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Published online 12 October 2005
Published in J Environ Qual 34:1964-1971 (2005)
DOI: 10.2134/jeq2005.0014
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Organic Compounds in the Environment

Oxytetracycline Sorption to Organic Matter by Metal-Bridging

Allison A. MacKay* and Brian Canterbury

Environmental Engineering Program, Univ. of Connecticut, 261 Glenbrook Rd., Unit 2037, Storrs, CT 06269-2037

* Corresponding author (mackaya{at}engr.uconn.edu)

Received for publication January 16, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS AND MATERIALS
 RESULTS AND DISCUSSION
 REFERENCES
 
The sorption of oxytetracycline to metal-loaded ion exchange resin and to natural organic matter by the formation of ternary complexes between polyvalent metal cations and sorbent- and sorbate ligand groups was investigated. Oxytetracycline (OTC) sorption to Ca- and Cu-loaded Chelex-100 resin increased with increasing metal/sorbate ratio at pH 7.6 (OTC speciation: 55% zwitterion, 45% anion). Greater sorption to Cu- than Ca-loaded resin was observed, consistent with the greater stability constants of Cu with both the resin sites and with OTC. Oxytetracycline sorption to organic matter was measured at pH 5.5 (OTC speciation: 1% cation, 98% zwitterion, 1% anion). No detectable sorption was measured for cellulose or lignin sorbents that contain few metal-complexing ligand groups. Sorption to Aldrich humic acid increased from "clean" < "dirty" (no cation exchange pretreatment) < Al-amended < Fe(III)-amended clean humic acid with Kd values of 5500, 32000, 48000, and 250000 L kg–1 C, respectively. Calcium amendments of clean humic acid suggested that a portion of the sorbed OTC was interacting by cation exchange. Oxytetracycline sorption coefficients for all humic acid sorbents were well-correlated with the total sorbed Al-plus-Fe(III) concentrations (r2 = 0.87, log-log plot), suggesting that sorption by ternary complex formation with humic acid is important. Results of this research indicate that organic matter may be an important sorbent phase in soils and sediments for pharmaceutical compounds that can complex metals by the formation of ternary complexes between organic matter ligand groups and pharmaceutical ligand groups.

Abbreviations: MWCO, molecular weight cut-off • OC, organic carbon • OTC, oxytetracycline • TOC, total organic carbon


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
 RESULTS AND DISCUSSION
 REFERENCES
 
THE IMPORTANCE of natural organic matter as a sorbent for polar environmental contaminants, such as pharmaceutical compounds, has been overlooked somewhat in the effort to understand the mechanisms governing solid-water exchange of these compounds. The classic hydrophobic partition model, in which the compound octanol–water distribution coefficient is used as a proxy for partitioning between organic matter and water (Chiou et al., 1979; Karickhoff et al., 1979), fails to account for the full extent of polar pharmaceutical compound sorption to soil and sediment samples (Tolls, 2001). To explain the order of magnitude differences between observed sorption coefficients and those predicted using octanol–water distribution coefficients, researchers have investigated pharmaceutical sorption to other soil/sediment components, including various clay minerals (Martin and Gottlieb, 1952; Pinck et al., 1961; Porubcan et al., 1978; Figueroa et al., 2004; Kulshrestha et al., 2004) and oxide solids (Figueroa and MacKay, 2005; Gu and Karthekiyan, 2005) in isolated systems. Pharmaceutical interactions with clay minerals and oxide particles occur through electrostatic ion exchange and ligand complexation interactions. Such interactions may be possible mechanisms for pharmaceutical sorption to organic matter phases that contain cation exchange groups and complexed metals. Given the high organic matter content of manure lagoons and wastewaters that are the sources of many pharmaceutical compounds introduced to the environment, there is a need to understand the mechanisms by which such polar compounds may sorb to natural organic matter phases.

