A solvent extraction technique for the isotopic measurement of dissolved copper in seawater

Analytica Chimica Acta 775 (2013) 106–113

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A solvent extraction technique for the isotopic measurement of dissolved copper in seawater Claire M. Thompson ∗ , Michael J. Ellwood, Martin Wille 1 Research School of Earth Sciences, The Australian National University, Building 142, Mills Road, Canberra, ACT 0200, Australia

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• A new sample preparation method • • • •

for seawater copper isotopic analysis (␦65 Cu). Solvent-extraction was used to preconcentrate metals from seawater samples. Anion-exchange was used to purify copper from the metal-rich extract. ␦65 Cu was measured in the north Tasman Sea. Seawater ␦65 Cu may be linked to marine biological activity.

a r t i c l e

i n f o

Article history: Received 1 November 2012 Received in revised form 25 February 2013 Accepted 1 March 2013 Available online 16 March 2013 Keywords: Copper Isotope Speciation Solvent-extraction MC ICP-MS Seawater

a b s t r a c t Stable copper (Cu) isotope geochemistry provides a new perspective for investigating and understanding Cu speciation and biogeochemical Cu cycling in seawater. In this work, sample preparation for isotopic analysis employed solvent-extraction with amino pyrollidine dithiocarbamate/diethyl dithiocarbamate (APDC/DDC), coupled with a nitric acid back-extraction, to concentrate Cu from seawater. This was followed by Cu-purification using anion-exchange. This straightforward technique is high yielding and fractionation free for Cu and allows precise measurement of the seawater Cu isotopic composition using multi-collector inductively coupled plasma mass-spectrometry. A deep-sea profile measured in the oligotrophic north Tasman Sea shows fractionation in the Cu isotopic signature in the photic zone but is relatively homogenised at depth. A minima in the Cu isotopic profile correlates with the chlorophyll a maximum at the site. These results indicate that a range of processes are likely to fractionate stable Cu isotopes in seawater. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction In the marine realm, copper (Cu) plays a significant role in governing the rate of organic matter production. This is reflected in its nutrient-like distribution, which is depleted through biological uptake and scavenging in surface seawater and is enriched

∗ Corresponding author. Tel.: +61 2 6125 1894; fax: +61 2 6125 7739. E-mail addresses: [email protected] (C.M. Thompson), [email protected] (M.J. Ellwood), [email protected] (M. Wille). 1 Present address: Isotope Geochemistry, University of Tuebingen, Tuebingen 72076, Germany.

due to remineralisation in the deep ocean (e.g. 0.58 to ≥1.88 nM respectively) [1]. Micro-algal processes such as photosynthesis, denitrification and iron uptake are facilitated by Cu containing enzymes and proteins, including plastocyanin, nitrous oxide reductase and multi-Cu oxidase [2–4]. In contrast to its biological importance, Cu can also be highly toxic, even when bioavailable free Cu ion concentrations are as low as 10−12 M [5]. Copper toxicity is controlled in seawater via complexation to natural organic ligands, but the identities and exact roles of these ligands are difficult to discern using modern analytical techniques such as competitive ligand exchange–adsorptive cathodic stripping voltammetry (CLE–ACSV). The measurement of stable Cu isotopes (65 Cu and 63 Cu) in seawater is proposed to help contextualise results obtained using techniques including CLE–ACSV. The fractionation of Cu isotopes can be used to

0003-2670/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.03.020

C.M. Thompson et al. / Analytica Chimica Acta 775 (2013) 106–113

Here, we present results relating to development and application of this combined method using seawater samples collected from the Tasman Sea region.

Reduced minerals [32, 33, 34] Oxidised minerals [7, 11, 32]

2. Experimental

Cu(I) lab precipitates [6, 7] Cellular uptake [8]

2.1. Materials, reagents and sample preparation

Cellular adsorption [8] Protein incorporation [7] Organic adsorption [8, 35] Oxide adsorption [36, 37, 38]

-5

107

-4

-3

-2

-1 0 1 δ65Cu (‰) Δ65Cu (‰)

