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A New Purification Method for Ni and Cu Stable Isotopes in Seawater 2Shows Widespread Ni Isotope Fractionation by Phytoplankton 3 4Shun-Chung Yang1*, Nicholas J

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A New Purification Method for Ni and Cu Stable Isotopes in Seawater 2Shows Widespread Ni Isotope Fractionation by Phytoplankton 3 4Shun-Chung Yang1*, Nicholas J 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
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