1A new purification method for Ni and Cu stable isotopes in seawater 2shows widespread Ni isotope fractionation by phytoplankton 3 4Shun-Chung Yang1*, Nicholas J. Hawco1*, Paulina Pinedo Gonzalez2, Xiaopeng 5Bian1, Kuo-Fang Huang3, Ruifeng Zhang4, Seth G. John1 6 71 Department of Earth Sciences, University of Southern California, Los Angeles, CA, 8USA 92 Lamont Doherty Earth Observatory, Columbia University, Palisades, NY, USA 103 Institute of Earth Sciences, Academia Sinica, Taipei, Taiwan 114 School of Oceanography, Shanghai Jiao Tong University, Shanghai, China 12 13* Corresponding authors at: Department of Earth Sciences, University of Southern 14California, Los Angeles, CA, United States 15E-mail address: [email protected] (S.-C. Yang); [email protected] (N. J. Hawco) 16 17Keywords: δ60Ni; δ65Cu; chromatography; trace metal cycling; phytoplankton 18
11 2 19Abstract 20 Nickel and copper are cofactors in key phytoplankton enzymes and the stable 21isotope composition of Ni and Cu (δ60Ni and δ65Cu) in seawater have the potential to 22identify major processes that influence their biogeochemistry. However, accurate 23analysis of δ60Ni and δ65Cu is challenging because of the difficulties in separating the 24metals from interfering elements in the seawater matrix. Here we report a fast and 25simple method for purification of Ni and Cu from seawater samples that is able to 26completely remove major interfering elements Mn, Ti, Cr, and Fe. Our methods were 27verified by analyzing four reference materials (powdered plankton, natural soils, 28marine sediments) that contain significant levels of interfering elements. Using this 29technique, we generated a dataset of 49 seawater δ60Ni and δ65Cu measurements from 30the upper water column of the North Pacific, which show isotopic fractionation 31associated with phytoplankton uptake of Ni but not Cu. This new method simplifies 32treatment of seawater samples for Ni and Cu isotope analysis, accelerating the 33investigation of δ60Ni and δ65Cu throughout the global ocean. 34
32 4 35 1. Introduction 36 The biogeochemical cycles of nickel (Ni) and copper (Cu) in the ocean remains 37mysterious. Both metals are nutrients to phytoplankton, but are not fully depleted in 38the surface ocean (Boyle et al., 1981; Moore et al., 2013). Both elements are 39regenerated in the water column, but are decoupled from major nutrients like 40phosphorus (Bruland, 1980; Boyle et al., 1977). Relative to other divalent metals, Ni 41and Cu also form the strongest complexes with organic ligands (Irving and Williams, 421953), but whereas most Ni is found in labile, presumably inorganic, species 43(Achterberg and Van Den Berg, 1997; Saito et al., 2005; Boiteau et al., 2016), Cu is 44almost completely bound by strong organic ligands (Coale and Bruland, 1990; 45Jacquot and Moffett, 2015; Whitby et al., 2018). Because of their strong binding, both 46metals may be toxic under certain conditions (Huertas et al., 2014), often because they 47outcompete other metals for protein binding sites (Sunda and Huntsma, 1996; 48Macomber and Imlay, 2009; Hawco and Saito, 2018). As a result, efflux pumps and
49sensor proteins for Cu and Ni are widespread. Whether the nutritional or toxic 50properties of Ni and Cu affect phytoplankton growth in the oceans is also unknown. 51 Over the past two decades, ion chromatography and multi-collector ICP-MS 52techniques have been developed to measure Ni and Cu isotopes in seawater (Bermin 53et al., 2006; Cameron et al., 2009; Takano et al., 2017), which has raised additional 54questions about their biogeochemistry. Initial reports highlighted somewhat 55homogenous profiles of 65Cu and 60Ni, despite significant concentrations gradients 56between surface and deep waters (Vance et al., 2008; Cameron and Vance, 2014), and 57the heavy 60Ni and 65Cu composition of the oceans compared to crustal material – 58roughly +1.4 ‰ and +0.65 ‰, respectively – has also emphasized the lack of isotope 59mass balance in the marine Ni and Cu cycles, suggesting the existence of unidentified 60sources or sinks of these metals (Vance et al., 2016; Ciscato et al., 2018; Little et al., 612014; 2017). 62 More recently, deviations from mean ocean 65Cu and 60Ni have been identified. 63Takano et al. (2017) observed 60Ni up to +1.7 ‰ in South Pacific surface waters, 64suggesting biological uptake of isotopically light Ni. This conflicts with the 65observations of Wang et al. (2019), who found no evidence for Ni isotope 66fractionation during phytoplankton Ni uptake in Southern Ocean waters. Variations in 6765Cu have also been observed in the upper ocean, and have been hypothesized to 68reflect biological uptake, Cu ligand production, or new Cu sources (Takano et al.,
53 6 692014; Little et al., 2018; Baconnais et al., 2019). However, interpretation of these 7065Cu observations has been complicated because of incomplete Cu extraction and 71insufficient replication. 72 The speed of sample purification is a major limitation on the generation of 73isotopic data from high resolution transects (e.g. GEOTRACES). Purification of Ni 74and Cu from seawater has proven particularly challenging. Successful purification 75protocols must separate Ni and Cu from other inorganic elements in the seawater 76matrix which may interfere the analysis of 60Ni and 65Cu, especially Na, Mg, Ca and 77S. The utilization of Nobias-PA1 resin, which can extract 99% of Ni and Cu from 78the seawater matrix, has partially succeeded in addressing this problem (e.g. Takano et 79al., 2017; Little et al., 2018). However, micro-gram level of primary matrix elements 80are still present, usually at around one order of magnitude higher concentrations than 81Ni and Cu (Takano et al., 2017). Minor elements, including Mn, Ti, Cr and Fe, can 82also interfere 60Ni and 65Cu measurements and also bind to Nobias-PA1 resin. 