2

3

Fig. 1. Natural Cu isotope fractionating processes include changes in redox state (green, ␦65 Cu scale – defined in Eq. (2), Section 2.3), biological uptake (red, 65 Cu scale) and equilibrium partitioning (blue, 65 Cu scale) where 65 Cu = ␦65 Cuprecipitate − ␦65 Cuparent solution. ␦65 Cu values are reported relative to SRM NIST 976. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

track Cu transport in the environment. Natural Cu isotope fractionation mechanisms (Fig. 1) include changes in redox state, biological uptake, surface adsorption and aqueous complexation [e.g. 6–8], which are all significant processes in marine Cu cycling. Measurement of the changes in the Cu isotope composition of seawater may provide a new perspective on the marine biogeochemical cycling of Cu which may ultimately help to improve our understanding of its role in regulating organic matter production in the ocean. Measurement of the Cu isotope composition of seawater is analytically challenged by its low ambient concentration and the complexity of the seawater matrix. Pre-concentration, followed by Cu-purification, is required to enable interference free measurement of samples using multi-collector inductively coupled plasma mass-spectrometry (MC ICP-MS). Initial Cu pre-concentration techniques have employed Mg(OH)2 co-precipitation and Chelex100 cation exchange [9,10] followed by purification using anion-exchange [11,12]. To achieve sufficient purity using these pre-concentration techniques, sub-sampling and/or re-processing of samples using anion-exchange is required. This requires large sample volumes and multiple analytical steps which increases the risk of isotope fractionation [e.g. 13]. Anion-exchange purification of Cu for isotope analysis was first reported by Marechal et al. [11] using macroporous analytical grade resin (AG MP-1). This technique has been progressively optimised to accommodate smaller sample sizes with lower analytical blanks [12]. Most recently, Borrok et al. [14] revised the anion exchange elution scheme to exploit the complex speciation of Cu2+ Cln 2−n in a chloride media, which enables rapid Cu elution with a very low analytical blank. This work attempts to optimise the pre-concentration technique for the extraction of Cu from seawater. Solvent extraction, using ammonium pyrrolidine dithiocarbamate/diethyl-dithiocarbamate (collectively termed APDC) as chelating agents, is used here to preconcentrate Cu prior to isotope determination. APDC has long been used for the extraction of trace metals from seawater to the exclusion of many major spectral interference ions such as Na, Ca and Mg [e.g. 15, 16–18]. This extraction technique has been coupled with Cu2+ Cln 2-n anion-exchange purification [14] to produce a method that achieves Cu-purity with a single pass over an anion exchange resin and accommodates small seawater sample sizes (≤500 mL).

All sample purification work was performed in a class 100 clean laboratory. All plastic labware was rigorously acid cleaned before use. Hydrochloric (HCl) and nitric acid (HNO3 ) reagents (Scharlau Australia) were purified by sub-boiling distillation using a Teflon elbow and PFA still, respectively. Chloroform (Scharlau Australia) was cleaned by triple extraction through 10% v/v HCl. The combined APDC reagent was prepared by dissolving 0.5 g of ammonium pyrrolidine dithiocarbamate (Fluka) and 0.5 g of diethyl-dithiocarbamate salts (Sigma Aldrich) in 4 mL of ammonia (Fluka) which was then diluted with 96 mL of Milli-Q water (Millipore, Australia). This solution was triple-extracted with clean chloroform and stored for up to one month in a Teflon bottle. An ammonium acetate buffer was prepared by dissolving 92 g of ammonium acetate (Sigma Aldrich) in 100 mL of Milli-Q water. The buffer was mixed with 1 mL of APDC reagent and triple-extracted with clean chloroform and stored in a Teflon bottle. Seawater samples were collected from the north Tasman Sea (GEOTRACES station P1, 30◦ S, 165◦ E) on 30th January 2010 on board the RV Southern Surveyor (Primary productivity induced by Iron and Nitrogen in the Tasman Sea voyage). The bulk seawater sample used throughout the method development experiments was collected from a depth of 500 m. A full water column profile (bottom depth 3396 m) was also collected over 9 depths at this site, with sample depths ranging between 15 and 3000 m. Seawater was collected using an autonomous trace-metal clean rosette (General Oceanics, USA), hung from a 6 mm Dynex cable (Hampidjan). The rosette was equipped with 12 × 10 L Niskin X bottles for sample collection. All samples were 0.2 ␮m filtered using acid-washed capsule filter cartridges (Pall, USA) in the CSIRO Gen 2 clean container under HEPA filtered air. Samples were acidified to pH 1.6 with Teflon distilled nitric acid. Acidified samples were double bagged in zip-lock plastic bags and stored in plastic tubs for a minimum of 3 months prior to analysis. 2.2. Analytical procedure 2.2.1. Solvent extraction pre-concentration The low concentration of many trace metals in seawater requires them to be pre-concentrated to enable detection by ICP-MS. This was achieved for Cu by organic solvent extraction using APDC and chloroform with HNO3 acid back-extraction. The trace metal pre-concentration procedure closely followed that of Danielsson et al. [18]. Briefly, up to 500 mL of acidified seawater was weighed into an acid-cleaned Teflon (Nalgene) separatory funnel and buffered to pH 4.5 using 12 M ammonium acetate solution. One mL of APDC solution was added and the sample was homogenised by shaking for 10 s. The metal–carbamate complexes were extracted into a hydrophobic phase by adding 15 mL of chloroform and vigorously shaking the funnel for 2 min. The chloroform phase was removed into a 30 mL Teflon vial (Savillex) and a further 5 mL of chloroform was added to the separatory funnel and shaken again for 2 further minutes. The second chloroform addition was removed and combined with the initial chloroform aliquot. Seawater droplets that settled on top of the chloroform were carefully removed by pipetting off using a 1 mL Teflon tip (Savillex). To liberate Cu from the dithiocarbamate complex, 100 ␮L of concentrated HNO3 was added to the extract and shaken for around 3 s. After