A review of literature studies suggests that metal-bridging may be the most important mechanism of interaction for pharmaceutical compounds with organic matter. Several pharmaceutical sorption studies conducted using isolated organic matter phases from soils or manure have found measured organic carbon (OC)–normalized sorption coefficients that were up to hundreds or thousands of times greater than estimates using octanol–water distribution coefficients, even after accounting for pH effects on octanol–water partitioning (Sithole and Guy, 1987; Schmitt-Kopplin et al., 1999; Holten Lutzhøft et al., 2000; Loke et al., 2002). For example, norfloxacin sorption to soil humic acid at pH 9.2 had a Kocobs of 1050 L kg–1 C (Schmitt-Kopplin et al., 1999), whereas the predicted Kocest was only 0.11 L kg–1 C (log Kocest = 0.84 log Kow + 0.41; Karickhoff, 1981) using an octanol–water distribution coefficient of 0.02 at this pH (Takács-Novák et al., 1992). Similarly, OTC sorption to manure particles at pH 7.8 occurred with a Kocobs of 200 L kg–1 C, which was much greater than the Kocest of 0.3 L kg–1 C calculated using an octanol–water distribution coefficient of 0.08 (Herbert and Dorsey, 1995). Most of these studies applied no specific treatments to remove strongly bound cations from the organic matter sorbents, and most of the sorbates are known to complex metals in solution (Abd El Wahed et al., 1984; Machado et al., 1995; Turel and Bukovec, 1996; Djurdjevic et al., 2000; Schneider, 2001). Thus, it is possible that formation of ternary complexes between the pharmaceutical sorbate and strongly bound cations in the organic matter could explain the high amounts of sorption observed, relative to estimates using only hydrophobic partitioning.

Cation cross-linking is known to alter the hydrophobic nature of natural organic matter (Yuan and Xing, 2001; Lu and Pignatello, 2004); however, this effect does not appear to be of sufficient magnitude to account for differences between observed and estimated sorption coefficients of pharmaceutical compounds for isolated organic matter phases. Lu and Pignatello (2004) found that sorption of nonpolar organic compounds to soil humic acid shifted from partitioning into rubbery regions to hole-filling of glassy regions with Al3+–saturation of humic acid by the same Al treatment method used in our study. Overall OC-normalized sorption coefficients for naphthalene and 1,2,4-trichlorobenzene decreased as much as 30% for Al3+–saturated humic acid compared with H+-saturated humic acid. Similar trends were suggested in the work of Yuan and Xing (2001). Schlautman and Morgan (1993) reported no change in polycyclic aromatic hydrocarbon sorption to Suwanee River humic acid with 1 mM Ca2+ at pH 4 and a slight increase with 1 mM Ca2+ at pH 7. Although these studies do not indicate clearly which sorbates will have decreased or enhanced hydrophobic interactions with cross-linked organic matter, they do suggest that cation cross-linking produces relatively small changes to hydrophobic interactions of sorbates with organic matter. Thus, the order of magnitude greater Kocobs than Kocest for pharmaceutical compounds must result from interactions different than from strict hydrophobic partitioning with organic matter.

Additional evidence in support of a metal-bridging mechanism for pharmaceutical sorption to organic matter is provided by pH studies of organic matter sorption and by drug pharmacokinetics. Holten Lutzhøft et al. (2000) showed increased flumequine and oxolinic acid sorption to humic acid with increasing pH, despite the unfavorable electrostatic repulsion expected from the increased deprotonation of both the sorbate and the sorbent. Such a trend of increased sorption to organic matter with increasing pH has been observed for metal complexation to deprotonated ligand groups (Dzombak and Morel, 1990) and thus, formation of a cation bridge between the sorbate and sorbent could be favored at high pH values. Furthermore, ternary complex formation between pharmaceuticals, metal cations, and proteins or nucleic acids are known to be important for antibiotic activity (Schneider, 2001; Turel, 2002) and thus, it is reasonable to hypothesize that similar complexes could form with soil, sediment, and manure organic matter.

The purpose of this research was to investigate whether OTC sorption to organic matter includes a metal-bridging mechanism. Several authors have alluded to possible ternary complex formation between pharmaceutical compounds, metal cations, and organic matter in their studies (Sithole and Guy, 1987; Tolls, 2001); however, no metal concentrations were reported for the sorbents or systems in any of the reviewed studies. Metal-bridging can only occur for pharmaceutical compounds that have functional groups that can complex metal ions in solution. Thus, we chose OTC (Fig. 1) , a high-use veterinary antibiotic, as our test sorbate because tetracyclines are known to complex with divalent and trivalent cations (Abd El Wahed et al., 1984; Machado et al., 1995). Our test sorbents were metal-loaded cation exchange resin, cellulose, lignin, and humic acids with varying degrees of metal loading. If metal-bridging is an important sorption mechanism for pharmaceutical compounds that can complex metals in solution, we expect to observe increased sorption to cation exchange resin as: (i) the metal concentration was increased, and (ii) as the stability of the metal–cation complex increased. We also expect higher sorption coefficients for OTC using organic matter with bound metals than for organic matter that could not complex metals.