83Further purification is therefore necessary. However, successful purification methods 84require high quantities of AG-MP1 anion exchange resin (1-2 mL) and high aspect 85ratio columns (lengths 5 cm) (e.g. Maréchal et al., 1999; Borrok et al., 2007; Takano 86et al., 2017), resulting in low throughput. Even with such modifications, it can be 87difficult to fully remove interfering elements without compromising Ni and Cu 88recovery, requiring that each column be carefully tuned. Other non-reusable resins 89like Eichrom TRU Spec Resin or Nickel Resin have allowed researchers to further 90remove interfering elements (e.g. Archer and Vance, 2004; Cameron and Vance, 912014), but are expensive. In summary, recent methods for Ni and Cu purification are 92time-consuming and expensive, which may limit the throughput of sample handling, 93and are prone to error either through incomplete removal of interfering elements from 94the matrix, or by incomplete recovery of Cu or Ni. 95 Here we describe a new purification method that is fast, simple, and easy to 96couple with existing methods to purify other metals in interest (e.g. Fe, Zn and Cd). 97This procedure effectively separate Ni and Cu from other elements which may 98interfere with ICPMS analysis, while also saving time, money and effort compared to 99current techniques. We applied these techniques to measure 60Ni and 65Cu in 49 100seawater samples, but this purification approach is also suitable for other geochemical 101applications. Furthermore, the simplicity of this method highlights its potential for 102automation by robotic sample processing systems (e.g. prepFAST), that can greatly
74 8 103increase the number of high quality 60Ni and 65Cu measurements that can be made. 104 105 2. Experimental 1062.1 Reagents, standards, reference materials and samples 107 All sample preparation work was carried out in flow benches with Ultra Low 108Particulate Air (ULPA) filtration within a class 100 clean room at the University of 109Southern California. Ultrapure reagents were purchased from the manufacturer (BDH
110Aristar Ultra HF, HBr, HN4OH and H2O2; Fluka traceSELECT methanol) or 111purchased in a lower grade and further purified by sub-boiling distillation in a PFA
112still (BDH Aristar Plus: HCl, HNO3, and acetic acid). Ultrapure water (Milli-Q; 18.2 113M) was used throughout. All steps involving concentrated acids were performed in 114acid-cleaned PFA (Savillex), and purified sample solutions were stored in acid- 115cleaned polyethylene (LDPE, VWR Metal-free centrifuge tubes). In order to minimize 116Zn contamination, all clean equipment was handled with polyethylene gloves. Before 117use, batches of Nobias-PA1 resin were cleaned in 3M nitric acid for several days and 118rinsed with ultrapure water. Likewise, AG-MP1 resin was cleaned in 10% 119hydrochloric acid solution and stored in ultrapure water until use. 120 For the assessment of column elution procedures, Ni and Cu isotope ratios were 121measured in the reference materials including BCR-414 (European Commission; 122powdered and freeze-dried plankton), Arizona Test Dust (Powder Technologies 123Incorporated); natural soils collected in the southwestern US and sifted to include 124particles only in the 1–100 μm range; lot 10386BK), PACS-2 (National Research 125Council of Canada; marine sediments collected in the harbor of Esquimalt) and 126MESS-3 (National Research Council of Canada; marine sediments collected from the 127Beaufort Sea). 128 The 49 seawater samples reported in this study were collected during the 129Gradients expedition in the North Pacific aboard the R/V Ka`imikai-O-Kanaloa in 130April 2016 during a latitudinal transect along 158° W from 23° to 38° N. Samples 131were collected from the upper 600 m using 8L external-spring Niskin bottles modified 132for trace-metal sampling with titanium and Delrin brackets (Ocean Test Equipment) 133mounted on an epoxy-coated rosette and deployed on a polypropylene (Amsteel) line. 134After collection, seawater was filtered through 0.2 μm Acropack cartridge filters (Pall) 135into acid-washed 4 L low density polyethylene bottles (Nalgene). 136
95 10 1372.2 Sample pretreatment and preconcentration 138 For reference materials, approximately 100 g of sample was digested by heating
139overnight on a hotplate at 120-130 C with 1.7 mL of 14 M HNO3, 1 mL of 11 M HCl 140and 1 mL 28 M HF in a 7 mL PFA vial, and then evaporated to dryness at 120-130 C. 141To further decompose organic matter, samples were re-dissolved with 1 mL of 14 M
142HNO3 and 1 mL of 35% H2O2 and heated overnight at 120-130 C, and evaporated to 143dryness at 120-130 C. Samples were then re-dissolved with 0.5 mL 0.05 M 144ammonium acetate buffer (pH = 6.0 0.2) for Ni and Cu preconcentration with 145Nobias-PA1 resin. Nobias PA1 resin (0.6 mL) was loaded in Bio-Spin® Disposable 146Chromatography Columns (#7326008; id ~6.4 mm) and sample solution was loaded 147onto the resin. The resin was then rinsed with 5 mL 0.006 M ammonium acetate 148buffer (pH = 6.0 0.2) and eluted with 5 mL 1 M nitric acid (Table 1). 149 Nobias-PA1 resin was used to extract Ni and Cu as well as remove primary 150matrix in seawater (Table 1). Seawater samples (4L) were acidified to pH = 1.8 with 151concentrated distilled HCl (0.1% v/v), and left to equilibrate for several months. Ni 152and Cu were extracted from seawater using the two-step protocol described by 153Conway et al., 2013. 2.5 mL pre-cleaned Nobias PA1 resin was added to pH = 1.8 154seawater and shaken overnight on a shaker table. The pH of the solution was 155increased to 6.0 0.2 by addition an ammonium acetate buffer and shaken for ~5 156hours. Afterward, the resin was filtered from the solution by pouring through an acid- 157washed, 8 m polycarbonate filter (Whatman), rinsed thoroughly with Milli-Q water 158and eluted in ~20 mL 3M nitric acid (Conway and John, 2013). 159 Preconcentrated seawater samples were evaporated to dryness on a hot plate
160overnight, digested with 200 L 1:1 HCl:HNO3 for >2 hours, and evaporated to 161dryness again. To separate samples in series with published Fe, Zn, and Cd protocols 162(Conway and John 2013), the seawater sample was redissolved in 10M HCl with