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Table 1 MC ICP-MS cup configurations. Cup Detected mass (amu)

L4

L3 60

L2 61

L1 62

C 63

H1 64

H2 65

H3 66

H4

5 min, 500 ␮L of Milli-Q water was added and again shaken for 3 s. The extract was left overnight to ensure complete breakdown of the Cu–carbamate complex. The acidified layer was transferred using a Teflon pipette tip into a clean Teflon vial where it was evaporated to dryness on a Teflon-coated hot-plate. 2.2.2. Anion-exchange separation The anion-exchange Cu purification method of Borrok et al. [14] was used in this work with some minor changes to the methodology. The bulk AG MP-1 resin (100–200 ␮m mesh-size, BioRad) was pre-cleaned by rinsing 10 times with 10% v/v HCl before loading into anion-exchange columns. All columns were prepared by trimming acid-washed disposable 1 mL plastic pipettes at the base and bulb top to accommodate a 5 × 0.5 cm resin bed which was supported by a polyethylene frit. When not in use all columns were stored in ∼1% v/v HNO3 . Prior to use, the anion-exchange columns were individually cleaned by eluting with 2 mL of 1% v/v HNO3 followed by 3 mL of Milli-Q water. The resin was then pre-conditioned using 4 mL of 11 M HCl before samples were loaded in 11 M HCl. Interfering ions were eluted by passing a further 2 mL of 11 M HCl over the column before Cu was eluted with 5.5 mL of 5 M HCl. In preparation for MC ICP-MS determination, Cu eluents were evaporated to dryness and re-dissolved with dilute nitric acid (2% v/v) containing nickel (NIST SRM 986). 2.3. Mass spectrometry Two MC ICP-MS instruments were used at The Australian National University to make Cu isotope measurements: a ThermoFinnigan Neptune and a Neptune Plus. Both instruments were operated in low resolution mode with identical cup configurations (Table 1), APEX desolvating sample inlet systems (IR type, Elemental Scientific Inc., ESI), H-type Ni sample and skimmer cones, 100 ␮L min−1 uptake nebulisers (ESI MicroFlow PFA) and quartz sample injectors. The main difference between the two systems was that the Neptune operated using a higher than standard capacity interface pump (Trivac S 65 B) while the Neptune Plus used a jet interface pump with standard cones. Each instrument was tuned daily to maximise signal intensity and peak shape (Table 2). Isotopes of 63 Cu and 65 Cu, as well as 60 Ni, 61 Ni and 62 Ni for external normalisation, were typically measured at a Cu concentration of 10 ␮g L−1 and a Ni concentration that ranged between 200 and 600 ␮g L−1 . A normal analytical sequence consisted of a 2% v/v HNO3 blank measurement followed by an in-house Cu standard (ACS), the sample and the standard again with instrumental baselines measured by ion beam deflection. Washouts in 2% v/v HNO3 were monitored in real-time to ensure that isotope intensities returned to background levels between analyses. Each measurement consisted of 60 4-s integrated measurements. Table 2 Typical tuning parameters for the Neptune Plus MC ICP-MS.

