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Jul 3, 2014 - ABSTRACT: As copper (Cu) stable isotopes emerge as a tool for tracing Cu biogeochemical cycling, an understanding of how. Cu isotopes ...
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Copper Isotope Fractionation during Equilibration with Natural and Synthetic Ligands Brooke M. Ryan,*,† Jason K. Kirby,‡ Fien Degryse,† Kathleen Scheiderich,‡ and Mike J. McLaughlin†,‡ †

Soil Sciences, University of Adelaide, Adelaide, SA 5064, Australia CSIRO Land and Water, Contaminant Chemistry and Ecotoxicology Program, Waite Campus, Adelaide, SA 5064, Australia

‡

S Supporting Information *

ABSTRACT: As copper (Cu) stable isotopes emerge as a tool for tracing Cu biogeochemical cycling, an understanding of how Cu isotopes fractionate during complexation with soluble organic ligands in natural waters and soil solutions is required. A Donnan dialysis technique was employed to assess the isotopic fractionation of Cu during complexation with the soluble synthetic ligands ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA) and desferrioxamine B (DFOB), as well as with Suwannee River fulvic acid (SRFA). The results indicated enrichment of the heavy isotope (65Cu) in the complexes, with Δ65Cucomplex‑free values ranging from +0.14 to +0.84‰. A strong linear correlation was found between the logarithms of the stability constants of the Cu complexes and the magnitudes of isotopic fractionation. These results show that complexation of Cu by organic ligands can affect the isotopic signature of the free Cu ion. This free Cu is considered the most bioavailable species, and hence, our results highlight the importance of understanding fractionation processes in the uptake medium when using Cu isotopes to study the uptake mechanisms of organisms. These data contribute a vital piece to the emerging picture of Cu isotope cycling in the natural environment, as organic complexation plays a key role in the Cu cycle.

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−1‰ (Δ65Cuplant‑growth medium),7−9 while Navarrete et al.10 noted a bacterial consortium favored light isotope absorption by up to −4.4‰. Redox cycling of Cu induces the most significant fractionations, with reduced Cu minerals enriched in light Cu by up to −4‰, and oxidized Cu minerals enriched in heavy Cu up to +5.3‰.4,11,12 Organic matter (OM) is a key variable controlling Cu speciation in both the terrestrial and aquatic environments, due to the strong inner sphere complexes formed by Cu with OM functional groups, even at low pH.13,14 Little research has been done to examine the isotopic fractionation of Cu during OM complexation, either dissolved (DOM) or solid-phase OM.15 Given the importance of Cu−OM complexes in the environment, an understanding of how OM complexation fractionates

INTRODUCTION Copper (Cu) is an element of key environmental importance as it is an essential micronutrient for plants and many microorganisms. At elevated concentrations Cu can cause toxicity with impacts on the growth, reproduction and survival of aquatic and terrestrial organisms.1,2 The biogeochemical cycle of Cu in aquatic and terrestrial environments can be influenced by numerous biological (e.g., root rhizosphere) and physicochemical (e.g., redox, pH) properties.2,3 There is an increasing need for the development of new analytical tools that can be used to provide further insights into the biogeochemical cycles of elements such as Cu in complex environmental systems. Copper stable isotope analysis has the potential to provide significant information on the biogeochemical cycling of Cu in the environment.4−6 Recent research has examined the effect of mineral adsorption, redox reactions and plant uptake on the isotopic signature of Cu. Plant uptake has been shown to fractionate Cu, preferentially incorporating light Cu by up to ca. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8620

February 13, 2014 July 1, 2014 July 3, 2014 July 3, 2014 dx.doi.org/10.1021/es500764x | Environ. Sci. Technol. 2014, 48, 8620−8626

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solution via a negatively charged membrane over which only cations can cross. We assessed the effects of pH on Cu binding to natural DOM, in the form of Suwannee River fulvic acid (SRFA), as well as the effect of binding strength on fractionation factor in synthetic DOM complexes to gain a better understanding of Cu isotope fractionation in natural systems.