1630.011% H2O2 and loaded onto microcolumns containing AG-MP1 resin (100–200 164mesh). Cu and Ni were eluted into the same vial with successive elutions of 10M HCl 165(150 l total) and 5M HCl (300 l total) and dried down at 105 C. We note that this 166first chromatography step is only necessary for obtaining purified Fe, Zn and Cd 167fractions; investigators interested only in the purification of Ni and Cu should skip 168this step. 169 Samples were then processed with the protocol shown in Fig. 1 and Table 1-4 to 170separate Ni and Cu and remove the elements which are potential bias the isotopic
116 12 171analysis. AG-MP1 exchange resin (100–200 mesh) and Nobias-PA1 resin was used in 172Bio-Spin® Disposable Chromatography Columns. After purification, the Ni and Cu 173elution fractions were collected and evaporated to dryness in 7 mL PFA vials, then
174refluxed in 1 mL of 14 M HNO3 and 0.1 mL of 35% H2O2 at 160 °C for at least 6 hours 175to decompose any leftover organic matter (Yang et al., 2019). The samples were
176evaporated to dryness again and were re-dissolved with 0.1 M HNO3 to achieve a final 177concentration of approximately 300 ppb Ni or 50 ppb Cu for isotopic analysis. To 178measure the elemental composition of purified Ni and Cu samples, a small portion of
179sample aliquot was taken and diluted in 0.5 M HNO3. The protocol of sample 180handling and analysis is shown by a flowchart in Fig. 1. 181 1822.3 Instrumentation and mass spectrometry 183 Elemental analysis was carried out by a Thermo Element 2 ICPMS using a 100 184μL min−1 Teflon nebulizer, glass cyclonic spraychamber with a PC3 Peltier Cooled 185inlet system (ESI), standard Ni sampler and Ni ‘H-type’ skimmer cones at the 186department of Earth Sciences of University of Southern California. The sensitivity 187and stability of the instrument was tuned to optimal conditions before analysis. The 188analysis was conducted with sensitivity around 106 counts s−1 for 1 ppb In. Both the 189standard and samples were doped with 1 ppb In to correct for shifts in instrumental 190sensitivity and matrix. Elemental concentrations in samples were determined by their 191signal intensity compared to a 10 ppb multi-element standard, which was diluted from 192a certified standard (Santa Clarita). 193 The isotopic composition of Ni and Cu (δ60Ni and δ65Cu) were measured on a 194Thermo Neptune Plus MC-ICPMS at the institute of Earth Sciences, Academia Sinica, 195Taiwan. Jet sampler and an ‘x-type’ skimmer cones were used to maximize sensitivity. 196Standards and samples were introduced through a 100–150 L min-1 Teflon nebulizer 197using a double-pass cyclonic spray chamber. 198 Instrumental settings and data acquisition method followed Takano et al. (2017). 199Briefly, Ni and Cu were measured in ‘high resolution’ mode, using a high resolution 200slit (25 m), in order to resolve polyatomic isobaric interferences. For Ni 201measurements, four Ni isotopes 58Ni, 60Ni, 61Ni and 62Ni were simultaneously 202measured in each analysis, along with 57Fe to correct the interference from 58Fe and 20363Cu and 65Cu to account for instrumental mass bias. δ65Cu was based on 63Cu and 65Cu 204signals, along with 69Ga and 71Ga for instrumental mass bias correction. Data
137 14 205collection consisted of 30 4.2 s cycles. Pure 0.1 M HNO3 solution was measured 206every 4–6 samples to determine background signals, which were then subtracted from 207sample intensities. Standards were also measured every 4–6 samples routinely to 208correct potential systematic drift in each occasion. Isotope ratios are expressed in per 209mil (‰) relative to the average of the standards NIST 986 Ni and NIST 976 Cu, 210respectively, using delta notation calculated as follows; 60 58 60 ( ❑¿/ ❑¿)sample 3 211δ ∋¿ 60 58 −1 ×10 [ ( ❑¿/ ❑¿)NIST SRM 986 ] 65 63 65 ( ❑Cu/ ❑Cu)sample 3 212δ Cu= 65 63 −1 ×10 [ ( ❑Cu/ ❑Cu)NIST SRM 976 ] 213 2142.4 Mass bias correction 215 We used Cu and Ga to correct the artificial isotopic fractionation on sample Ni 216and Cu caused by mass spectrometry in isotopic analysis. NIST SRM 976 Cu standard 217was added in Ni samples to achieve a ratio of 300 ppb Ni: 300 ppb Cu, and an in- 218house Ga solution (VWR Chemicals BDH®) was used for Cu at a ratio of 50 ppb Cu: 219100 ppb Ga. We note that because the method cannot correct potential isotopic 220fractionation by chromatographic separation on Ni and Cu samples, quantitative 221recovery during sample processing is essential. 222 2233. Result and discussion 224 Below, we describe our new Ni and Cu purification protocols and their 225performance, including the separation of matrix elements and potential interfering 226elements, recovery of Ni and Cu and procedure blanks. This procedure is compatible 227with methods for separating Fe, Zn and Cd within a single seawater sample described 228in Conway et al. 2013 (Fig. S1). Finally, we present the isotope analyses for various 229reference materials and seawater samples purified with this new method. 230 2313.1 Nobias-PA1 preconcentration 232 Before examining Cu and Ni recovery from seawater samples, we first tested 233Nobias-PA1 extraction of a multi-element standard with 100 ng of each element, 234which recovered 99.80.3% Ni and 99.70.1% Cu and removed over 99% of Na, 235Mg, Ca, and S, which made up the primary matrix in most natural samples (Fig. 2). 236The Cu and Ni recovery for large volume seawater samples by Nobias-PA1 was