Cool gas Aux gas Sample gas RF power Extraction

L/min L/min L/min W V

Neptune

Neptune Plus

15 1.0 0.90 1200 −2000

16 1.1 1.02 1200 −2000

There is a range of isobaric (e.g. Sn2+ , Te2+ , Xe2+ ) and poly-atomic (e.g. NaCl, ArNa, NaOH, ArMg, POO, ScO, TiO, TiOH) species that can cause interferences on the Ni (60–62) and Cu (63, 65) masses during isotopic analysis. Most of these species have average concentrations in seawater that are significantly lower than Cu and Ni and so are not of concern during sample preparation and MC ICP-MS. However, major seawater cations such as Na and Mg may introduce interference effects. To investigate their removal from samples prepared using this technique, scans over masses 23 (Na) and 24 (Mg) were performed on fully prepared samples. Instrumental and matrix induced mass fractionation was corrected using an externally normalised standard-sample-standard bracketing technique [6,19]. Nickel was preferred over Zn as the external dopant due to the similar first ionisation potentials of Cu and Ni (7.73 eV and 7.64 eV, respectively) compared to the high value of Zn (9.39 eV) [6]. Briefly: all samples were doped with NIST SRM 986 Ni standard. Short term instrumental fractionation was corrected with reference to fractionation of the doped Ni standard according to the exponential form of the generalised power law for mass fractionation [11,19,20]: R=r

 M ˇ 2

(1)

M1

where r is the measured isotopic ratio of two isotopes with mass M2 and M1 , R is the true isotopic ratio and ˇ is the exponential law mass fractionation coefficient. Eq. (1) was solved for ˇ using the measured Ni isotopic ratio and the certified masses (61.92835 and 59.93079 amu for 62 Ni and 60 Ni respectively) [21] and isotopic ratio of 62 Ni/60 Ni (0.1386) [22]. The value for ˇ was then inserted back into Eq. (1) which was solved for R using the measured Cu isotopic values and certified masses of 65 Cu and 63 Cu (64.927792 and 62.929598 respectively) [21]. Corrections using the 62 Ni/60 Ni ratio were preferred, with results corrected using this pair generally being the most consistent given the similar mass difference between 62 Ni and 60 Ni and 65 Cu and 63 Cu. All Cu isotope values reported here were obtained using the 62 Ni/60 Ni ratio unless stated otherwise. Longer-term instrumental fractionation was corrected by standard bracketing where:

 65

ı Cu (‰) =

(RCu sample ) (RCu mean−ACS )

 − 1 × 103

(2)

where the RCu ratios were calculated from the measured Cu isotope values in Eq. (1) and the mean − ACS RCu value is the averaged Cu isotope ratio from the in-house standards measured immediately prior to and following the sample. The Cu concentration in method development samples was measured using MC ICP-MS and calculated using a standard intensity curve. Copper concentrations from north Tasman Sea samples were measured using isotope dilution with a 65 Cu spike on a high resolution ICP-MS (Element XR, ThermoFinnigan) at the University of Canberra.

2.4. In-house copper standard A Fluka Analytical Cu standard (Sigma Aldrich Australia, TraceCERT Copper Standard for AAS, product # 38996, Lot # 1414402) was characterised against an international Cu isotope standard, NIST SRM 976, for use as an in-house Cu isotope standard, ACS. All samples were measured relative to ACS. Seawater ␦65 Cu values are corrected and reported relative to NIST SRM 976.

C.M. Thompson et al. / Analytica Chimica Acta 775 (2013) 106–113

109

1.0 Data Model Data regression 95% Confidence Band 95% Prediction Band

0.8

δ65CuNIST976 (‰)

0.12 0.08 0.04

15 Jun 12

26 Apr 12

07 Mar 12

17 Jan 12

09 Oct 11

04 Aug 12

2a

0.00

28 Nov 11

δ65CuACS (‰)

0.16

0.6

0.4

0.2

0.0

Analysis date

0.0

0.2

0.4

0.6

0.8

1.0

0.16

δ65CuACS (‰)

[Cu]spike/[Cu]total 0.12 Fig. 3. Mixing between a seawater end-member and pure ACS is modelled as a function of ␦65 Cu with respect to the proportion of ACS spike in the sample (black, Eq. (3)). Corresponding experimental values from 6 analytical sessions are shown. Linear regression of the experimental results shows a close fit to modelled values while hypothesis testing shows that there is no significant difference between the model and linear regression lines. ␦65 Cu is reported relative to ACS.

0.08 0.04 2b

0.00

0

5

10

15

20

25

30

35

40

Number of analyses (n) Fig. 2. Long term variability of Cu standard NIST SRM 976 relative to ACS. The mean (0.11 ‰) is bounded by the 2 standard deviation (0.03 ‰ band for the dataset. Error bars on individual data points represent instrumental error for that measurement. 2a shows the inter session variability. 2b shows the variability within the combined dataset. ␦65 Cu is reported relative to ACS.

