bioassay studies, in which trace-metal salts are added to the test water, there is an implicit assumption that the reaction of the added metal with materials in the water is rapid. In this study, however, we showed that the toxicity of copper to C. dubia decreased when added copper was given longer to equilibrate with DOM in the test water. This indicates that most laboratory toxicity testing protocols, which provide only a few minutes for reaction with the organic components in the test water prior to metal introduction into the test chamber, may expose organisms to a higher proportion of metal (in more potent forms) than would be present if the water were given a longer time to equilibrate. Thus, testing protocols that do not consider the reaction kinetics would overestimate metal toxicity in natural environments that afford considerable time for equilibration. Conclusions from our study include the following: the toxicity of copper to C. dubia decreased with increasing copper DOM reaction time. The toxicity of copper to C. dubia was closely related to the measured free copper concentration (Cu 2 ). The LC50 of copper for C. dubia increased (i.e., toxicity decreased) linearly with increasing total available binding sites. Copper binds more strongly to commercial HA than to the natural DOM. Acknowledgement This work was supported by the International Copper Association. The authors thank Douglas J. Baker for making the mixing reactors and the bioassay chambers used in the bioassay system. Fig. 5. The relationship between 24-h LC50 and total available binding sites in humic acid (HA) and dissolved organic matter (DOM) with four different hydraulic retention times. Total available binding sites were calculated by multiplying organic binding capacity (DOM, 3.91 mmol/g C; HA, 3.6 mmol/g C) and measured carbon concentrations. REFERENCES 1. Brand LE, Sunda WG, Guillard RRL Reduction of marine phytoplankton reproduction rates by copper and cadmium. J Exp Mar Biol Ecol 96: Borgmann U, Ralph KM Complexation and toxicity of copper and the free metal bioassay technique. Water Res 17: Anderson DM, Morel FMM Copper sensitivity of Gonyaulax tamarensis. Limnol Oceanogr 23: Campbell PGC Interactions between trace metals and aquatic organisms: A critique of the free-ion activity model. In Tessier A, Turner DR, eds, Metal Speciation and Bioavailability in Aquatic Systems. John Wiley & Sons, New York, NY, USA, pp Sunda WG, Engel DW, Thuotte RM Effect of chemical speciation on toxicity of cadmium to grass shrimp, Palaemonetes pugio: Importance of free cadmium ion. 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