Cu isotopes is necessary if stable isotopes are to be used to track the sources and fates of Cu in the environment. Under equilibrium conditions, heavier isotopes are known to preferentially partition to the strongest bonding environment, as the heavier isotopes form complexes with the lowest vibrational energies.16,17 For iron (Fe), it has been shown that Δ56Fecomplex‑solution values increase with increasing binding strength of soluble organic complexes under equilibrium conditions.18 Fractionation of Zn isotopes during complexation with dissolved humic acid (HA) has been shown to be pH dependent.19 No fractionation was observed at pH ≤6, while at pH >6, an enrichment of +0.24‰ (Δ66Zncomplex‑free) was found, and this was attributed to more binding with high affinity sites on the HA at high pH.19 To date, studies quantifying Cu isotope fractionation with OM have been limited to adsorption onto microorganisms and insolubilized OM, and these have yielded mixed results. Adsorption of Cu to various microorganisms at circumneutral pH was found to induce insignificant fractionation of Cu isotopes (Δ65Cu solid‑solution = 0.0 ± 0.4‰), while at acidic pH a large enrichment in the light 63Cu isotope on the cell surface was observed (Δ65Cusolid‑solution = −1.2‰).20 The large observed fractionation at low pH was attributed to a weaker bonding environment for Cu in outer-sphere complexes on the membranes compared to stronger bonds in octahedral aqueous species. In contrast, a study of Cu complexation to insolubilized HA showed a small but significant enrichment in the 65Cu isotope that was independent of pH (Δ65Cusolid‑solution = +0.26‰).15 Copper can be complexed to a large extent by DOM in soil solutions,13 hence DOM is likely to play a key role in determining the fate and bioavailability of Cu in soil environments. The free Cu ion is considered to be the most bioavailable species for uptake by biota,21 hence, an understanding of Cu partitioning between free and Cu−DOM complexes and the resulting isotope fractionations will be important. The isotopic signature of the total soluble Cu may not accurately reflect that of the bioavailable pool being accessed directly by plants. In hydroponic studies where organic complexation with root exudates was minimized, Strategy I plants (dicotyledons and nongraminaceous monocotyledons) were shown to significantly fractionate Cu during uptake (Δ65Curoot‑solution = ca. −1 ‰), likely because of reductive uptake of Cu.7 However, strategy II (graminaceous monocotyledons) plants showed minimal Cu isotopic fractionation (Δ65Curoot‑solution = ca. 0.1 ‰).7 Strategy II plants are known to excrete complexing ligands (phytosiderophores) into soil solutions, to assist with the mobilization of insoluble Fe nutrients and these ligands may also be involved in Cu uptake. This phytosiderophore complexation may possibly lead to fractionation of Cu isotopes in the soil solution. The isotopic fractionation process for Cu−OM binding will not only affect the isotopic signature of bioavailable Cu in the uptake medium, but may also affect the isotope distribution within the plant.7 For instance, it has been suggested that Cu complexation to nicotianamine (NA) may explain the significant isotope fractionation observed during translocation (Δ65Cushoot‑rootca. +0.8‰) in strategy I plants.7 In this study, we examined the isotope fractionation of Cu during equilibrium complexation with synthetic and natural organic ligands. A Donnan dialysis technique was used to separate free Cu from soluble complexes. During Donnan dialysis, a donor solution is equilibrated with an acceptor