3. Results 3.1. In-house copper standard The NIST SRM 976 Cu standard was measured in every analytical session relative to the ACS (Fig. 2). The long-term average ␦65 Cu value for the NIST SRM 976 was 0.11 ‰ ± 0.03 ‰ (2 sd, n = 34). 3.2. Solvent-extraction pre-concentration 3.2.1. Reproducibility and fractionation A number of seawater and standard mixing experiments were undertaken to test the performance of the APDC solvent extraction procedure for Cu pre-concentration, isolation and ␦65 Cu determination (Fig. 3). Mixing between the bulk seawater sample and ACS can be modelled using the following equation: 65

ı Cusample =

 [Cu]

seawater

[Cu]total

+

 [Cu]

ACS

[Cu]total





65

× ı Cuseawater

× ı65 CuACS



 (3)

where [Cu] denotes the Cu concentration of the respective total, seawater and ACS portions of the sample. For different [Cu]seawater /[Cu]total ratios, ␦65 Cu measurements that correlate with the model are assumed to be free of analytical fractionation. A linear regression line fit to the experimental data shows a slight deviation from the modelled ␦65 Cu values as the total spike fraction in the sample increases. However, the modelled values fall well within the 95% prediction band of the data and hypothesis testing at the 95% confidence level (tslope = 0.9669, n = 49) indicates that the slope of the data regression and modelled lines are not significantly different, thereby giving us confidence in the method.

The APDC technique for seawater ␦65 Cu measurement was further tested for improved isotopic reproducibility and yield. Tests included the addition of H2 O2 prior to and/or following extraction to oxidise organics and liberate bound Cu and Cu(I) species, increasing the pH during extraction by the addition of ammonia, pre-extraction sample exposure to UV light for 3 h for intense oxidisation of the sample and rinsing the chloroform extract with buffered water to remove unwanted seawater cations before metal liberation. None of the tests produced significant variations in ␦65 Cu or yield from un-manipulated samples. 3.2.2. Yield Eight seawater samples were processed in duplicate over 3 different days through both stages of the method: the preconcentration and anion exchange procedures. The measured Cu isotope composition of all duplicates agreed within error (±0.10 ‰, see Section 3.4.2) and the percentage yield for the 16 samples was calculated to be 98 ± 11% (1 sd). The Cu concentration in each sample was calculated by comparing the measured voltage against a standard intensity curve measured on the day and does not account for subtle variations associated with evaporation, sample dissolution and standard preparation etc. 3.3. Anion-exchange separation Anion-exchange chromatography, employing the AG MP-1 resin, provides excellent purification of Cu from other elements extracted from seawater during the sample preconcentration step. Most ions elute during the separation phase (Fig. 4). When present in high concentrations, matrix cations such as Na and Mg can leak through the resin and contaminate the Cu eluate (Fig. 5). However, this only occurred when the leftover seawater matrix floating on top of the chloroform phase was not removed before acidification of the trace-metal enriched extract. Fig. 5 shows the variability in the proportion of Na and Mg, relative to 63 Cu, present for 7 processed samples. Rarely was the concentration of the contaminant greater than the concentration of the Cu analyte in the sample. Rinsing the chloroform extract with buffered Milli-Q water prior to the metal liberation from the acidified chloroform phase did not appear to improve the purity of that acidified phase over that of pipetting off the excess seawater (Section 3.2.1). Possible interference effects, induced by Na and Mg contamination, were investigated using a

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b

5M HCl

11M HCl

δ65Cu (‰)

1

103

40 102

101

100

Ni Cu

Fe

-1

30

-2 20

δ65CuACS (‰)

0

Mg

-3

Al

10

Mg

Element mass (ng)

50

Milli-Q H2O

Cu eluted (ng)

Element mass (ng)

a

-4

103

0 102

Ni

Zn Fe

100

Al

0

4

5

6

7

8

Cumulative elution volume (mL)

Cu 101

-5 3

Fig. 6. Elution of Cu (solid line) from 1 mL × 112 mg L-1 multi-element standard. Corresponding ␦65 Cu values (diamonds) show a range of almost 4 ‰ during elution. ␦65 Cu is reported relative to ACS. Symbols are larger than error bars.

