Fink-Jensen, Peter

Hüssy, Karin

Nielsen, Kristian Ege

Serre, Simon Hansen

Thomsen, Tonny Bernt

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetAnalysis of cod otolith microchemistry by continuous line transects using LA-ICP-MSSerre, Simon Hansen; Nielsen, Kristian Ege; Fink-Jensen, Peter; Thomsen, Tonny Bernt;Hüssy, KarinPublished in:Geological Survey of Denmark and Greenland Bulletin (GEUS)Publication date:2018Document versionPublisher's PDF, also known as Version of recordDocument license:UnspecifiedCitation for published version (APA):Serre, S. H., Nielsen, K. E., Fink-Jensen, P., Thomsen, T. B., & Hüssy, K. (2018). Analysis of cod otolithmicrochemistry by continuous line transects using LA-ICP-MS. Geological Survey of Denmark and GreenlandBulletin (GEUS), 41, 91-94.Download date: 03. Feb. 202091Fish otoliths, also called ear stones or statoliths, are calcified structures functioning as movement and equilibrium indica-tors in the inner ear of fish (Fig. 1). From hatching to death these structures grow incrementally, with new material ac-creted daily (Pannella 1971) in successive layers of protein (1–8%, Degens et al. 1969) and calcium carbonate. The ac-cretion rate of otoliths varies with fish growth, and in tem-perate species it is usually lowest during the winter season (Hüssy et al. 2010). This results in concentric growth resem-bling the ringed structure in trees (Fig. 1D), enabling the use of dendrochronological techniques to approximate the age and growth history of fish. During growth, certain elements are incorporated into the otolith structure, some associated with proteins and some with the calcium carbonate compo-nent (Thomas et al. 2017), supplying a valuable record of dif-ferent aspects in fish life history and serving as a potential environmental record. Previous studies show that trace element and isotopic compositions of otoliths can be used as a proxy for recon-structing water chemistry, temperature and salinity (Pat-terson et al. 1993; Thorrold & Shuttleworth 2000). Other studies demonstrate that elemental histories can be used to investigate fish spawning and migration patterns (e.g. Stur-rock et al. 2012), and more recent studies use elements such as Zn, Cu and Mg as indicators of seasonality (Hüssy et al. 2016; Limburg et al. 2018). Combining this knowledge of elemental variation with the micro-beam capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) turns otolith microchemistry into a powerful tool for studying important parameters fundamental for estab-lishing modern, sustainable fisheries management policies (e.g. stock identification, migration, pollution indicators, spawning habitats, duration of larval and juvenile stages, and magnitude and timing of spawning). We present an analytical method developed by the Geo-logical Survey of Denmark and Greenland (GEUS) in col-laboration with the National Institute of Aquatic Resources, Technical University of Denmark (DTU Aqua), for element abundance analysis in otoliths. Analyses of otoliths from Baltic Cod (Gadus morhua; Fig. 1) are used as an example for its application.Analytical approachThe microchemical analysis of otoliths focuses on Mg, P, Ca, Mn, Cu, Zn, Sr and Ba, as these elements are typically incor-porated into otoliths, and are either subject to environmental control (e.g. Sr and Ba correlate with water salinity) or physi-ological control (e.g. Zn, Cu and Mg are useful to the inter-pretation of otolith growth history; Hüssy et al. 2016 and references therein; Limburg et al. 2018). The LA-ICPMS fa-cility at GEUS employs a NWR213 laser system coupled to an ELEMENT 2 double-focusing, single-collector magnetic sector field ICPMS. Operating conditions, data acquisition and processing parameters are listed in Table 1. LA-ICPMS is often the preferred technique for rapid, in-situ analyses of trace elements and isotopes obtained from natural samples, Simon Hansen Serre, Kristian Ege Nielsen, Peter Fink-Jensen, Tonny Bernt Thomsen and  Karin HüssyAnalysis of cod otolith microchemistry by continuous  line transects using LA-ICP-MS5 mmCDA2 mmBFig. 