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MATERIALS AND METHODS Chemicals and Reagents. All reagents used in this study were prepared in ultrapure deionized water (Milli-Q, Millipore), and the nitric (HNO3) and hydrochloric (HCl) acids used were distilled in DST-1000 acid purification systems (Savillex). Suwannee river fulvic acid (IHSS) with 53% carbon was used as a natural DOM ligand, and all synthetic ligands were of analytical grade purity. Strontium nitrate (Sigma) was of trace analysis quality, and the Cu stock used for all experiments was from our in-house Cu wire isotopic standard. Free Cu Separation Using Donnan Dialysis. The Donnan dialysis procedure used in this study was a modification of that previously published by Nolan et al.22 to determine free divalent cationic metal (Cd, Cu, Pb, and Zn) concentrations in pore waters of agricultural and contaminated soils. Donnan dialysis was performed using custom-designed Teflon cells that contained a strong-acid cation-exchange membrane (Nafion-117, E.I. Dupont de Nemours). The membranes separated a donor solution (30−40 mL) containing a mixture of free Cu(II) and complexed Cu(II) from an initially pure solution of Sr(NO3)2 (acceptor, 300 μL). The acceptor volume was a small percentage (1%) of the donor solution volume so it would not alter the equilibrium composition of the donor solution. The acceptor solution rests on the top surface of the membrane that is contained in the top part of the exchange cell. The sample solution was continuously circulated past the bottom of the membrane by a Teflon pump at a rate of 100 mL min−1 for 3 h. The pH of the donor solutions was determined at the start and end of the equilibrations. The ionic strengths of the acceptor solutions were matched to the donor solutions using Sr(NO3)2 as a background electrolyte.23 The ionic strengths of the donor solutions for samples containing synthetic ligands and natural DOM were calculated from the measured electrical conductivities using the procedure outlined by Nolan et al.22 The Donnan membranes were prepared by washing in high purity deionized water, followed by soaking overnight in 2% HNO3 to remove any contaminant cations from the membrane surface. Membranes were then thoroughly rinsed in ultrapure deionized water, and soaked for 3 h in 50 mL of 10 mM Sr(NO3)2. Following this the membranes were twice soaked for 2 h in 1 mM Sr(NO3)2 solutions, and finally soaked in the acceptor solution matched in ionic strength to that of the donor solution to be analyzed. The Donnan cells, pumps and tubing were acid washed between samples with 2% HNO3 to prevent cross-contamination and thoroughly rinsed with ultrapure deionized water to prevent pH change in the donor solutions during the dialysis. All donor solutions contained Cu(II), from an in-house Cu wire isotopic standard, at a concentration of ca. 2 mg L−1, and 5 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer adjusted to pH 5 using 1 M sodium hydroxide (NaOH). The MES buffer was selected because of its negligible complexation with divalent cationic metals such as Cu.24 All donor solutions containing synthetic ligands contained 1 mM Sr(NO3)2 as a 8621

dx.doi.org/10.1021/es500764x | Environ. Sci. Technol. 2014, 48, 8620−8626

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resolution mode using wet plasma conditions with a 100 μL min−1 PFA nebulizer and Ni sampler and skimmer cones. NIST 986 Ni standard (62Ni/60Ni = 0.138600) was used for instrumental mass bias correction of the measured 65Cu/63Cu isotope ratios, using an external normalization method, coupled with sample-standard bracketing using an in-house Cu wire standard.15,27The instrument’s accuracy during each analytical session was confirmed by measuring this Cu wire standard bracketed by NIST 976 Cu at the beginning and end of each analytical session, and confirming the known δ65Cu of 0.45 ± 0.04‰ (mean ±2sd, n = 50, measured over 24 months) for the Cu wire standard. If the measured δ65Cu value for the Cu wire standard deviated from this value of 0.45‰ during these quality control checks by more than 0.02‰, the analytical run was repeated. This Cu-wire standard was used for samplestandard bracketing, with delta values reported relative to NIST 976 Cu, according to eq 1, taking into account the difference of 0.45‰ between the Cu wire standard and the NIST 976 standard:

background electrolyte. A Cu treatment without ligands (i.e., 100% free Cu at 2 mg L−1) (n = 3) was also examined in the Donnan system to ensure the system reached equilibrium within 3 h. This test also assessed whether dialysis induced any artificial Cu fractionation (e.g., due to binding of free Cu on the strong-acid cation-exchange membrane). Copper fractionation was examined using the synthetic ligand ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA) and desferrioxamine B (DFOB), as well as natural SRFA. Donor solutions containing synthetic ligands were made at an approximate 2:1 Cu:ligand molar ratio (ca. 30 μM:15 μM) in order to achieve donor solutions that theoretically contained ca. 50% free Cu. The actual free Cu concentration was determined from the measured acceptor solution concentration and this measured value was used in the final mass balance calculations. The SRFA donor solutions contained 5 mg L−1 SRFA and 2 mg L−1 Cu, as at pH 5 this yielded a suitable percentage of complexed Cu (ca. 39%, as determined by the NICA-Donnan model in Visual Minteq25 calculations). The effect of pH on natural DOM binding was assessed in donor solutions of SRFA adjusted to pH 3 or pH 5 with 1 M NaOH. A minimum of three experimental replicates per ligand were run through the dialysis system. The acceptor solutions were recovered from the cell into PTFE vials, dried down and resuspended in 1 mL of 10 M HCl for Cu purification. The small volume of the acceptor solution meant that each replicate could only be analyzed for isotope ratios once. The free Cu(II) concentrations (used in the mass balance calculations) were measured by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7700) after diluting 0.1 mL of the 1 mL resuspended acceptor solution up to 4 mL using 2% HNO3. Copper Purification. Copper was purified from 0.9 mL of resuspended acceptor solution using AG-MP-1 anion exchange resin (BioRad), following the procedure described by Borrok et al.26 However, a smaller volume of 10 M HCl was used for matrix flushing due to lower concentrations of matrix ions in our experimental samples. Columns were conditioned with 5 mL of 10 M HCl, samples were loaded in 1 mL of 10 M HCl, matrix elements were rinsed through with 3 mL of 10 M HCl and Cu was eluted in 8 mL of 5 M HCl. The Cu fraction from the columns was collected in PTFE vials and evaporated to dryness on a hot plate. Samples were redissolved in 2% HNO3, and 0.1 mL aliquots were taken and diluted to 4 mL for total Cu analysis using ICP-MS to determine Cu recoveries from the column purification procedure. Our previous work with standard reference materials (NIST SRM 1573a tomato leaves) showed no significant difference in δ 65 Cu values for experimental replicates with column recoveries between 100 ± 10%.7 Also in this study, experimental replicates that were purified separately that had varied column recoveries within 100 ± 10% did not differ in isotopic composition outside of the analytical uncertainty. Samples that failed to meet the target recovery (100 ± 10%) were not analyzed further. Isotope Analysis. Copper isotope ratios (65Cu/63Cu) of purified acceptor solutions were determined using a multicollector-inductively coupled plasma-mass spectrometer (MCICP-MS) (Neptune, Thermo Scientific). Copper isotope ratios were measured by methods previously outlined by Ryan et al.7 Briefly, the samples and standards were measured at concentrations between 150−300 μg Cu L−1 in 2% HNO3 with NIST 986Ni spiked into samples at 1:3(v/v) Cu:Ni ratio for mass bias correction. Measurements were made in low

⎛ ⎡ (65Cu/63 Cu) ⎤⎞ sample ⎥⎟ + 0.45 1 δ 65Cu = ⎜⎜1000·⎢ 65 − 63 ⎢ ⎥⎦⎟⎠ ( Cu/ Cu) ⎣ Cuwire ⎝

(1)

The reliability of the mass bias correction using the external normalization method was confirmed by checking for agreement between these values and those obtained by standardsample bracketing only, as well as the modified sample-standard bracketing technique reported by Peel et al.27 If the various correction methods varied by more than 0.16‰, the analytical uncertainty of the method,7 the analytical session was repeated. Copper Mass Balance. The free Cu in the donor solution equilibrates with the acceptor solution, whereas complexes do not pass the membrane. Hence, if the system has fully equilibrated, the δ65Cu of complexed Cu in the donor solution can be calculated from the following mass balance equation: δ 65Cucomplexed =

δ 65Cu total − (δ 65Cu free × Ffree) (1 − Ffree)

(2)

where δ Cucomplexed, δ Cutotal and δ Cufree are the delta values of the complexed Cu, total Cu (δ65Cu = 0.45‰) and free Cu (δ65Cu in the acceptor solution), respectively, and Ffree is the fraction of free Cu (corresponding to the ratio of acceptor to donor concentration). The Δ65Cucomplex‑free can then be calculated using the following equation: 65

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65

65

Δ65Cucomplexed − free = δ 65Cucomplexed − δ 65Cu free

(3)

RESULTS AND DISCUSSION Donnan dialysis of a solution with only free Cu showed that the dialysis system reached equilibrium, both with respect to the concentration and the isotopic signature, within 3 h (Table 1). Time series data using solutions with 100% or 50% free Cu Table 1. Equilibrium Cu Concentrations and δ65Cu values after Donnan Dialysis of a Donor Solution Containing 100% Free Cu (Mean ±2 SD, n = 3)

donor acceptor 8622

Cu (μg L−1)

δ65Cu (‰)

1809 ± 230 1785 ± 102

0.45 ± 0.04‰ 0.46 ± 0.10‰

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Table 2. Summary of Donnan Dialysis Measurements to Assess Cu Isotope Fractionation Induced by Complexation with Organic Ligands: Stability Constant (Log K) of the Dominant Complex, Measured % Free Cu (±SD), Measured δ65Cu of the Free Ion (In the Acceptor Solution), Calculated δ65Cu of the Complex (eq 2), the Calculated Fractionation Factor Δ65Cucomplex‑free (±2SD), and the Number of Experimental Replicatesa ligand b