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  		Fig. 1. Structure and chemistry of oxytetracycline (OTC). Bar shows dominant species charge as a function of pH where vertical bars represent pKa values (Tavares and McGuffin, 1994).

 

   METHODS AND MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials
Oxytetracycline hydrochloride was used as received from USB Corporation (Cleveland, OH). Chelex-100 resin (Na+ form, 300–1180 µm) was obtained from Bio-Rad (Hercules, CA). Cellulose (microcrystalline, colloidal), lignin (organosolv), and humic acid were all from Aldrich (Milwaukee, WI). Chloride salts of Ca and Fe(III) and sulfate salts of Al and Cu were from Fisher Scientific (Fair Lawn, NJ). pH adjustments were made with hydrochloric acid, sodium hydroxide, and sodium acetate from Fisher and MOPS (4-morpholinepropane sulfonic acid, sodium salt) from Aldrich. High purity water (18 M{Omega}-cm) was prepared on-site with a Barnstead NANOpure Diamond low-TOC purification system.

Analytical
Oxytetracycline concentrations were quantified by HPLC using a LiChrospher 100 RP-18 endcapped column (5 µm, 4.6 mm i.d., 150 mm) and HP1050 with diode array detector. Isocratic elutions were performed using 80% phosphate buffer (20 mM, pH 2.5)/20% acetonitrile at a flowrate of 1 mL min–1. Compound concentrations were quantified by absorbance at 360 nm wavelength. Peak identities were confirmed by comparison of peak spectra with metal-free and metal-containing spectra obtained with a CARY 50 UV-Vis spectrophotometer (Varian) operated in scan mode (200–600 nm).

Quantities of organic matter in solution were determined by high-temperature combustion and CO2 detection (Shimadzu TOC-5000A with high sensitivity catalyst).

Metal concentrations in aqueous solutions were quantified by inductively coupled plasma–mass spectrometry (ICP–MS) (PE SCIEX, Elan 6000, Environmental Research Institute, Storrs, CT) following USEPA methods 3010A (Acid Digestion of Aqueous Samples) and 6020A (Inductively Coupled Plasma–Mass Spectrometry).

Sorbent Preparation
Humic acid suspensions were created by adding dry powder to 0.01 M NaOH and mixing for 24 h. The suspension was centrifuged at 8000 x g for 30 min to remove mineral particles. An aliquot of the supernatant was saved as "dirty" humic acid. The remaining supernatant was further treated by acidifying to pH 2 with HCl for 24 h and centrifuging at 8000 x g for 30 min to precipitate the humic acid. The precipitated humic acid was resuspended in 0.01 M NaOH and the acid–base treatment sequence was repeated twice more. The precipitated humic acid was resuspended in pH 7 water and dialyzed (1000 MWCO) against high purity water containing strong cation-exchange resin (Dowex 50W8–100, H-form, Aldrich) until the external solution TOC concentration was <0.5 mg L–1 C. The dialyzed suspension was designated as "clean" humic acid. The dirty humic acid, cellulose, and lignin were also dialyzed against high purity water until the external solution had TOC < 0.5 mg L–1 C, but no cation exchange resin was added. No other treatments were applied to the cellulose and lignin. Dialyzed organic matter suspensions were stored at 4°C and used within 2 d.

Sorption Experiments
Chelex-100 sorption experiments were prepared by adding 0.36 g (dry wt.) of resin to 40 mL of 10 mM MOPS buffer containing different concentrations of CaCl2 (up to 4 mM) or CuCl2 (up to 0.8 mM). pH was adjusted to 7.6 and tubes were mixed for 1 wk. Oxytetracycline was added from a freshly prepared stock solution to an initial concentration of 0.086 mM (40 mg L–1). Several tubes were used for kinetic monitoring of equilibrium sorption conditions. Two types of controls tubes were included: (i) metal-free controls to account for nonspecific interactions of OTC with Chelex-100 resin, and (ii) resin-free controls to account for nonsorptive compound losses. Resin-free controls were prepared without metals and also with metal-containing supernatant that had been pre-equilibrated with resin for 1 wk. All experiments were conducted at 26°C. The strong buffer capacity of the resin resulted in some initial trials with final pH values of 9 or 10. These data were used to evaluate pH effects.