1. A: Cod specimen caught in the Baltic Sea. B: Removal of otoliths, the cut is situated just above the eyes. C: Otolith, with dotted line show-ing the direction of a cross-section. D: Photograph (reflected light) of a polished cross-section of an otolith. The red line shows the position of the laser scan.© 2018 GEUS. Geological Survey of Denmark and Greenland Bulletin 41, 91–94. Open access: www.geus.dk/bulletin92as it requires little sample preparation and can produce high sample throughput, extracting elemental and isotopic infor-mation at a micrometre scale. Most conventional LA-ICPMS analysis is performed by spot analyses, following a bracketing analysis protocol using well-characterised standard materials. This is a powerful method when studying specific areas in solid materials. However, for compositional variations along millimetre- to centimetre-scale transects, the spot approach becomes very time-consuming. For example, a 5 mm long transect requires about one hundred spots 40 µm in diam-eter, taking 3–4 hours to complete. Spot analysis also intro-duces difficulties like downhole-element fractionation when the laser drills into the material. Instead, research of fish oto-lith microchemistry favours faster sampling approaches such as line scans across the sample to acquire continuous time-resolved compositional profiles (a 5 mm long line takes c. 17 min, using a fixed scan speed of 5 μm s-1; e.g. Søndergaard et al. 2015; Hüssy et al. 2016). This approach is rapid, suppress-es depth heterogeneity and avoids downhole elemental frac-tionation, as it ablates only to a depth of a few microns. The potential interfering effects of varying scan speeds, washout times and debris blanketing from the ablation are not yet studied, but this is intended in the near future. For ongoing otolith studies, we used a line-scan LA-ICPMS approach to measure 325 cod otoliths (15–30 otoliths per day). The otoliths were embedded in epoxy resin and cut along the dorsoventral profile to expose the core and annuli. For streamlined, high-throughput analyses, custom-made epoxy mounts 7 × 0.8 × 0.8 cm large were cast, each holding 10 otoliths. A custom-made 10 cm2 sample holder with four mounts allows for analysis of 40 otoliths per day. For each otolith, a reflected light optical photo depicting a line from core to edge (Fig. 1D) was imported as an overlay image into the laser ablation software. The image was fitted to the oto-lith position in the live image, using reference points to facili-tate the exact positioning of the line scans.  The ICPMS analysis was optimised for dry plasma condi-tions through continuous linear ablation of the NIST 612 standard. The signal-to-noise ratios were maximised for the isotopic mass range from Mg to Ba, while opting for low ele-ment-oxide production levels by minimising the 254UO2/238U ratio. Instrumental drift was minimised by following a stan-dard–sample–standard analysis protocol, bracketing every sample analysis by line analyses of the NIST-612 and NIST-614 glass standards (Jochum et al. 2011), while the FEBS-1 otolith (Sturgeon et al. 2005) and the MACS-3 carbonate powder tablets (Jochum et al. 2012) provided quality con-trol of the NIST-612 standard measurements. The averaged 2σ accuracy and uncertainty of the standards were typically <5% for element abundances >1–3 ppm. Data processing was done with software Iolite v. 2.5 (Hellstrom et al. 2008; Paton et al. 2011) using the Trace Elements IS data reduction rou-tine. Calculation of abundances were based on 43Ca isotope as the internal standard, assuming 38.3 wt% Ca in all oto-liths, comparable to the certified Ca concentration reported for the FEBS-1 otolith standard (Sturgeon et al. 2005).Application of the analytical approach on Baltic codThe main purpose of the analyses is to answer research ques-tions important to the fisheries management in Denmark and Greenland. The specific approach presented here and modified versions of this method are used in several projects addressing a range of management questions relating to stock structure, migrations and age determination. Here, we high-light some preliminary results of the most advanced project.  