DFOB CDTAb EDTAb NTAb Suwannee River FAc

log K

% free

SD

δ65Cufree

δ65Cucomplex (calculated)

24.7 23 20.5 14.4 8

53 49 51 41 18

5 15 28 16 5

0.05 0.14 0.24 0.24 0.33

0.89 0.76 0.76 0.69 0.48

Δ65Cu(complexed‑free) ± 2sdd

no. replicates

± ± ± ± ±

4 3 4 3 4

0.84 0.62 0.51 0.44 0.14

0.30a 0.33ab 0.32b 0.40b 0.11c

All values presented as averages of experimental replicates. No 2σ values are reported for the δ65Cu Free and δ65Cu Complex values as the δ65Cu values varied with % free Cu, and hence, 2σ values here give no information on the isotope measurement reproducibility. The fractionation factor was calculated from the free ion fraction and δ65Cufree of each individual replicate. bLog K stability constants taken from GEOCHEM31 cLog K stability constants taken from Hirose et al.32 and Vance et al.6 dDifferent letters indicate significant (p < 0.05) differences in Δ65Cucomplex-free values (one-way ANOVA, Tukey’s posthoc test) a

CHEM.32 The dominant complex was the 1:1 metal:ligand for all ligands (M+L → ML), except for DFOB for which the 1:1:1 (metal:ligand:H) is the dominant complex, i.e., the complex with the monoprotonated ligand. The log K values for SRFA were taken from Hirose et al.33 and Coale and Bruland.40 The ligand strength of binding with Cu is dictated by the number, type and length of bonds. DFOB binds with Cu through O atoms only, while CDTA and EDTA bind through a combination of O and N atoms.29−31 A notable decrease in stability constant is observed going from the Cu−EDTA to Cu−NTA complex, as NTA typically forms a tetradentate complex,31,34 while EDTA and CDTA form hexadentate complexes. More information will be required to validate the relationship between bond strength and isotope fractionation, however, these data provide evidence that it may be possible to predict isotope fractionation between free and complexed Cu if stability constants are known. The complex of Cu with SRFA showed a small enrichment in the heavier 65Cu isotope (Δ65Cucomplex‑free = +0.14 ± 0.11‰) (mean ±2sd) with no effect of pH on isotope fractionation (Table 3). When the pH was decreased from 5 to 3, the free Cu

showed that the concentration equilibrium was achieved after about 1 h (Supporting Information Figure S1). This fast equilibrium time is consistent with previous work by Fitch and Helmke23 using a similar Donnan dialysis system. All complexes of Cu with synthetic and natural ligands showed a significant enrichment in the 65Cu isotope, relative to the free Cu pool, as evident from the lower δ65Cu value of the acceptor solutions (free Cu) compared with the initial total Cu δ65Cu value of 0.45‰ (Table 2). The calculated fractionation factor (Δ65Cucomplex‑free) between complexed and free Cu ranged from +0.14 to +0.84‰ (Table 2). This positive fractionation factor for organically complexed Cu relative to free Cu2+ (i.e., the hexaaqua copper complex) agrees with expectations, since, in general, the species with the stronger bond is enriched with the heavy isotope under equilibrium conditions.28 There was a strong positive correlation observed between stability constants (log K) and Δ65Cucomplex‑free for all ligands examined (Figure 1). This relationship between bond strength

Table 3. Suwannee River Fulvic Acid Fractionation at pH 3 and pH 5a pH 4.8 ± 0.16 3.0 ± 0.21

n

% free Cu

δ65Cufree

δ65Cucomplex

Δ65Cu(complex−free)

4 3

18 ± 10 41 ± 6b

0.33 0.37

0.48 0.50

0.15 ± 0.11ns 0.13 ± 0.14ns

b

a Percentage of free Cu and δ65Cufree measured from the acceptor solution and the δ65Cucomplex from eq 2. (mean ±2SD). NB: No 2σ values are reported for the δ65Cu Free and δ65Cu Complex values as the δ65Cu values varied with % free Cu, and hence, 2σ values here gives no information on the isotope measurement reproducibility. The fractionation factor was calculated from the free ion fraction and δ65Cufree of each individual replicate. bdenotes significant differences at the 0.05 significance level. ns = no significant difference between pH values.