Organic matter sorption experiments were conducted using the dialysis tube method (Carter and Suffet, 1982). Dialysis tubing (1000 MWCO, Spectra/Por 6, Spectrum Laboratories, Rancho Dominguez, CA) was pretreated according to the manufacturer's instructions by soaking in high purity water for 2 h, then heating to 60°C in 1 mM EDTA–2% NaHCO3 solution for 3 h. This process was repeated a second time and followed by extensive high purity water rinsing. The treated dialysis tubing was stored in high purity water at 4°C until use. Samples were assembled in triplicate by first preparing 200 mL of organic matter suspension (pH 5.5, 10 mM acetate buffer) in brown light-block polyethylene plastic bottles to give final concentrations of 53 mg C L–1 cellulose, 108 mg C L–1 lignin, 19.5 mg C L–1 clean humic acid, or 21 mg C L–1 dirty humic acid. Additional clean humic acid samples were prepared to contain organic matter complexes with individual cations and minimal formation of cation hydrolysis species. Following the method of Masion et al. (2000) and Vilgé-Ritter et al. (1999a), metal salts were added to clean humic acid to give 0.146 or 1.46 mM total Ca, Al (1.46 mM only), or Fe(III). The organic matter suspensions were mixed rapidly (140 rpm) for 3 min followed by slow mixing for 30 min. Formation of only mono-, di-, or trimer metal polymer complexes with organic matter functional groups after following this experimental protocol has been verified with spectroscopic observations in previous studies (Vilgé-Ritter et al., 1999b; Masion et al., 2000). Oxytetracycline was then added to the bottles to give an equivalent initial concentration of 0.084 mM. A 20-mL aliquot of freshly prepared OTC stock solution at pH 5.5 in 10 mM acetate buffer was placed in a knotted length of dialysis tubing and transferred to the bottles containing the organic matter suspensions. The sample bottles were mixed end-over-end gently for 5 d at 26°C, as determined in preliminary equilibration studies. An aliquot of the dialysis bag contents was analyzed for OTC concentration by HPLC. The use of an organic buffer and a chromophore-containing sorbate prohibited TOC or UV-Vis spectroscopy analyses from being used to quantify sorbate transfer across the dialysis bag. Consequently, dialysis pretreatment of the sorbents (1000 MWCO, see Sorbent Preparation) was conducted to minimize the amount of organic matter (<0.5 mg C L–1) that would pass into the bag during the sorption equilibration. No organic matter transfer into the bags was observed during the sorption experiments. Several control tubes were assembled by the same procedure, but contained only pH 5.5 acetate buffer to account for nonsorptive OTC losses. Sorption losses to the polypropylene sample vessels were negligible (Figueroa et al., 2004). An aliquot of the dialysis bag contents was also analyzed for total metal concentrations by ICP–MS. These measurements were assumed to represent the total dissolved metal concentrations. The external organic matter suspension was analyzed for total metal concentrations so that sorbed metal concentrations in the organic matter could be calculated from the difference between the metal concentrations in the external solution and the dialysis bag contents.

Assuming that the dissolved OTC concentration inside the dialysis bag (no organic matter) was equal to the dissolved OTC concentration outside of the bag (with organic matter) at the end of the experiment, sorbed OTC concentrations, Cs (mol kg–1 C) were calculated as follows:

[1]
where Cctl (mol L–1) is the OTC concentration in a sorbent-free control prepared by the same procedure; Cw (mol L–1) is the total aqueous phase concentration of dissolved OTC species inside the bag at the end of the experiment; Vw (L) is the total volume of water in the bottle and dialysis bag, and Ms (kg C) is the mass of organic matter in the bottle. Desorption steps were not performed because the total sorption-plus-desorption time would have resulted in unacceptable (>40%) mass loss of OTC. Sorbed OTC concentrations were used to calculate sorption coefficients as follows:

[2]
where Kd has units of L kg–1 C because our measure of sorbent mass was by total organic C analysis. Note that the Kd values calculated from Eq. [2] are effective OTC distribution coefficients because the total dissolved OTC concentrations were used in all calculations, without accounting for aqueous phase speciation. Thus, Kd values are specific to the total metal, sorbate, and sorbent masses used in these experiments.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS AND DISCUSSION
REFERENCES
 