158 16 237determined in two steps. First, 15 mL aliquots were taken from 4 L seawater samples, 238spiked a solution containing 62Ni and 65Cu, automatically preconcentrated using a 239seaFAST Pico system (Elemental Scientific) with Nobias PA-1 resin and analyzed by 240an Element 2 ICP-MS to determine total dissolved Ni and Cu concentrations. Using 241this method, Cu and Ni concentrations for GEOTRACES reference seawater GSP was 242determined to be 2.53 nM Ni and 0.60 nM Cu GSP, which agrees with average values 243from a recent intercomparison (2.60 0.14 nM Ni and 0.53 0.11 nM Cu (J. 244Moffett, personal communication). 245 Ni and Cu in 4 L samples were then preconcentrated by the batch extraction 246protocol described in section 2.2. A small portion of the extracts were taken and 247spiked with 62Ni and 65Cu spikes and measured by Element 2 ICP-MS. The 248comparison between extractable (large volume samples) and total dissolved 249concentrations (extracted by seaFAST) yields recoveries of 1023% for Ni and 250863% for Cu (2). Incomplete extraction of Cu may result from strong ligands in 251seawater can introduce bias into Cu isotopic measurements if Cu bound to strong 252ligands is isotopically distinct from extractable Cu. We note that UV oxidation of 253large volume samples is extraordinarily cumbersome, but ultimately needed to 254evaluate whether or not isotopic disequilibrium exists between Cu bound to strong 255ligands and the remainder of the Cu pool. However, because our measurements 256overlap with published measurements of seawater δ65Cu, we believe that incomplete 257sample recovery has not affected out results (see Section 3.7). 258 Besides Ni and Cu, the extracts also contain 28-865 g Na, 9-191 g Mg, 23- 2591073 g S, 1-109 g Ca, 11-224 ng Fe, 22-176 ng Mn, 2-136 ng Ti and 2-13 ng Cr. 260Each of these elements can cause isobaric interferences on Ni and Cu isotopic 261analysis. As a result, we designed the following procedure to further remove the 262elements from Ni and Cu samples. 263 2643.2 Ni and Cu separation 265 From seawater extracts, Ni and Cu must first be separated from one another, 266before they can be purified of interfering elements. Our previous study has shown that 267anion exchange chromatography using AG-MP1 resin with either 15 M acetic acid + 2681.2 M HCl or 11 M acetic acid + 4 M HCl is able to separate Ti, Cr and Fe from Ni 269and Cu (Yang et al., 2019). Both protocols can yield >98% recovery for Ni and >99% 270recovery for Cu with <1% Na, Mg and Ca in Cu fraction (Fig. 3). We adopted the 15
179 18 271M acetic acid + 1.2 M hydrochloride acid protocol to process samples (Table 2) 272because it was more effective at removing Ti from the Ni fraction (Fig. 3). Although 273this protocol results in higher Ti, 100% Mn and ~4% S recoveries in the Cu fraction, 274these elements can be quantitatively separated by a second column chemistry 275described below (Table 3). This protocol can be performed in approximately 1 hour. 276 2773.3 Cu purification 278 We developed a novel protocol by using AG-MP1 resin to further isolate Cu 279from undesirable elements based on two key observations. First, in a solution of 22 M 280methanol + 0.9 M HBr, the resin will retain Cu but not Mn and S. Second, AG-MP1 281will retain Ti in a 1M HF solution, but Cu will be eluted. This was inspired by the 282studies done over half century ago which explored trace metal adsorption onto 283strongly basic anion-exchange resins in various organic and non-organic solvents 284including methanol and hydrochloric, hydrofluoric and hydrobromic acid. Studies on 285Dowex 1 X8 resin observed that the resin could tightly bind Cu in methanol- 286hydrobromic mixture (distribution coefficient > 1000) meanwhile there was almost no 287adsorption to Mn, Ti and Cr (Korkisch and Hazan, 1965; Klakl and Korkisch, 1969). 288A similar resin Dowex 1 X10 was also studied and found to retain Ti but not Cu in 1 289M hydrofluoric acid (Faris, 1960; Nelson et al., 1960). 290 The Cu purification procedure is described below. Aliquots of the Cu fraction 291(the product of Section 3.2) were evaporated to dryness and redissolved in 1 mL 22 M 292methanol + 1.2 M HCl. After cleaning and condition the AG-MP1 resin with 5 mL 2
293M HNO3, 0.5 mL Milli-Q water and 0.8 mL 22 M methanol + 1.2 M HCl, the sample 294was loaded onto the column and resin was then rinsed with 5 mL 22 M methanol + 1.2 295M HCl to fully remove Mn and S. Cu was then eluted with 5 mL 1 M HF (Table 3). 296The protocol takes 1 hour to finish and can yield 99.70.2% Cu recovery with ≤ 1% 297Mn and Ti and ~6% S based on testing on a multi-element standard 298(Fig. 4). Ignoring the incomplete recovery of Cu from seawater 299during Nobias-PA1 preconcentration, the combined Cu recovery of 300the protocols for seawater samples was 105±7%. The larger uncertainty 301here derives from reduced precision associated with external calibration of Cu 302concentration by MC-ICPMS compared to isotope dilution via Element 2. 303 3043.4 Ni purification
1910 20 305 As described in section 3.1, Nobias-PA1 preconcentration of seawater can still 306allow g levels of major matrix elements existing in trace metal extracts. In the Ni 307and Cu separation protocol (Section 3.2), these elements are partitioned into Ni 308fraction (Fig. 3). To avoid their interference on Ni isotopic analysis, a second Nobias- 309PA1 purification was carried out to isolate Ni from the elements (Table 4), which 310takes around 1 hour. Because extended processing can lead to inadvertent 311contamination of Fe and other metals that are also extracted by Nobias-PA1, a final Ni 312separation procedure was performed. This follows the same AG-MP1 separation 313protocol from Section 3.2 (Table 4) and takes ≤ 1 hour to perform. External 314calibration (beam intensity matching) during 60Ni analysis indicated that 315overall Ni recovery from these protocols was 96±11%. 316 3173.5 Blanks 318 Overall procedural blanks (including Nobias-PA1 extraction, Ni and Cu 319separation and purification) were ≤ 1 ng Ni and Cu, which is equivalent to ≤4 pM Ni 320and Cu in a 4 L seawater samples. Compared to the concentrations of samples we 321processed, 2.1-6.3 nM Ni (120–375 ng) and 0.5-1.7 nM Cu (30 – 110 ng), these 322blanks are insignificant. 