Figure 1. Relationship between the binding constant K (as log K) and the isotopic fractionation factor between complexed and free (aqueous) Cu (Δ65Cucomplex‑free).

concentration in solutions increased, on average, from 19% to 41%, with no significant change in Cu isotope fractionation (p = 0.60) (Table 3). This finding supports work previously published by Bigalke et al.15 who found no significant effect of pH on Cu isotope fractionation during Cu binding to insolubilized humic acid. However, Zn has been found to have pH dependent isotope fractionation during humic acid binding, with no fractionation observed at pH < 6 and a heavy enrichment at pH > 6 (Δ66Zncomplexed‑free = +0.24‰), as

and Δ65Cucomplex‑free was highly significant (p = 10−7) (Supporting Information Table S1), despite the variability in replicate measurements. The relationship remained significant even when FA was excluded from the analysis and only the synthetic ligands were considered (p = 0.01) (Supporting Information Table S1). The log K values of the dominant complex with the synthetic ligands were taken from GEO8623

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determined by Donnan dialysis.19 The pH effect on Zn isotope fractionation was attributed to an increase in high affinity binding sites at higher pH, creating a bonding environment that was significantly stronger than the Zn−hexaaquo complex that exists in solution.19 The observed behavior may also be related to the presence of Ca(NO3)2 as a background electrolyte in the donor solution in the Zn study; Zn may not have been able to effectively compete with Ca for high affinity sites until more became available at pH > 6.35 However, Cu(II) has a greater affinity for humics than Zn(II) (and most other metal ions),35,36 suggesting Cu can associate with strong binding sites across a larger pH range, and may explain why no effect of pH on fractionation was seen in the current study. Due to the potential for Cu precipitation at pH > 6, as predicted by Visual MINTEQ,25 Cu fractionation by OM complexation was not examined at pH > 5 in this study or at pH > 7.0 in the study by Bigalke et al.15 Other techniques have been used to investigate the nature of Cu-OM bonding and have suggested that Cu forms strong bonds with OM across a wide pH range. Extended X-ray absorption fine structure (EXAFS) analysis of Cu−OM complexes support the hypothesis that Cu contained in multidentate humic organic ligands has a stronger bonding environment than the hexaaquo complex. Karlsson et al.37 showed that Cu binding in humics was dominated by Cu−O bonds and potential chelate ring structures, and Korshin et al.38 noted that the Cu−O bond distances for Cu-humics were significantly shorter than those of the Cu−hexaaquo complex. EXAFS have shown no detectable change in bond length or geometry for Cu during OM complexation for the pH range 4− 6 and isotopic data also suggest no change in Cu binding with pH changes (across the restricted pH range studied).39 Copper in natural water bodies is usually >99% complexed with soluble organic ligands.6,33 Organic ligands found in natural waterways can be separated into two main groups; high affinity ligands with log K values up to ca. 16, known as ligand class 1 (L1), and low affinity fulvic acid (FA) type ligands with log K values down to ca. 8, known as ligand class 2 (L2).6,40−42 The L1 ligands found by Coale and Bruland,42 Moffett et al.41 and Shank et al.43 were found to have the same Cu complexing stabilities as those released by phytoplankton or benthic organisms and are likely to be analogous to siderophore complexes. These L1 ligands are thought to be secreted from microorganisms in response to Cu concentrations in waters to control Cu free ion activity, and hence Cu toxicity.40−42 Our results suggest that L1 type ligands, such as siderophores present in natural waters, would cause significantly more fractionation between bioavailable free Cu(II) and the soluble complexed Cu than FA-type ligands (L2). In a large scale study of natural waters by Vance et al.,6 soluble Cu ( 12) in oceanic waters.40−42 Coale and Bruland42 found Cu concentrations of 0.5−1.5 nM in the North Pacific, with L1 ligands at a concentration between 1.5−3 nM in the surface waters, and L2 ligands at a concentration of 5−10 nM. Thus, it was expected that almost all the Cu at this site would be complexed by the stronger binding L1 ligands. Given the complexity of natural systems and the fact that only total dissolved δ65Cu values were measured by Vance et al.,6 it is not possible to draw any direct comparisons between their study