Sorption to Metal-Chelating Resin
Ternary complex formation by metal-bridging between OTC and sorbent ligand groups was demonstrated using Chelex-100 resin. Experiments were performed at pH 7.6 ± 0.1 to minimize electrostatic attraction between OTC (Fig. 1) and negatively charged resin sites. In the absence of complexing metal cations, OTC concentrations in resin tubes were 6 to 10% lower than concentrations in resin- and metal-free controls, indicating that cation exchange still accounted for a small amount of OTC sorption to the beads at pH 7.6 (Fig. 2a , Na data). The addition of Cu or Ca to the resin resulted in >10% decreases in OTC concentrations, relative to controls, with more OTC sorption at increasing metal/sorbate molar ratios (Fig. 2a). For each metal/sorbate ratio, OTC sorption was greater for the Cu systems than for the Ca systems. Greater OTC sorption to Cu-containing resin than to Ca-containing resin is consistent with a metal-bridging mechanism of sorption: Cu forms stronger complexes both with the resin sites and with OTC than does Ca. Distribution coefficients for Cu and Ca with Chelex-100 resin have been reported to be 105 L kg–1 and 103 L kg–1 (Leyden and Underwood, 1964), respectively, above pH 6 for systems with metal/resin molar ratios similar to our system. Stability constants for 1:1 complexes of Cu and Ca with OTC are 1012.4 (Jezowska-Bojczuk et al., 1993) and 104.5 (Lambs et al., 1988), respectively. Consequently, the trends of OTC sorption with Chelex-100 suggest that OTC sorption to ligand-rich sorbents will occur in systems that have metal cations complexed to sorbent ligand sites.



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  		Fig. 2. Sorption of oxytetracycline (OTC) to Chelex-100 resin with presorbed Cu, Ca, or Na cations: (a) at pH 7.6, and (b) variation with pH where numbers denote metal/OTC ratio. Note that metal/sorbate ratios were computed from the initial masses added to the tubes.

 
pH trends in OTC sorption to Chelex-100 resin were also consistent with ternary complex formation between OTC, metal ions, and resin sites, rather than OTC interactions by cation exchange (Fig. 2b). No significant difference in OTC concentration was observed between Na-resin tubes and resin-free controls at pH 9, indicating that cation exchange was not an important contributor to OTC sorption at high pH (Fig. 1). Oxytetracycline mass sorbed to Cu- and Ca-containing resin increased when the pH was raised. At pH 10.5, the mass fraction of OTC sorbed at a Cu/sorbate ratio of 1 was 45% and at a ratio of 3 was 77%, compared with 22 and 38%, respectively, at pH 7.6. Similar increases in OTC sorption were observed for Ca-containing resin at pH 9 (Fig. 2b). Such trends of increased sorption with pH would not be observed for cation exchange interactions of OTC with the Chelex-100 resin, especially at pH values >9.4 (Tavares and McGuffin, 1994) when the majority of OTC molecules are deprotonated at the dimethyl amine group (Fig. 1). Increased metal complexation, and hence ternary complex formation, is expected at high pH because of decreased competition from H+ both for OTC acid groups and for resin sites. In contrast, at the lower pH conditions of 5 to 8 that are typical of environmental systems, ternary complex formation between OTC sorbate and sorbent ligand groups would be attenuated by competition from protons, resulting in a decrease of sorption by metal bridging to some extent.

Sorption to Organic Matter
Oxytetracycline sorption was measured for a variety of organic matter types characterized by differing abilities to complex metals and different concentrations of metals complexed to organic matter ligand groups. Although ternary complex formation between OTC, metal cations, and organic matter ligand groups is expected to be greatest around pH 8 (lowest competition from protons or hydroxide ions), these experiments were conducted at pH 5.5, which is more representative of typical soil or sediment conditions and where the dominant aqueous phase species was the zwitterion (Fig. 1). Previous experiments with clays have shown that cation exchange interactions may still be significant at pH 5.5, despite the low abundance of cation species (Figueroa et al., 2004). The use of pH 5.5 enabled these observations to complement the expanding study of OTC interactions with other sorbents that have been conducted at pH 5.5 (Figueroa et al., 2004; Figueroa and MacKay, 2005; Jones et al., 2005).