1

2

3

4

5

6

7

8

9

10

11

Elution volume (mL) Fig. 4. Anion exchange separation of a bulk seawater sample effectively purifies Cu during elution using 5 M HCl. Measured elements with a cumulative mass of less than 10 ng are not shown. Note the exponential y-axis. Duplicates of the same sample are shown, prepared using different anion exchange columns. The Cu yields were 99 and 98% respectively.

multi-elemental standard (Perkin-Elmer P/N N812-5034). Signals measured at masses 63 and 65 had the same ratio (63 Cu/65 Cu) as the absolute isotopic abundance ratio of Cu (2.2). The measured 2.8 V 23 Na and 0.32 V 25 Mg signals did not appear to induce polyatomic interferences (23 Na40 Ar and 25 Mg40 Ar) on either of the Cu masses. The 23 Na/63 Cu (4.58) and 35 Mg/63 Cu (0.51) ratios from this experiment are thus considered as a minimum threshold for interference free ␦65 Cu measurement. Careful preparation of samples almost always resulted in 23 Na/63 Cu and 35 Mg/63 Cu ratios lower than these values (Fig. 5). Typically, our samples are run at ∼4 V for 63 Cu. This would require interference voltages of greater than 18 V and 2 V, for Na and Mg respectively, to cause significant interference during ␦65 Cu measurement.

3.3.1. Resin induced copper fractionation Copper isotope fractionation can occur during anion exchange purification when the amount of sample exceeds about 40 ␮g of Cu [e.g. 11, 13]. Samples with up to 112 ng of Cu in this study were found to fractionate in a similar way with incomplete yields dramatically influencing the Cu isotopic composition of the sample (Fig. 6). In Fig. 6 for example, if the last 5% of the Cu eluate is not collected then the sample will be 0.06 ‰ heavier in its isotopic composition. Thus it is important that a 100% Cu yield is obtained during the anion exchange process. To ensure that this was achieved within our characterised elution scheme, NIST SRM 976 Cu samples were run over every column and measured against a non-column treated NIST SRM 976 standard. If the isotope composition for NIST SRM 976 passes over each column was not within the error margin for the standard, those columns were discarded. 3.3.2. Anion-exchange blank The Cu contribution in the anion-exchange blank was measured by processing a 1 mL sample of distilled 11 M HCl through the anion-exchange procedure. The Cu concentration in the anionexchange blank was 0.6 ± 0.6 ng (1 sd, n = 18). 3.4. Procedural performance

Beam intensity (relative to 63Cu)

100

3.4.1. Procedural blank The procedural blank for the combined Cu extraction and purification scheme was determined by re-extracting stripped seawater samples (∼500 mL) and purifying as per normal. The Cu contribution from the procedural blank was 1.6 ± 1.2 ng (1 sd, n = 31).

e1

10

e1

b

1

b

e2

e2 d1

a d2 c

0.1 a c

0.01 22.95

d1

d2

23.95

23

24

3.4.2. Reproducibility Twenty measurements of the bulk seawater sample, measured over a 17 month period, had a reproducibility of ± 0.13 ‰ (2 sd, Fig. 7). The average repeatability of the Tasman Sea replicates (Fig. 8), prepared and measured over a 5 month period, was ± 0.10 ‰ (2 sd, n = 9) and is reported as the reproducibility on all measurements unless the instrumental error is greater.

Atomic mass Sample a Sample b Sample c

Sample d replicate 1 Sample d replicate 2

Sample e replicate 1 Sample e replicate 2

Fig. 5. Mass scans of 7 seawater samples, processed using APDC extraction and anion exchange purification for Cu, show the presence of Na and Mg relative to sample Cu.

3.5. Instrumental precision The standard error reported by the instrument for the mean raw 65 Cu/63 Cu and 62 Ni/60 Ni isotope ratios was consistently around 0.02 ‰. Error propagation of the raw

C.M. Thompson et al. / Analytica Chimica Acta 775 (2013) 106–113

Sea (Fig. 8). The total Cu concentration was depleted in surface waters (0.5 nmol kg−1 ) but increased to 3.3 nmol kg−1 with depth (Fig. 8a). The Cu isotopic profile showed the greatest compositional variability in the upper water column but was homogenised below 300 m (Fig. 8b and d). The oligotrophic nature of the site was reflected in a deep chlorophyll a maximum located at 95 m depth (Fig. 8e). A minima in ␦65 Cu occurred at this chlorophyll a maximum (Fig. 8d).