and ours. However, the literature on the abundance of strong Cu complexers and the isotopic data measured to date suggest that complexation by soluble ligands in natural waters may be driving significant isotope fractionation of Cu. This has consequences for the δ65Cu of the free Cu ion, which is the most bioavailable Cu species taken up by organisms, and exists in equilibrium with labile Cu pools. Hence, the organic complexation of Cu affects the way in which Cu isotopes can be used as a tracer of uptake processes in the natural environment.6 When assessing Cu isotope fractionation during biogeochemical cycling, both biologically induced fractionations and chemically induced fractionations (i.e., driven by bond strength) need to be considered. Copper uptake into plants and microorganisms has been shown to potentially induce significant isotope fractionation. Light isotope enrichment of greater than 1‰ has been found for Cu taken up by bacteria and strategy I plants, while no or minimal fractionation has been observed for strategy II plants.7−10 The preference for light isotopes in the strategy I uptake mechanism was attributed to a reductive uptake process, as reduction is known to favor light isotopes. Ryan et al.7 found that Cu translocated to the shoot was enriched in heavy Cu relative to the Cu in the roots, and suggested that Cu(I) taken up by strategy I plants may be oxidized to Cu(II) within the plant and complexed with nicotianamine (NA) when it is translocated. This is supported by the isotope data as oxidation and complexation are both processes that favor the heavy isotope. Copper has been shown to have a strong affinity with NA in plants, with a stability constant of log K = 18.6.44 Assuming the trend shown with the examined synthetic ligands extends to biological ligands, a fractionation factor of approximately +0.5‰ is estimated for Cu−NA complexation. This agrees reasonably well with the fractionation factor of +0.85‰ observed between shoots and roots Cu of strategy I plants.7 However, this is a rather straightforward interpretation of a complex biological system where there will be other competing ligands and reactions affecting Cu isotope fractionation. For example, there is not just free Cu(II) and NA, but also Cu(I) and other ligands present, and not only complexation but also redox cycling and various other fractionating mechanisms may occur. Our results show that Cu complexation with siderophores (DFOB) induces significant isotopic fractionation, with the siderophore complexes being strongly enriched in 65Cu. Hence, heavy Cu isotope enrichment in strategy II plants, relative to free Cu in the growth medium, would be expected if phytosiderophore (PS) complexed Cu was directly absorbed by the plant roots. However, no evidence of heavy isotope enrichment has been observed in strategy II plants,7−9 possibly due to the presence of other fractionating interactions occurring in the growth medium. Ryan et al.7 showed that oats (strategy II plant) had a more positive δ65Cu value than tomatoes (strategy I plant) grown under similar conditions, however, there was no significant difference in the δ65Cu value between the solution and the oat plant. The observed increase in uptake of Cu under Fe-deficiency, when PS excretion is enhanced, did suggest PSs assisted in the uptake of Cu in strategy II plants. Moynier et al.45 recently showed that different species of strategy II plants can fractionate Fe to varying degrees, and suggested that growth conditions and translocation pathways may play a key role in Fe isotope fractionation, suggesting fractionation is not solely dependent on Fe uptake strategies. This may also be the case for Cu 8624

dx.doi.org/10.1021/es500764x | Environ. Sci. Technol. 2014, 48, 8620−8626

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uptake and translocation in strategy II plants, although this is yet to be determined. As more in situ plant stable Cu isotope studies are conducted, the results presented here showing the strong heavy Cu isotope enrichment during Cu complexation by PSs may help to elucidate conditions where direct Cu-PS uptake and translocation are important. Understanding the potentially significant fractionations that Cu may undergo in the soil environment is very important if Cu isotopes are to be used for biogeochemical tracing. This paper provides evidence that Cu can undergo significant isotope fractionation in natural waters, with organic ligands preferentially complexing the heavy Cu isotope and with the magnitude of fractionation increasing with increasing bond strength. Given that the majority of Cu in soil solutions and natural waters exists in organically complexed species, it would be expected that the isotopic signature of Cu found in the DOM pool will closely reflect that of total dissolved Cu, while the “free” Cu pool will be enriched in light Cu isotopes. This has significant implications for the use of Cu isotope fractionations to track the sources and fates of Cu in natural environments, especially Cu uptake in plants, as the isotopic signature of the bioavailable pool is not necessarily identical to that of the total soil or soil solution Cu. This could lead to fractionations observed in plants being attributed to uptake processes, when in fact they reflect the fractionation processes that occurred within the uptake medium. Armed with this new knowledge, more accurate assessments of the biogeochemical cycling of Cu can be made.

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S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Phone: +61 8 83136787; e-mail: [email protected]. au. Notes

The authors declare no competing financial interest.

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