The lowest OTC sorption coefficients for organic matter samples were measured for those sorbents with the poorest abilities to complex metals (Table 1). Cellulose structure has repeating cellobiose saccharide units with functional groups that are poor ligands, and thus little sorption to this sorbent would be expected if metal-bridging were an important interaction mechanism. Sorbed metal concentrations were below detection limits for this sorbent (Table 1). There was no significant difference (95% CI) between the dissolved OTC concentrations in cellulose-containing samples (64 µM) and sorbent-free controls (64 µM, Table 1, footnote {dagger}); therefore, no sorption coefficient Kd was calculated for cellulose. Lignin also has a poor ability to complex metals due to the lack of good chelating groups on the cross-linked phenylpropanoid monomers that make up this polymer. Since lignin could be chemically altered to contain metal-complexing thiol groups during the Kraft extraction process (Lin and Lebo, 1996), solvent-extracted "organo-solv" lignin was chosen for this study. Concentrations of metals sorbed to lignin were below the detection limits at the end of the equilibration time. No significant difference (95% CI) in dissolved OTC concentrations was observed between the lignin-containing samples and sorbent-free controls (Table 1). Note that a Kocest of 0.4 L kg–1 C was estimated for OTC at pH 5.5 (98% as neutral zwitterion) using the relationship log Kocest = 0.84 log Kow + 0.41 (Karickhoff, 1981) with a log octanol–water distribution coefficient of –0.96 previously measured at this pH by the shake-flask method (Krach, 2002). Actual hydrophobic partitioning of OTC to cellulose is likely somewhat less given that cellulose is a poor sorbent phase, even for nonpolar sorbate compounds (Rutherford et al., 1992; Xing et al., 1994). Possible interactions of OTC with cellulose or lignin by hydrophobic partitioning were below the levels of quantitation at the sorbent concentrations used (53 mg C L–1 for cellulose, 108 mg C L–1 for lignin).


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  		Table 1. Oxytetracycline sorption coefficients and measured metal concentrations for systems with different organic matter types.

 
Detectable OTC sorption was observed for humic acid (19.5–21 mg C L–1), a sorbent with functional groups that are known to complex metals (Table 2). Although humic acid chemical structures are less well-defined than for cellulose and lignin, it is generally believed that a distribution of metal-complexing phenol and carboxylic acid ligand groups are present at an abundance of about 1 functional group per 10 C atoms (Morel and Hering, 1993). There is debate in the literature as to whether or not this model is appropriate for Aldrich humic acid, since complexation constants and Koc values for this sorbent differ widely from those of organic matter isolated from well-classified soils or aquatic sources. Aldrich humic acid was used for this study instead of organic matter from a well-classified source because several-gram quantities of humic acid were required and the Aldrich material was available in semi-purified form to demonstrate the concept of OTC sorption by metal-bridging without labor intensive extraction steps. Additional treatments, including with strong cation-exchange resin, gave clean humic acid that had significantly reduced, but still measurable, metal concentrations of Al and Fe (Table 1). Clean humic acid sorbed between 8 and 10% of the added OTC mass giving a Kd of 5500 ± 600 L kg–1 C. The OTC sorption coefficient for clean humic acid was significantly less than the Kd of 32000 ± 190 L kg–1 C measured for dirty humic acid that had no acidification or cation-exchange resin treatments. Clearly, manipulating the metal content in the humic acid had large effects on the sorption of OTC; thus, OTC sorption to organic matter was consistent with a metal-bridging mechanism of interaction.


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  		Table 2. Logarithms of the formation constants for the 1:1 metal–ligand complexes with organic ligand analogues of humic acid functional groups and oxytetracycline (25°C, zero ionic strength).

 
Sorption to Metal-Amended Humic Acid
Clean humic acid was amended with common soil cations (Ca, Al, or Fe) to investigate how OTC sorption to organic matter would be affected by metals with differing organic ligand formation constants. By analogy to the Chelex-100 resin, Al and Fe(III) exhibit much greater formation constants with organic matter ligand analogs, than does Ca (Table 2), and thus might be expected to control OTC sorption to humic acid through ternary complex formation. Aluminum and Fe(III) formation constants have not been reported for OTC; however, the strong complexes formed between OTC and these cations have been exploited in analytical methods (Monastero et al., 1951; McCracken et al., 1995). Aluminum and Fe(III) formation constants for anhydrotetracycline reported for 1:1 metal/ligand complexes with various protonated ligand species suggest several orders of magnitude stronger interactions between Al and Fe(III) than between Ca and Mg (Machado et al., 1995; Schneider, 2001). Thus, it was expected that OTC would also have a more favorable interaction in ternary complexes with Al and Fe(III) than with Ca.

Low and high metal amendments to clean humic acid were tested using initial metal salt concentrations of 0.146 and 1.46 mM, respectively. These concentrations were chosen to maintain the organic matter/metal ratio that was employed by Masion et al. (2000) and Vilgé-Ritter et al. (1999a) in their careful study of Al and Fe complex formation with organic matter. These researchers demonstrated that the experimental protocol yielded mono-, di-, and trimer metal polymer complexes with organic matter functional groups. We assume that by following the same protocol, similar complexes between the added metal cations and organic matter functional groups in our systems were achieved. Iron- and Al-amended clean humic acid suspensions were slightly cloudy compared with unamended clean humic acid; however, no flocculation was observed during our experiments. Possible analytical interferences from humic acid colloids were minimized by the dialysis pretreatment of clean humic acid that removed the <1000 MW fraction before metal amendment.