323 3243.6 Validation of purification method 325 The accuracy of our analytical conditions was verified by determining the 326isotope composition of multiple international standards for each element on the same 327day. Ni standard Wako Ni was -0.27±0.02 ‰ compared to NIST SRM 986 (2, n = 32810). NIST SRM 3114 Cu was -0.07±0.04 ‰ (2, n = 6) and Cu standard Wako Cu 329was +0.27±0.02 ‰ compared to Cu standard NIST SRM 976 (2, n = 5). These 330values are statistically consistent with reported values previously (Takano et al., 2017; 331Baconnais et al., 2019) (Table 5). The analytical reproducibility for the reported 332isotopic composition of each sample was obtained by combining the internal precision 333of both sample and standard measurements (Conway et al., 2013). The precision of 334single measurement (~30 cycles) ranged from ±0.05 to ±0.08 ‰ for Ni and from 335±0.05 to ±0.19 ‰ for Cu. 336 We used reference materials including BCR-414 (plankton), Arizona Test Dust 337(continental sediments), PACS-2 and MESS-3 (marine sediments) to test our 338purification method (Table 5). The high Fe, Mn, Ti and Cr content of these samples
2111 22 339allows the merit of this method to be demonstrated. For example, the marine plankton 340sample BCR-414 contains around 2000 ppm Fe, 300 ppm Mn, 100 ppm Ti and 25 341ppm Cr while the Ni and Cu contents are approximately 20 and 30 ppm, respectively. 342Following purification, these elements were all <1 ppb while recovery of Ni and Cu 343were over 99%. Isotopic analysis yielded values of 60Ni = +0.060.13 ‰ and 65Cu 344= -0.320.05 ‰ (2) in BCR-414. These are consistent with the values of 60Ni = 345+0.110.06 ‰ and 65Cu = -0.270.05 ‰ obtained independently by S. Takano at 346Kyoto University, Japan (personal communication). 347 Cu isotope values for marine sediment standards PACS-2 and MESS-3 were 348determined to be -0.030.04 ‰ and -0.100.06 ‰, respectively. These results agree 349the values published in Araújo et al. (2019) (+0.050.06 ‰ for PACS-2 and 3500.000.10 ‰ for MESS-3). Furthermore, we report 65Cu of Arizona Test Dust to be 351+0.160.04 ‰. We are unaware of published measurements of this material, although 352our determination overlaps with 65Cu values for igneous rocks and clastic sediments 353(+0.080.17 ‰ and +0.080.20 ‰, respectively; compiled by Moynier et al., 2017). 35460Ni values for PACS-2, MESS-3 and Arizona Test Dust were determined to be 355+0.190.10 ‰, -0.040.09 ‰, and -0.090.11 ‰, respectively, and are reported for 356the first time in this study. The values overlap with the isotopic ranges of continental 357sediments (soil, loess and river sediment; -0.040.02 ‰ to +0.230.08 ‰) and deep- 358sea sediments (clay; +0.020.03 ‰ and +0.040.04 ‰) (Cameron et al., 2009; 359Cameron and Vance, 2014; Ratié et al., 2015 ). As PACS-2, MESS-3 and Arizona Test 360Dust have different origins (harbor sediments, offshore sediments and nature soils, 361respectively), the slight differences in 60Ni among the materials might reflect 362different sources. 363 3643.7 Dissolved Nickel and Copper isotopic composition in the North Pacific 365 Seawater samples from the upper water column of the North Pacific were 366collected on the Gradients cruise in April 2016 and were analyzed for Ni and Cu 367concentration and isotopic composition (Fig. 5 and 6; Table S1). Both elements have a 368similar distribution with an upwelling source from deep waters leading to elevated 369concentrations at the northern end of the transect (36N), and lower concentration in 370subtropical surface waters further to the south. The general features of their 371distributions along this transect are similar to nitrate, phosphate, and other 372phytoplankton macronutrients. In surface waters, however, latitudinal gradients of Ni
2312 24 373and Cu are less intense than observed for macronutrients, indicating slower rates of 374uptake and/or export (Pinedo-Gonzalez et al., in prep). 375 Dissolved Ni in the deepest samples (600 m) has a uniform isotopic composition 376of +1.370.07 ‰ (2; n = 3). Toward the surface, 60Ni increases in all subtropical 377stations, reaching a maximum of +1.730.07 ‰ in the surface waters where 378dissolved Ni is at a minimum. With the exception of a few outliers, this pattern of 379higher 60Ni for decreasing dNi holds for the entire dataset. We observe a correlation 380between 60Ni and the natural log of dissolved nickel (R2 = 0.59; Fig. 7a), suggesting 381biological Rayleigh fractionation associated with phytoplankton uptake. Least squares 382regressions indicate a fractionation factor of 0.99971 (2) best explains the change 383in 60Ni along the section. 384 The biological removal of light Ni isotopes which can be inferred from our 385dataset is consistent with a profile of 60Ni in the South Pacific Gyre (Takano et al. 3862017), which shows a decrease of 0.4 ‰ associated with a 6 nM decrease in dissolved 387nickel and a surface 60Ni of +1.7 ‰, similar to our measurements (Fig. 5a and b). It 388is interesting that such fractionation is not observed further north in the Subarctic 389North Pacific (Cameron and Vance, 2014), or in other upwelling regions such as the 390Southern Ocean (Wang et al. 2019). In these cases, 60Ni is uniform in the water 391column at approximately +1.4 ‰, which overlaps with deepest samples, even though 392partial depletion of Ni in the surface ocean was observed. While an ongoing Ni 393isotope intercalibration will add confidence to these isotopic differences, it is possible 394that Ni isotope fractionation may be localized in subtropical environments. 395Widespread Ni enzymes such as Ni superoxide dismutase and urease are likely more 396important in lower latitudes where both solar irradiation and nitrogen recycling is 397intense. These environments also harbor diazotrophic cyanobacteria such as 398Trichodesmium, which have a much higher demand for Ni than other phytoplankton 399groups, due to the use of NiFe nitrogenase, uptake hydrogenase, as well as increased 400demand for Ni superoxide dismutase. We note that heavy Ni use associated with 401methanogens also results in a strong Ni isotope fractionation ( = ~0.9992; Cameron 402et al. 2009). 403 Unlike Ni, the 63Cu of dissolved Cu is relatively uniform and does not show 404strong evidence for fractionation during biological uptake. Despite a two-fold 405difference in deep and shallow Cu concentrations, the 63Cu of the 10 lowest Cu 406samples (+0.590.16, 2) is statistically identical to the 63Cu of the 10 highest