1.1 1.0

δ65CuNIST976 (‰)

111

0.9 0.8 0.7 0.6

4. Discussion 050810 -01 050810 -02 240810 -20 310810 -01 310810 -02 310810 -03 310810 -04 310810 -05 310810 -06 031110 -01 271010 -01 271010 -01 140411 -01 140411 -02 140411 -05 061011 -02 130112 -01 130112 -02 130112 -05 130112 -06

0.5

Sample Fig. 7. Twenty replicates of our bulk seawater sample measured over 17 months have a mean ␦65 Cu of 0.82 ± 0.13 ‰ (2 sd). Instrumental errors (2 se) are shown on each measurement while the mean (solid line) and 2 standard deviation band is also shown. ␦65 Cu values are reported relative to SRM NIST 976.

standard-sample-standard datasets used to calculate final ␦65 Cu values also produced instrumental error values of 0.02 ‰ for both the 65 Cu/63 Cu and 62 Ni/60 Ni isotope ratios. 3.6. Tasman Sea ı65 Cu profile This ␦65 Cu pre-concentration and purification technique has been applied to sea water samples collected in the north Tasman

δ65CuNIST976 (‰)

[Cu] (nmol/kg) 0

0

1

2

3

4

0.6

0.8

1.0

0.8

1.0

Fluorescence

500

Depth (m)

1000 1500 2000 2500 3000 0

8a 0

8b 0.5

1

0.6

0

5

10 15

40

Depth (m)

80 120 160 200

8c

8d

8e

Fig. 8. North Tasman Sea Cu concentrations, Cu isotopic compositions and fluorescence (in situ fluorometer measurements reported relative to fluorescence at 1000 m). Panels 8a and b show the full depth profile. Panels 8c–e are for values between 0 and 200 m depth. ␦65 Cu values are reported relative to SRM NIST 976. Unless the instrumental error is greater, the procedural reproducibility of ±0.10 ‰ is reported as the error on all measurements.

4.1. Analytical performance This combined solvent extraction/anion-exchange technique provides an efficient and reproducible method for the measurement of Cu isotopic compositions in complex aqueous media such as seawater. The procedure is efficient over a range of Cu concentrations and pH and residual organics and/or Cu(I) species do not alter the behaviour of the Cu-dithiocarbamate complex during the extraction nor the behaviour of AG MP-1 resin during anion exchange. The APDC extraction procedure is highly efficient with yields close to 100% which is required for the measurement of stable Cu isotopes. The blank from the combined APDC extraction and anion-exchange separation procedure is low and compares well with the blank values for the previously published Chelex-100 cation exchange (2.2 ± 1.2 ng, n = 9, 1) and Mg(OH)2 co-precipitation (0.35 ng) techniques [10]. Other advantages of this technique includes the relatively low sample volume, <0.5 L, for pre-concentration making sample handling easy. The lower Cu requirement of the procedure also minimises the risk of Cu pre-elution during the anion-exchange phase. Using this combined technique, initial sample volumes are only limited by the sensitivity of the ICP-MS and typically ranged from 0.2 to 0.5 L per extraction. The high level of sample purity achieved after a single pass on the anion exchange column also reduces the risk of contamination and resin-induced fractionation, when compared with other techniques. Routine testing of the anion-exchange columns for reproducibility identified two processes that each had the effect of increasing the measured ␦65 Cu value in samples. Broad elution curves of Cu were obtained from columns that had a high level of use. This resulted in fractionated Cu yields due to Cu elution outside of the characterised elution scheme. Compaction in the resin was probably responsible for these uncharacteristic Cu elution curves and regular backwashing of the resin columns may be a useful approach to avoid this issue. Despite the storage of the columns in 1% v/v HNO3 acid to oxidise residual organics between uses, organic contamination in the AG MP-1 resin was still experienced following the processing of an organic rich set of seawater particulate samples. Extensive rinsing of the resin columns with Milli-Q H2 O, 50% v/v methanol and 1% v/v HNO3 was ineffective at removing the contamination. Organic contamination in the resin had the dual effect of fractionating Cu in situ via preferential complexation of 63 Cu, and fractionating Ni in the samples via leaked organics which either complexed Ni or caused a mass interference on Ni during analysis. The effects of this contamination in the resin combined to inflate the calculated ß values (Eq. (1)) which produced artificially heavy ␦65 Cu values in samples. In each case, regular comparisons of ␦65 Cu in untreated and column treated samples identified columns that produced incorrect ␦65 Cu values. 4.2. Seawater ı65 Cu composition Most of the measured variability in the Tasman Sea ␦65 Cu composition occurred within the top 300 m of the profile. It therefore

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1.6

δ65CuNIST976 (‰)

1.4 1.2 1.0 0.8 0.6 0.4 0

0.5

1

1.5

2

2.5

3

1/[Cu] (nmol/kg) Tasman Sea 30°S, 165°E English Channel ::: 50°N, 1°W Riverine Cu Source :::