The project Tagging Baltic Cod (TABACOD) is a joint Baltic collaboration aiming to develop a new age-estimation method based on seasonal variations in element concen-trations along a gradient from the core (birth) to the edge (death) of otoliths. Fish age is one of the key variables in stock Table 1. Instrument operating conditions,data acquisition and processing parametersInstrumentation:Thermo-Fisher Scientific Element 2 double focusing SF-ICP-MS Forward power: 1470 W Cones: Ni Plasma gas: 16 l min-1 Auxiliary gas (He): 0.85 l min-1 Nebuliser gas (Ar): 0.95 l min-1New Wave Research NWR 213 solid state Nd:YAG laser ablation system Laser wavelength: 213 nm Laser fluence: ~ 9.5 J cm-2 Spot size: 40 µm Repetition rate: 10 Hz Scan speed: 5 µm s-1Data acquisition and processing:Analyte isotopes: 25Mg, 31P, 43Ca, 44Ca, 55Mn, 65Cu, 66Zn, 88Sr, 137BaSampling time, ms: 10 10 10 10 10 10 10 10 10Samples per peak: 10 10 10 10 10 10 10 10 10Acquisition: Time resolved (continuous analysis) along transectsMass resolution: 300 (low)    Oxide production rate tuned to ≤0.3% UO2 (254UO2/238U) Single analysis duration and setup: 30 s blank, 2–20 min ablation(sample dependent), 30 s washoutSoftware for data reduction: Iolite version 2.5 (Paton et al. 2011; Hellstrom et al. 2008)Standards: Internal standard isotope: 43Ca      External standardisation: NIST-612 glass    Secondary standards: NIST-614 glass, FEBS-1 otolith powder  and MACS-3 carbonate powder pressed as tablets93assessment and has traditionally been obtained by visual ex-amination of otolith cross-sections, where seasonal fluctua-tions in growth are visible as optically contrasting growth zones much like the rings in cross-sections of trees. In recent years, this traditional method has failed to provide reliable age information, thus posing severe management problems for the Eastern Baltic cod stock. Initial results from the TA-BACOD project on the seasonality in the otolith chemical composition are presented as an example of the application of the LA-ICPMS approach. All analysed otoliths were ac-quired from cod that were subjected to a mark-recapture experiment. A total of c. 500 cod specimens were captured, externally marked, injected with SrCl2 and released again. When the cod were recaptured they were sent to DTU Aqua for analysis. The SrCl2 is incorporated into the otolith as it grows and acts as an internal timestamp. The chemical sig-nals from timestamp to edge corresponds to the time the fish spent at sea between capture and recapture. Combining in-formation on how many days the fish had been at sea, what time of year it was released/recaptured and how much it had grown since tagging allows us to validate our hypotheses on seasonally varying element concentrations. Concentrations of elements like Mg and Zn vary with season (Hüssy et al. 2016). Figure 2 depicts core-to-edge compositional profiles of Mg, P, Mn, Cu, Zn and Sr from the otolith shown in Fig. 1D, corresponding to the entire life of the fish. The red line in Fig. 2 marks where the Sr concentration dramatically in-creases, representing the SrCl2-tagging event. The Mg, P and Mn concentrations show clear and similar variations throughout the otolith structure on a scale of tens to hun-dreds of ppm. Zn and Cu concentrations vary around our analytical resolution threshold of c. 1–3 ppm and do not show significant systematic variations.  