2513 26 407samples (+0.680.21, 2). Our measurements of 63Cu overlap with published 408measurements, which mostly fall between +0.4 and +0.8 ‰ (Fig. 7b; Thompson and 409Ellwood, 2014; Takano et al, 2014; 2017; Little et al., 2018; Boyle et al., 2012; 410Baconnais et al., 2019), despite incomplete extraction of Cu from seawater with 411Nobias PA-1 resin (average recovery from this sample set is 86%). Recently, Little et 412al. (2018) reported no significant difference in 63Cu between samples with ‘good’ and 413‘poor’ and recovery. However, extraction of Cu by magnesium hydroxide co- 414precipitation appears to result in systemically heavier 63Cu (Vance et al. 2008) 415compared to Nobias PA1 extracted samples at similar concentration (Fig. 7b). 416 Limited variability in 65Cu in our dataset and other from other ocean basins 417indicates that net isotopic fractionation associated with biological uptake, 418remineralization, and scavenging is probably insignificant. This may reflect the 419important role of organic ligands in the marine Cu cycle, which probably have masses 420>500 Da, and the fact that phytoplankton may take up ligand-bound Cu directly. 421Complexation with ligands makes the relative mass difference between 63Cu and 65Cu 422extremely small, making mass dependent fractionation insignificant. In contrast, 423electrochemical studies indicate that only a fraction of dissolved nickel is bound by 424ligands, with a large concentration of inorganic Ni species. Therefore, isotopic 425fraction associated with Ni uptake may indicate direct uptake of Ni2+ and inorganic 426species, while the lack of fraction uptake associated with Cu uptake may indicate 427uptake by organic Cu complexes. 428 Despite the lack of systematic change in 65Cu, there is some evidence in our 429dataset for coherent variation in Cu isotopic composition near the surface ocean. 430Negative excursions in 63Cu at 100m to ~0.4‰ are observed at the two northern- 431most stations of the Gradients transect. Slight decreases in 65Cu are also found at the 432southern end of our transect (23N). Hints of this effect are also observed in published 433Cu isotope profiles (Takano et al., 2014; 2017; Little et al., 2018). We anticipate that 434high resolution studies associated with GEOTRACES sections will provide more 435conclusive documentation of these features and perhaps identify their origin. 436 437Conclusion 438 We have developed a new method for purification of Ni and Cu from seawater 439samples for determination of their stable isotope ratios. The method successfully 440recovers Ni and Cu after preconcentration from seawater matrix, while effectively
2714 28 441separating the metals from other major and minor matrix elements which might 442interfere with analysis. The most notable improvement in this method compared to 443previous techniques is the ability to easily and effectively purify Ni and Cu with 444separation of Ti, Mn and Cr, ensuring the accuracy of measurements of 60Ni and 44563Cu. This method can be also utilized for environmental and geological samples 446which typically contain high levels of Ti, Mn and Cr. 447 Application of this method to 49 samples in the North Pacific has revealed 448widespread fractionation of 60Ni due to biological Ni uptake, confirming initial 449observations from a single profile by Takano et al. (2017). Tandem Ni and Cu isotope 450measurements also highlights the lack of Cu isotope fraction in these waters, despite 451similar magnitudes of Ni and Cu uptake by phytoplankton. 452 This new method has the potential to greatly increase the throughput of Ni and 453Cu isotope measurement and is suitable for sectional studies, such as GEOTRACES 454transects. Each column protocol takes only about 1 hour to process and the resin is 455reusable. This procedure is not only fast, but simple enough to be automated (Bian et 456al., in prep). Furthermore, this technique can be coupled with purification of Fe, Zn, 457and Cd (Conway et al. 2013), allowing isotopic measurements of metals to be 458performed from in a single seawater sample (Table 2). 459
2915 30 460Acknowledgement 461We would like to thank Shotaro Takano for providing pure Ni and Cu isotope 462standards, Irit Tal and Wen‐Hsuan Liao for technical support. We acknowledge the 463Captain and Crew of the R/V Ka`imikai-O-Kanaloa, Chief Scientist E. Virginia 464Armbrust and Ryan Tabata for sampling support. This work was supported the Simons 465Foundation (SCOPE award 329108 to S.G.J., Gradients award 426570SP to S.G.J., 466and 602538 to NJH), the National Science Foundation (NSF-OCE 1736896 to S.G.J.), 467and the Taiwan-USC Postdoctoral Fellowship Program (S.C.Y). We would also like to 468thank Editor xxx and xxx anonymous referees for their helpful comments. 469
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3920 40 606
4121 42 607Tables 608
Table 1. Protocol for preconcentration of Ni and Cu by Nobias-PA1 resin. For seawater For reference material Collection a. Adding 2.5 mL pre-cleaned resin a. Loading 0.6 mL resin into a in seawater; shaking overnight column, cleaning resining
with 5 mL 1 M HNO3, rinse with Milli-Q water, and conditioning with 0.05 M
CH3COONH4 (pH = 6) b. Adjusting sample pH to 6 with b. Loading sample onto resin
CH3COONH4 and HN4OH; shaking (in 0.05 M CH3COONH4, pH = for ~5 hours 6) c. Filtering resin c. Loading sample onto resin Na, Mg, Ca, P, S d. Rinsing with ~125 mL Milli-Q d. Rinsing with 5 mL 0.006 M Na, Mg, Ca, P, S water CH3COONH4 (pH = 6) e. Eluting with ~20 mL 3 M HNO3 e. Eluting with 5 mL 1 M HNO3 Ni, Cu, other trace metals
609 610
4322 44 611
Table 2. Protocol for separating Ni and Cu in trace metal extracts by AG-MP1 resin. Step Reagent Volume Collection Loading AG-MP1 resin into columns 0.25 mL