Several checks were conducted to evaluate whether dissolved metal–OTC complexes in any of our test systems could cause overestimation of the fraction of compound sorbed to metal-amended organic matter. Early eluting complexes of OTC with metals could not be quantified reliably by HPLC using external standards. Furthermore, co-elution of metal complexes with uncomplexed OTC could introduce quantification errors by causing shifts in the peak spectra to lower absorbances at the wavelengths monitored for uncomplexed OTC. Thus, peak area response factors in solutions with OTC and Ca (up to 1 M) were compared with those of OTC in Ca-free solutions and showed <5% variation, indicating that our HPLC method yielded accurate measures of total dissolved OTC in systems with Ca. Such agreement between metal-containing and metal-free systems was not observed for the Al and Fe systems and so a UV-Vis technique was used to quantify the total dissolved OTC (Fig. 3) . In these cases, sorbent-free standards were prepared at the same total dissolved metal concentration as measured in the dialysis bag contents, but containing differing amounts of OTC up to the initial sorbate concentration of 84 µM. The absorbance spectra of these Al- or Fe-containing standards were used to develop OTC-metal complex response factors for each test system. Figure 3b shows example standards spectra for the Al case (solid lines) with the characteristic shift of absorbances to longer wavelengths (Machado et al., 1995), relative to the no-Al case (dotted line). The spectra of OTC in the metal-containing controls showed no shifts when normalized to the total OTC concentration, indicating that the same OTC metal complex(es) was dominant over the full range of OTC concentrations. Total OTC concentrations in the samples from the sorbent-containing tubes were then obtained from adsorption measurements (e.g., Fig. 3a) using the system-specific response factors (e.g., solid lines in Fig. 3b). Iron-containing controls showed the expected shift in absorbance to shorter wavelengths (Machado et al., 1995) from 365 to 354 nm and were used to calculate dissolved concentrations of OTC in dialysis bags of the high Fe samples. The solution phase spectra for the low Fe case showed a shift to 317 nm such that sample spectra did not match that of Fe-containing controls; therefore, Kd was not calculated. Aluminum was omitted from the low concentration tests because of limited quantities of clean humic acid prepared from the same batch.



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  		Fig. 3. Schematic of oxytetracycline (OTC) concentration measurements for metal-amended humic acid at pH 5.2: (a) sample spectrum of bag contents with dissolved OTC and 80 µM dissolved Al, and (b) comparison spectra of OTC standards prepared with 80 µM total Al. The dotted line in (b) shows the OTC spectrum in the absence of Al.

 
The Ca amendments of the clean humic acid suggested that a portion of the sorbed OTC was interacting with the humic acid by cation exchange at the pH 5.5 used in the organic matter studies. The factors of sorbed Ca concentration and pH must both be considered to evaluate the significance of OTC cation interactions with organic matter. For cation exchange, lower sorbed OTC concentrations would be expected for organic matter samples with higher sorbed Ca concentrations, while lower pH favors high sorption by OTC. Therefore, according to the sorbed Ca concentration (Table 1), sorption coefficients for OTC should follow the order clean > "low Ca2+" > "high Ca2+" humic acid. High Ca2+ humic acid had the lowest OTC sorption coefficient by a factor of about 2; however, low Ca2+ humic acid and the clean humic systems had similar sorption coefficients (Table 1). The pH of the low Ca2+ humic acid system was lower than clean humic acid, which may have offset some of the competition between Ca2+ and OTC resulting in coincidently similar sorption coefficients. At pH 4.9 (low Ca2+ system) and pH 5.4 (clean) approximately 4.8 and 1.5%, respectively, of the dissolved OTC exists as a cation. Note that solution phase complexes between OTC and 1060 µM Ca2+ in the case of high Ca2+ humic acid were insignificant at pH 5.5 (Table 2).