The LA-ICPMS data are currently undergoing statistical analysis to quantitatively identify seasonal variations. Howev-er, some analysed elements indicate clear patterns resembling seasonality. If a seasonality in element concentration occurs, superimposing individual transects (like the ones shown in Fig. 2) of all cod in one plot will result in a generic signal. If element signals are random in relation to time, no such signal will be evident. Figure 3 shows P concentrations from all ana-lysed otoliths, standardised by dividing each measured value by the mean profile P concentration to remove the effect of differences in average P levels between individuals. All pro-files were centred at the Sr peak, since all cod were tagged during the same season (April to May of the same year). The time scale on the x-axis is estimated, assuming linear growth within years, similar growth in individuals across the year, and that all specimens were tagged and released on the same date of a given year. Although there are individual differences between fish, Fig. 3 indicates a general seasonal variation in Distance to core μm25Mg31P55Mn65Cu66Zn88Sr204060ppm2505007501000051015200.00.40.8012345000100000 1000 2000 3000 4000Fig. 2. Trace-element concentration profiles in ppm of the cod otolith shown in Fig. 1D. The x-axis indicates the concentration along the profile from 0 µm, when the fish was born, to 4300 µm, when it died. The red line marks the position of the Sr peak induced by SrCl2 injection. Data from Nielsen et al. (2018). As this cod was tagged in April, the Sr timestamp corresponds to the coldest water temperatures experienced by the cod over a year, where Mg and P concentrations are at a minimum.−4000400800−100 0 100 200Days around centered Sr peak positionsMean centered 31 P ppm from all otolithsFig. 3. Variations of phosphorous concentration in all measured otoliths c. 200 days before and after the SrCl2 tagging and release experiment. On the x-axis, measurements are centred on the injection-induced Sr peak. On the y-axis, measurements have been centred on the mean P concentra-tion of each otolith transect. The red line is a Generalised Additive Model smoothed curve.94P concentrations. The total number of minima occurring in the profiles from birth to death thus corresponds to the num-ber of winters the fish has experienced and hence its age.Other ongoing projectsMigration patterns of the Kattegat cod: This project seeks to map migration patterns of cod captured in the Kattegat, which were genetically identified to belong to the North Sea or western Baltic stock. Elemental profiles of 400 cod cap-tured along a geographic gradient covering the entire Kat-tegat will be compared with baseline samples from adjacent areas. Comparing the results to information about the oto-liths’ annual growth zones can reveal at what time in its life the cod has migrated to and from the Kattegat. Stock structure in capelin: With partners from Greenland’s fishing industry this project investigates stock structure, mi-gration and natal homing (the return to a birthplace to repro-duce) of capelin (Mallotus villosus) from 18 areas along the coasts of South and West Greenland. The aim of this project is to provide counsel on sustainable management of a species that plays a vital role in the marine food chain. Final remarksA LA-ICPMS approach for quantitative, high-throughput transect measurements of otoliths was successfully set up at GEUS. Data from 325 otoliths are being thoroughly exam-ined, and only an ‘appetizer’ of the data is presented here. The LA-ICPMS approach is adaptable for most solid car-bonate (e.g. bivalves) and phosphate (e.g. teeth, horn) materi-als showing cross-surface compositional variations. Analyte isotopes include most major, minor and trace elements and acquisition parameters are easily optimised for the specific sample type, thus providing a rapid and extremely versatile in-situ analytical approach for comparable natural materials.AcknowledgementsWe thank BalticSea2020 for financial support and Mojagan Alaei, GEUS, for laboratory assistance.ReferencesDegens, E.T., Deuser, W.G. & Haedrich, R.L. 1969: Molecular structure and composition of fish otoliths. Marine