Cleaning resin 2 M HNO3 2 mL Rinsing Milli-Q 0.5 mL
Conditioning 15 M CH3COOH + 1.2 M HCl + 0.003% H2O2 0.4 mL 2
Sample (15 M CH3COOH + 1.2 M HCl + Sample loading 0.5 mL Ni 0.003% H2O2) 0.25 mL Ni elution 15 M CH3COOH + 1.2 M HCl + 0.003% H2O2 Ni, Cr 4
Matrix removal 15 M CH3COOH + 1.2 M HCl + 0.003% H2O2 0.5 mL 8 Ti, Cr 0.4 mL Cu elution 5 M HCl + 0.003% H2O2 Cu, Ti, Mn 16 0.2 mL Fe elution* 1 M HCl Fe, Mo 14 0.2 mL Zn elution* 2 M HNO3 + 0.1 M HBr Zn, Sn 12 0.2 mL Cd elution* 2 M HNO3 Cd 10 * Steps following Yang et al. (2019).
4523 46 612
Table 3. Protocol for final purification of Cu by AG-MP1 resin. Step Reagent Volume Collection Loading AG-MP1 resin into columns 0.25 mL
Cleaning resin 2 M HNO3 2 mL Rinsing Milli-Q 0.5 mL Conditioning 22 M methanol + 0.9 M HBr 0.4 mL 2 Sample loading Sample (22 M methanol + 0.9 M HBr) 1 mL Ti, Mn, Cr Matrix removal 22 M methanol + 0.9 M HBr 1 mL 5 Ti, Mn, Cr
Cu elution 1 M HF + 0.003% H2O2 1 mL 5 Cu
Leftover 2 M HNO3 1 mL 5 Ti 613 614
4724 48 615
Table 4. Protocols for final purification of Ni. Step Reagent Volume Collection Loading Nobias-PA1 resin into columns 0.6 mL
Cleaning resin 1 M HNO3 5 mL Rinsing Milli-Q 0.5 mL
Conditioning 0.05 M CH3COONH4 (pH = 6) 0.5 mL 2
Sample loading Sample (0.05 M CH3COONH4, pH = 6) 0.5 mL Na, Mg, Ca, P, S
Matrix removal 0.006 M CH3COONH4 (pH = 6) 1 mL 5 Na, Mg, Ca, P, S
Ni elution 1 M HNO3 1 mL 5 Ni
Loading AG-MP1 resin into columns 0.25 mL
Cleaning resin 2 M HNO3 2 mL Rinsing Milli-Q 0.5 mL
Conditioning 15 M CH3COOH + 1.2 M HCl + 0.003% H2O2 0.4 mL 2
Sample (15 M CH3COOH + 1.2 M HCl + Sample loading 0.5 mL Ni 0.003% H2O2) 0.25 mL Ni elution 15 M CH3COOH + 1.2 M HCl + 0.003% H2O2 Ni 4
616 617
4925 50 618
Table 5. 60Ni and 65Cu in pure standards and reference materials. Material Result in this Reference value study Ni type 60Ni ±2 n 60Ni ±2 (‰) (‰) Wako Pure standard -0.27±0.02 1 -0.28±0.03 a 0 BCR-414 Plankton +0.06±0.13 1 +0.11±0.06 b PACS-2 Marine sediment +0.190.10 1 MESS-3 Marine sediment -0.040.09 1 Arizona test dust Natural soil -0.090.11 1
Cu type 65Cu ±2 n 65Cu ±2 (‰) (‰) NIST SRM 3114 Pure standard -0.07±0.04 6 -0.06±0.02 c Wako Pure standard +0.27±0.02 5 +0.28±0.03 a BCR-414 Plankton -0.32±0.05 2 -0.27±0.05 b PACS-2 Marine sediment -0.030.04 2 +0.05±0.06 d MESS-3 Marine sediment -0.100.06 2 0.00±0.10 d Arizona test dust Natural soil +0.160.04 2 a Takano et al. (2017) b Obtained independently by S. Takano at Kyoto University, Japan (personal communication) c Baconnais et al. (2019) d Araújo et al. (2019) 619 620
5126 52 621Figures
Acidified seawater (pH 2)
Elemental analysis Isotopic analysis
Take 15 mL, add 50 μLbuffer Extract Ni, Cu and other metals (Fe, Zn, and Ni & Cu spikes Cd…) by Nobias-PA1 (table 1)
Separate Ni, Cu (& Fe, Zn, Cd) fractions seaFastpreconcentration (Fe, Zn, Cd) (Nobias-PA1) by AG-MP1 (table 2) Cu fraction Ni fraction Analysis of total dissolved [Ni] and [Cu] on Element 2 Further remove Mn, Ti Further remove major matrix by AG-MP1 (table 3) elements by Nobias-PA1 (table 4)
Cu isotopic analysis Further remove Ti, Fe by AG-MP1 (table 4)
Ni isotopic analysis 622
623Figure 1. Flowchart of the sample handling and analysis. Highlighted steps are 624outlined in Tables 1-4. 625
5327 54 100% 80% 60% Ni 40%
e Cu c
n 20% a 0.2% Ni 0.3% Cu d
n 0% u
b Sample loading & 5mL 0.006 M 5 mL 1M HNO3 elution a buffer rinse e v i t a l 100% e R 80% Na 60% Mg 40% Ca 20% <1% Na Mg Ca S S 0% Sample loading & 5mL 0.006 M 5 mL 1M HNO3 elution buffer rinse 626
627Figure 2. Recovery of Ni, Cu and major elements during preconcentration with 628Nobias-PA1 resin. A multi-element standard containing 100 ng Ni, Cu, Na, Mg, Ca, P 629and S was dried and re-dissolved in in 0.5 mL 0.05 M ammonium acetate (pH = 6), 630loaded onto Nobias-PA1 resin. Bars show relative abundance of elements eluted 631during a combined loading and rinsing step, and during acid elution. recoveries of 632duplicate tests. 633
5528 56 100% >98% Ni >99% Cu 80% 60% 40% Ni 20% Cu <2% Ni <0.2% Cu 0% Sample loading 4 mL 15 M 5 mL 15 M 6.4 mL 5 M HCl & 1 mL 15 M AA+1.2 M HCl AA+1.2 M HCl AA+1.2 M HCl e
c 100% n
a 80% d
n Mn u 60% b a 40% Ti e v i 20% t Cr a l
e 0% Fe R Sample loading 4 mL 15 M 5 mL 15 M 6.4 mL 5 M HCl & 1 mL 15 M AA+1.2 M HCl AA+1.2 M HCl AA+1.2 M HCl
100% 80% 60% Na 40% Mg 20% S 0% Ca Sample loading 4 mL 15 M 5 mL 15 M 6.4 mL 5 M HCl & 1 mL 15 M AA+1.2 M HCl AA+1.2 M HCl 634 AA+1.2 M HCl
635Figure 3. Separation of Ni and Cu fractions. Recoveries of Ni, Cu, interfering 636elements Mn, Ti, Cr, Fe, and matrix elements Na, Mg, Ca and S following the 637purification outline in Table 2 with AG-MP1 resin. A multi-element standard with 100 638ng of each element was resuspended in 15 M acetic acid (AA) + 1.2 M HCl eluent.
639All reagents used contain 0.003% H2O2. 640
5729 58 100% >99% Cu 80% 60% 40% Cu 20% e c
n 0% a
d Sample loading & 5 mL 22 5 mL 1 M HF + 0.003% 5 mL 2 M HNO3 n
u M MeOH + 0.9 M HBr H2O2 b a
e v
i 100% t a l
e 80%
R Ti 60% Mn 40% Cr 20% ≤1% Ti Mn Cr, 6% S S 0% Sample loading & 5 mL 22 5 mL 1 M HF + 0.003% 5 mL 2 M HNO3 M MeOH + 0.9 M HBr H2O2 641 642Figure 4. Final copper purification. Recoveries of Cu, Ti, Mn, Cr, and S from a multi- 643element standard containing 100 ng of each Cu purification protocol by using AG- 644MP1 resin. Bars represent average recovery from triplicate tests. Samples were 645dissolved in 0.5 to 1 mL 22 M methanol (MeOH) + 0.9 M hydrobromic acid (HBr). 646 647
5930 60
[Ni](nM) δ60Ni (‰) 0 4 8 12 1.0 1.5 2.0 0 0 A B 400 400 )
m 800 800 (
h t p
e 1200 1200 D
1600 N Pacific 1600 NE Pacific S Pacific 2000 2000
[Cu](nM) δ65Cu(‰) 0 1 2 3 0.0 0.5 1.0 1.5 0 0 C D 400 400
) 800 800 m (
h t 1200 1200 p e D 1600 1600 N Pacific NE & NW Pacific S Pacific 2000 2000 648 650Figure 5. Profiles of dissolved Ni concentration (A), dissolved 60Ni (B), dissolved Cu 651concentration (C) and dissolved 63Cu (D) from the North Pacific. Blue circle shows 652seawater samples collected on the Gradients expedition (April 2016). Black cross in A 653and B represents data of NE Pacific (Cameron and Vance, 2014) and red cross 654represents data of S Pacific (Takano et al., 2017). In C and D, black cross represents 655NE and NW Pacific data from Takano et al. (2014) and red cross represents S Pacific 656data from Thompson and Ellwood (2014) and Takano et al. (2017). 657