Although sorption observations for the low Ca2+ vs. high Ca2+ cases were consistent with a cation exchange interaction of OTC with humic acid, an additional interaction was required to resolve the dirty humic acid with the clean and low Ca2+ systems. The dirty humic acid sorption coefficient was six times greater than that of the clean humic acid, but the dirty humic acid had 2 orders of magnitude greater Ca concentration (plus other sorbed cations) (Table 1). The latter would have resulted in lower sorption coefficients in the dirty humic acid system, if cation exchange was the only sorption mechanism. Similarly, the dirty humic acid had a six times greater Kd than that of the low Ca2+ humic acid, even though the system pH was greater for the dirty humic acid, which is also inconsistent with a cation exchange mechanism. However, the trends in dirty vs. clean or low Ca2+ humic acid are consistent with the higher sorbed Al and Fe concentrations of the dirty humic acid; Al and Fe can enhance sorption by serving as metal bridges.

Addition of Al or Fe(III) salts to clean humic acid to increase the bound concentration of these potential bridging cations increased OTC sorption to this sorbent. Amendment of the clean humic acid with 1.46 mM of Al resulted in an increase in sorbed Al concentration by a factor of 2000 compared with a factor of 6 increase in sorbed OTC concentration, or a factor of 9 increase in Kd over the unamended clean humic acid case (Table 1). Amendment with Fe(III) increased sorbed Fe concentrations by a factor of 530 while the sorbed OTC concentration was 10 times greater and Kd was 45 times greater than for unamended clean humic acid. Such increases in sorbed OTC were not consistent with alterations of the hydrophobic nature of the organic matter by cation cross-linking since Al-saturation of humic acid actually decreased C-normalized nonpolar organic compound sorption, but only by 30% (Lu and Pignatello, 2004). The trends in OTC sorption to the Al- or Fe(III)-amended humic acid were consistent, however, with observations for the dirty humic acid. Log-log plots of OTC Kd vs. total metal concentration sorbed to the humic acid sample showed good correlation between the total sorbed Al-plus-Fe(III) concentrations (r2 = 0.87) (Fig. 4) , whereas inclusion of Ca and Mg in the calculation of total sorbed metal gave a much poorer correlation between log Kd and log sorbed metal concentration (r2 = 0.39). Similar trends are observed of the log distribution coefficient of complexed/dissolved ligand varying linearly with log total metal concentration for aqueous systems with varied metal/ligand ratios. The correlation between OTC log Kd and log of total sorbed Al-plus-Fe(III) for all humic acid systems suggests that manipulation of the metal content in humic acid by the addition of cations with high stability constants (Al and Fe vs. Ca) increases the extent of OTC sorption to natural organic matter by complexation at typical soil pH values (e.g., 5.5). These findings, when considered together with the tendency for OTC to sorb to metal-loaded cation exchange resin, indicate that an important mechanism for OTC interaction with natural organic matter is by ternary complex formation with tightly bound polyvalent metal ions.



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  		Fig. 4. Correlation between Kd and sorbed metal concentration for all humic acid sorbents. Solid dots are for total sorbed Al plus Fe concentrations (r2 = 0.87). Open triangles are for total sorbed Ca plus Mg plus Al and Fe concentrations (r2 = 0.39). Note that the high concentration points (105 mmol kg–1 C) are coincident for both data sets because sorbed Ca and Mg concentrations were negligible for these cases (high Al, high Fe; Table 1).

 
Environmental Significance
The results of our research demonstrate that organic matter phases in soils and sediments may be important sorbents for pharmaceuticals with metal-complexation chemistry. More research is required to quantify thermodynamic constants (K) for ternary organic matter–metal–sorbate complexes so that accurate a priori predictions of pharmaceutical distributions between sorbent and aqueous phases can be made. The introduction of polyvalent metal cations to soil or sediment pore waters could have two possible impacts on pharmaceutical sorption: If the ternary complex is more stable than dissolved metal–pharmaceutical complexes, sorption of the pharmaceutical compound would result, thereby limiting the compound mobility and bioavailability. Our results suggest that that this would be the case for polyvalent cations, such as Al and Fe(III), that are strongly complexed by both organic matter and sorbate ligand groups. Competition between sorbed and dissolved complexes with the pharmaceutical compound could occur for high concentrations of pore water cations, such as Ca, that are only weakly held by organic matter ligand groups. The exact distribution of pharmaceutical compounds between ternary- and dissolved complexes in each case will depend on the total concentrations of organic matter, sorbent ligand groups, and sorbate compound.


   ACKNOWLEDGMENTS
 
Funding for this research was provided by the National Science Foundation (BES-0225696). Wojciech Krach and Allison Leonard are thanked for their efforts in developing experimental methods for this study. We thank Dr. Linda Lee, Dr. Britt Holmén, and three anonymous reviewers for their thoughtful comments and discussion of this manuscript.


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C. Gu and K. G. Karthikeyan
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