NE Pacific Ocean ::: 48°N, 126°W NE Pacific Ocean ::: 50°N, 145°W S-central Indian Ocean ::: 23°S, 73°E S. Ocean, Chatham Rise ::: 42°S, 178°E

Fig. 9. Adapted from Vance et al. [9]. The global seawater ␦65 Cu dataset.::: [9].

seems likely that processes related to biological activity are altering the dissolved ␦65 Cu composition in the upper water column at this site. Major influences on the dissolved Cu pool at these shallow depths are likely to include biological uptake, organic complexation, scavenging and remineralisation, the effects of which may be preserved in the dissolved ␦65 Cu profile. Further characterisation of the natural processes that fractionate stable Cu isotopes and their specific effects on ␦65 Cu are required to properly interpret the results of this Tasman Sea ␦65 Cu profile. Two interesting features of the profile are the ␦65 Cu minima measured at the fluorescence maximum and the homogeneity of the ␦65 Cu signature in the deeper waters. Curiously, at the deep chlorophyll a maximum where the effects of the biological signal should be strongest, the ␦65 Cu results are opposite to what would intuitively be expected. That is, the dissolved ␦65 Cu signal is lighter than at other depths in the photic zone. Our tentative interpretation of this result is that other ␦65 Cu fractionating processes are overwhelming the biological uptake signal at this depth. Such processes could include the preferential regeneration of 63 Cu, particulate scavenging of 65 Cu or strong organic complexation of 63 Cu at this depth. The general uniformity of the Cu isotopic composition below the photic zone may be due to the higher total dissolved Cu concentrations experienced at such depths. The isotopic compositions of these Cu concentrated deep waters may be more resistant to fractionation caused by natural processes. The deep Cu isotopic signatures could reflect the original isotopic composition of the Cu sources to seawater which include atmospheric, riverine and hydrothermal inputs. Alternately, they may indicate a high degree of organic complexation in deep seawater that would be the result of long-lived organic Cu-binding ligands which stabilise the Cu isotopic signature. The ␦65 Cu data presented here appear to support the findings of Vance et al. [9] (Fig. 9) who made the first measurements of ␦65 Cu in riverine and sea water. They suggested that the Cu isotopic composition in marine systems was a reflection of the seawater Cu source overprinted with biological uptake and remineralisation processes. The smaller magnitude of variability in our photic zone ␦65 Cu values may be the result of low productivity in the north Tasman Sea.

4.3. Future work The measurement of ␦65 Cu in both natural and laboratory settings will be extremely useful for understanding how natural processes fractionate the stable isotopes of Cu. Furthermore, in oceanographic terms, it is important to better understand how primary producers metabolise Cu and the role they play in influencing Cu bioavailability and toxicity in seawater. For example, it is well accepted that Cu intolerant phytoplankton such as cyanobacteria may be important producers of organic chelates that reduce Cu toxicity in seawater [e.g. 27]. Consequently, this maintains a bioavailable Cu pool that is too low for phytoplankton with higher Cu requirements such as diatoms and coccolithophores [e.g. 8, 28]. Proposed mechanisms to account for this shortfall in bioavailable Cu include the exclusive internalisation of Cu(I) by phytoplankton from dedicated organic complexes that are produced specifically to support biological uptake [e.g. 25, 28, 29, 30]. Validating these conceptual models of Cu uptake is restricted by analytical limitations which include the determination of the oxidation state of Cu in seawater, during uptake and in organic complexes. Copper isotope geochemistry may provide a new tool and perspective from which to approach these issues. The importance of characterising such mechanisms increases when the current interest in marine Fe biogeochemistry is considered with respect to the importance of the Fe-replacing Cu enzyme, plastocyanin, for marine photosynthesis and the Cu-assisted assimilation of Fe which has been identified in certain phytoplankton [e.g. 2, 4, 31]. 5. Conclusion A new technique for the extraction and measurement of stable Cu isotopes in seawater is presented and compared to previously published methods [9,10]. Its advantages include a low blank, a high level of reproducibility, small sample sizes and a low risk of analytical Cu isotope fractionation and contamination. The seawater depth profile for samples collected from the north Tasman Sea is comparable to previously published ␦65 Cu values for seawater. Coherent changes in ␦65 Cu values for the upper portion of the profile suggest that natural processes dominate Cu isotopic fractionation. The identification of a probable biological influence on the dissolved seawater ␦65 Cu composition highlights the value of this technique.

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