6131 62 658
6332 64 [Ni] (nM) [Cu] (nM)
A B C ) m (
h t
p δ60Ni(‰) δ65Cu (‰) e D
D E
659 Latitude (°N)
660Figure 6. Sampling sites (A), dissolved Ni concentration (B), dissolved Cu 661concentration (C), dissolved 60Ni (D) and dissolved 63Cu (E) from the Gradients 662transect in the North Pacific. 663 664
6533 66 665 666Figure 7. Compilation of dissolved 60Ni and dissolved 63Cu measurements from in 667the global oceans (A and B, respectively). Data sources for Ni include Cameron and 668Vance (2014) (N Atlantic, Southern Ocean and NE Pacific), Takano et al. (2017) (S 669Pacific) and Wang et al. (2019) (Southern Ocean). Literature Cu data is from Vance et 670al. (2008) (NE Pacific and Indian Ocean), Thompson and Ellwood (2014) (S Pacific), 671Takano et al. (2014) (Southern Ocean and NW Pacific), Takano et al. (2017) (S 672Pacific), Little et al. (2018) (S Atlantic), Boyle et al. (2012) (NW Atlantic) and 673Baconnais et al. (2019) (NE Atlantic). 674 675
6734 68 676Supplementary information 677
Multi-element standard Metals preconcentrated from seawater by Nobias-PA1 (n=1) (n = 2, 1SD)
15M CH3COOH + 1.2 2 M HNO3 + 15M CH3COOH + 1.2 2 M HNO3 + M HCl + 0.003% H2O2, 5 M HCl + 0.003% H2O2, 1 M HCl, 0.1 M HBr, 2 M HNO3, M HCl+ 0.003% H2O2, 5 M HCl+ 0.003% H2O2, 1 M HCl, 0.1 M HBr, 2 M HNO3, 0.05 mL x 6 0.08 mL x 8 0.04 mL x 7 0.04 mL x 6 0.04 mL x 5 0.05 mL x 6 0.08 mL x 8 0.04 mL x 7 0.04 mL x 6 0.04 mL x 5
1 0 0 % 1 0 0 % 8 0 % 8 0 % 6 0 % 6 0 % 4 0 % 4 0 % 2 0 % 2 0 % 0 % 0 % 0 . 0 0 0 . 5 0 1 . 0 0 1 . 5 0 2 . 0 0 M a t r i x + N i C u F e Z n C d E l u e n t ( m L ) F r a c t i o n 678 N i C u F e Z n C d N i C u F e Z n C d
679Figure1 0 0 % S1. Separation of Ni, Cu, Fe, Zn and1 0 0 %Cd by AG-MP1 micro columns. Design of 8 0 % 8 0 % 680the6 0 % column and resin volume (20 L) are identical6 0 % with that of Conway et al. (2013). 4 0 % 4 0 % 681A2 0multi-element% standard with 100 ng of 2 each0 % element and a preconcentrated 0 % 0 % 682seawater0 . 0 0 trace0 . 5 0 metal1 . 0sample0 1(resuspended. 5 0 2 . 0 0 inM a 15t r i x + N Mi aceticC u acidF e + 1.2 Z nM HCl + C d0.003% E l u e n t ( m L ) F r a c t i o n 683H2O2) were usedM n forT i theM g test.C a M n T i M g C a 684
6935 70 Table S1. Concentrations and isotopic composition of Ni and Cu from the Gradients transect in the North Pacific Latitude Longitude Depth [Ni] δ60Ni 2 [Cu] δ65Cu 2 Station (°N) (°W) (m) (nM) (‰) (‰) (nM) (‰) (‰)
2 23.57 158.00 15 2.11 +1.59 ±0.07 0.52 +0.52 ±0.08 2 23.57 158.00 45 2.14 +1.69 ±0.06 0.49 +0.50 ±0.07 2 23.57 158.00 130 2.12 +1.64 ±0.07 0.53 +0.57 ±0.08 2 23.57 158.00 150 2.19 +1.61 ±0.06 0.56 +0.56 ±0.09 2 23.57 158.00 200 2.45 +1.62 ±0.07 0.69 +0.52 ±0.08 2 23.57 158.00 400 4.36 +1.51 ±0.07 1.13 +0.75 ±0.08
4 28.14 158.00 15 2.09 +1.31* ±0.08* 0.50 +0.63 ±0.08 4 28.14 158.00 45 2.13 +1.67 ±0.07 0.55 +0.99 ±0.10 4 28.14 158.00 120 2.16 +1.46 ±0.06 0.56 +0.62 ±0.07 4 28.14 158.00 150 2.19 +1.57 ±0.07 0.70 +0.65 ±0.07 4 28.14 158.00 200 2.45 +1.69 ±0.06 0.65 +0.59 ±0.08 4 28.14 158.00 400 3.44 +1.49 ±0.07 0.98 +0.67 ±0.09
5 29.45 158.00 15 2.36 +1.64 ±0.07 0.48 +0.74 ±0.08 5 29.45 158.00 15 2.02 +1.69 ±0.06 0.45 +0.62 ±0.08
6 32.68 158.00 15 2.19 +1.63 ±0.06 0.55 +0.60 ±0.08 6 32.68 158.00 45 2.14 +1.70 ±0.06 0.53 +0.59 ±0.09 6 32.68 158.00 70 2.23 +1.18* ±0.06* 0.56 +0.64 ±0.08 6 32.68 158.00 150 2.39 +1.63 ±0.05 0.68 +0.56 ±0.07 6 32.68 158.00 200 2.63 +1.58 ±0.05 0.75 +0.67 ±0.08 6 32.68 158.00 400 3.66 +1.51 ±0.05 1.13 +0.78 ±0.08 6 32.68 158.00 600 5.62 +1.39 ±0.07 1.54 +0.64 ±0.08
7 34.05 158.00 15 2.42 +1.56 ±0.08 0.71 +0.70 ±0.19
8 37.30 158.00 15 2.89 +1.57 ±0.06 0.91 +0.58 ±0.08 8 37.30 158.00 45 2.90 +1.55 ±0.07 0.93 +0.40 ±0.08 8 37.30 158.00 75 2.90 +1.46 ±0.06 0.88 +0.48 ±0.08 8 37.30 158.00 150 2.93 +1.51 ±0.06 0.87 +0.54 ±0.08 8 37.30 158.00 200 2.99 +1.40* ±0.06* 0.93 +0.64 ±0.09 8 37.30 158.00 400 4.31 +1.35 ±0.05 1.35 +0.70 ±0.08 8 37.30 158.00 600 6.25 +1.33 ±0.06 1.74 +0.58 ±0.09
7136 72 9 36.57 158.00 15 2.94 +1.53 ±0.05 1.02 +0.57 ±0.08 9 36.57 158.00 60 2.85 +1.59 ±0.05 1.00 +0.48 ±0.08 9 36.57 158.00 200 2.90 +0.87* ±0.07* 0.95 +0.60 ±0.09 9 36.57 158.00 400 4.23 +1.36 ±0.07 1.26 +0.59 ±0.09
10 35.46 158.00 15 2.65 +1.56 ±0.06 0.80 +0.56 ±0.10 10 35.46 158.00 45 2.66 +1.53 ±0.08 0.79 +0.59 ±0.07 10 35.46 158.00 75 2.75 +1.28 ±0.07 0.86 +0.61 ±0.07 10 35.46 158.00 150 2.74 +1.50 ±0.06 0.81 +0.62 ±0.07 10 35.46 158.00 200 2.93 +1.47 ±0.06 0.88 +0.66 ±0.07 10 35.46 158.00 400 4.11 +1.54 ±0.05 1.26 +0.89 ±0.08 10 35.46 158.00 600 5.85 +1.40 ±0.07 1.68 N/A** N/A**
12 33.09 158.00 15 2.29 +1.63 ±0.08 0.66 +0.74 ±0.08 12 33.09 158.00 400 3.72 +1.37 ±0.06 1.21 +0.59 ±0.07
13 29.70 158.00 15 2.11 +1.62 ±0.07 0.54 +0.59 ±0.09
14 26.36 158.00 15 2.08 +1.73 ±0.07 0.47 +0.69 ±0.07 14 26.36 158.00 45 2.13 +1.66 ±0.06 0.48 +0.56 ±0.07 14 26.36 158.00 80 2.05 +1.70 ±0.06 0.53 +0.48 ±0.07 14 26.36 158.00 150 2.33 +1.66 ±0.06 0.65 +0.69 ±0.09 14 26.36 158.00 200 2.71 +1.58 ±0.06 0.72 +0.71 ±0.08 14 26.36 158.00 400 4.04 +1.51 ±0.06 1.24 +0.71 ±0.08
* The four relatively light values are highly likely inaccurate. In figure S2 which compares 60Ni/58Ni and 62Ni/58Ni raw ratios analyzed from pure standards and samples, the ratios of the four samples deviate from the main fractionation pattern. ** Data not available due to that Ga was missed to add into Cu samples. 685
7337 74 0.0602
0.0601 ) d e r u s
0.0600 a e m (
i N
0.0599 8 5 / i N 2 6 0.0598
0.0597 0.4075 0.4080 0.4085 0.4090 0.4095 60Ni/58Ni (measured) 686 687Figure S2. 60Ni/58Ni and 62Ni/58Ni raw ratios analyzed from pure standards and 688samples. The symbols in red are raw data of the four abnormally light δ60Ni values in 689table S1. 690
7538 76