Biology 2, 105–113.Hellstrom, J., Paton, C., Woodhead, J. & Hergt, J. 2008: Iolite: software for spatially resolved LA-(quad and MC) ICPMS analysis. Mineralogi-cal Association of Canada short course series 40, 343–348.Hüssy, K., Hinrichsen, H.H., Fey, D.P., Walther, Y. & Velasco, A. 2010: The use of otolith microstructure to estimate age in adult Atlantic cod Gadus morhua. Journal of Fish Biology 76, 1640–1654. Hüssy, K., Gröger, J., Heidemann, F., Hinrichsen, H.H. & Marohn, L. 2016: Slave to the rhythm: seasonal signals in otolith microchemistry reveal age of eastern Baltic cod (Gadus morhua). ICES Journal of Ma-rine Science: Journal du Conseil 73, 1019–1032. Jochum, K.P. et al. 2011: Determination of reference values for NIST SRM 610–617 glasses following ISO guidelines. Geostandards and Geoanalytical Research 35, 397–429. Jochum, K.P., Scholz, D., Stoll, B., Weis, U., Wilson, S.A., Yang, Q., Schwalb, A., Börner, N., Jacob, D.E. & Andreae, M.O. 2012: Accurate trace element analysis of speleothems and biogenic calcium carbonates by LA-ICP-MS. Chemical Geology 318–319, 31–44. Limburg, K.E., Wuenschel, M.J., Hüssy, K., Heimbrand, Y.  & Samson, M. 2018: Making the otolith magnesium chemical calendar-clock tick: Plausible mechanism and empirical evidence. Reviews in Fisheries Sci-ence & Aquaculture 26, 1–15.Nielsen, K., Serre, S., Thomsen, T. & Hüssy, K. 2018: Using LA-ICPMS to investigate seasonality in cod otolith microchemistry. 33rd Nordic Geological Winter Meeting, DTU, Kongens Lyngby, Denmark. Ab-stract volume.Pannella, G. 1971: Fish otoliths: daily growth layers and periodical pat-terns. Science 173, 1124–1127.Paton, C., Hellstrom, J., Paul, B., Woodhead, J. & Hergt, J. 2011: Iolite: Freeware for the visualisation and processing of mass spectrometric data. Journal of Analytical Atomic Spectrometry 26, 2508–2518. Patterson, W.P., Smith, G.R. & Lohmann, K.C. 1993: Continental paleo-thermometry and seasonality using the isotopic composition of arago-nitic otoliths of freshwater fishes. In: Swart, P.K. et al. (eds): Climate Change in Continental Isotopic Records. American Geophysical Un-ion Monograph (1993), 191–202. Søndergaard, J., Halden, N., Bach, L., Gustavson, K., Sonne, C. & Mos-bech, A. 2015: Otolith chemistry of common sculpins (Myoxocephalus scorpius) in a mining polluted greenlandic fjord (Black Angel Lead-Zinc Mine, West Greenland). Water, Air and Soil Pollution 226(10) 336, 12 pp. Sturgeon, R.E., Willie, S.N., Yang, L., Greenberg, R., Spatz, R.O., Chen, Z., Scriver, C., Clancy, V., Lam, J.W. & Thorrold, S. 2005: Certifica-tion of a fish otolith reference material in support of quality assurance for trace element analysis. Journal of Analytical Atomic Spectrometry 20, 1067–1071. Sturrock, A.M., Trueman, C.N., Darnaude, A.M. & Hunter, E. 2012: Can otolith elemental chemistry retrospectively track migrations in fully marine fishes? Journal of Fish Biology 81, 766–795. Thomas, O.R.B., Ganio, K., Roberts, B.R. & Swearer, S.E. 2017: Trace element–protein interactions in endolymph from the inner ear of fish: implications for environmental reconstructions using fish otolith chemistry. Metallomics 9, 239–249. Thorrold, S.R. & Shuttleworth, S. 2000: In situ analysis of trace elements and isotope ratios in fish otoliths using laser ablation sector field induc-tively coupled plasma mass spectrometry. Canada Journal of Fisheries and Aquatic Sciences 57, 1232–1242.Authors’ addressesS.H.S. & T.B.T., Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K., Denmark. E-mail: shs@geus.dk.K.E.N., P.F-J. & K.H., National Institute of Aquatic Resources, Technical University of Denmark, Kemitorvet, Building 202, DK-2800 Kgs. Lyngby, Denmark.

Analysis of cod otolith microchemistry by continuous line transects using LA-ICP-MS

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Analysis of cod otolith microchemistry by continuous line transects using LA-ICP-MS

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