Heavy metal uptake of near-shore benthic foraminifera during 1 multi-metal culturing experiments

Heavy metal pollution originating from anthropogenic sources, e.g., mining, industry and extensive land 8 use, is increasing in many parts of the world and influences coastal marine environments for a long time. The 9 elevated input of heavy metals into the marine system potentially affects the biota because of their toxicity, 10 persistence and bioaccumulation. An emerging tool for environmental applications is the heavy metal 11 incorporation into foraminiferal tests calcite, which facilitates monitoring of anthropogenic footprints on recent 12 and past environmental systems. The aim of this study is to investigate whether the incorporation of heavy metals 13 in foraminifera is a direct function of their concentration in seawater. Culturing experiments with a mixture of 14 dissolved chromium (Cr), manganese (Mn), nickel (Ni), copper (Cu), zinc (Zn), silver (Ag), cadmium (Cd), tin 15 (Sn), mercury (Hg) and lead (Pb) in artificial seawater were carried out over a wide concentration range to assess 16 the uptake of heavy metals by the near-shore foraminiferal species Ammonia aomoriensis, Ammonia batava and 17 Elphidium excavatum. Seawater analysis exhibited the increasing metal concentrations between culturing phases 18 and revealed high metal concentrations in the beginning of the culturing phases due to the punctual metal addition. 19 Furthermore, a loss of metals during the culturing process was discovered, which lead to a deviation between the 20 expected and the actual concentrations of the metals in seawater. Laser ablation ICP-MS analysis of the newly 21 formed calcite revealed species-specific differences in the incorporation of heavy metals. The foraminiferal calcite 22 of all three species reveals a strong positive correlation with Pb and Ag concentrations in the culturing medium. 23 Ammonia aomoriensis further showed a correlation with Mn and Cu, A. batava with Mn and Hg and E. excavatum 24 with Cr and Ni, and partially also with Hg. Zn, Sn and Cd showed no clear trend for the species studied, which 25 may be caused by the little variation of these metals in seawater. Our calibrations and the calculated partition 26 coefficients render A. aomoriensis, A. batava and E. excavatum as natural archives that enable the direct 27 quantification of metals in polluted and pristine environments. This in turn allows monitoring of the ecosystem 28 status of areas that are potentially under the threat of anthropogenic pollution in order to evaluate contemporary 29 emission reduction measures. 30

and incorporate them into their calcium carbonate shells during calcification (e.g., Boyle, 1981 1967; Boltovskoy and Lena, 1969;Wefer, 1976;Murray, 1992) and thus, react immediately to changing 51 environmental conditions and contamination levels of the surrounding environment. 52 Species of the foraminiferal genera Elphidium and Ammonia are among the most abundant foraminiferal taxa in 53 near-shore environments worldwide. They are found from subtidal water depths to the outer continental shelves 54 (Murray, 1991). Furthermore, their calcite tests are often well preserved in the fossil record (Poignant et al., 2000;55 McGann, 2008; Xiang et al., 2008) and therefore provide the opportunity to assess past environmental conditions. 56 The combination of all these properties make foraminifera, and especially Elphidium and Ammonia species, 57 suitable indicators of anthropogenic pollution (e.g., Sen Gupta et al., 1996;Platon et al., 2005). As such, this group 58 of organisms are excellent candidates for monitoring the spatial and temporal distribution of heavy metals in 59 seawater to evaluate, for example, the effectiveness of contemporary measures of reducing emissions caused by 60 anthropogenic inputs. 61 The majority of culturing studies on heavy metal incorporation into benthic foraminifera were designed to assess 62 the influence and uptake of one particular metal, e.g., copper (Cu) (De Nooijer et al., 2007), chromium (Cr) 63 (Remmelzwaal et al., 2019), lead (Pb) (Frontalini et al., 2015), zinc (Zn) (e.g., Smith et al., 2020), mercury (Hg) 64 (Frontalini et al., 2018a) or cadmium (Cd) (Linshy et al., 2013). This approach is adequate to detail the effects on For extracting the foraminiferal specimens from the sediment, about 1 cm 3 of the 63 to 2000 µm size fraction was 139 transferred to a petri dish. For maintaining optimal conditions for the foraminifera, the petri dish was filled with 140 artificial seawater (ASW) with a salinity of 30 PSU. All living specimens were picked with a paint brush from this 141 subsample and collected in a small petri dish of 55 mm diameter with ASW. The procedure was repeated until the 142 whole sample residue was screened. Only specimens with a glossy, transparent and undamaged test were chosen. 143 Furthermore, only individuals with the cytoplasm present in more than just a couple of chambers that were 144 connected and included the innermost chambers were chosen. After picking, a drop of concentrated food (pure 145 culture of Nannochloropsis, green colored algae) was added and the foraminifera were left untouched for a night. 146 Specimens that met one or more of the following criteria were considered as living and used for further procedures: 147  Specimens showed a structural infill of cytoplasm with a bright green color, indicating they took up the 148 food over night, 149  they developed a film or strings of pseudopodia firmly sticking to sediment particles or food, 150  they had covered themselves or gathered a cyst of sediment or food particles. 151 https://doi.org/10.5194/bg-2021-158 Preprint. Discussion started: 12 August 2021 c Author(s) 2021. CC BY 4.0 License. Over the entire culturing period, both systems were exposed to a natural day and night cycle and the flow rate was 224 adjusted to 0.017 ml s -1 (one drop per second) within the culturing vessels. The foraminifera were fed with 225 Nannochloropsis concentrate twice a week (~ 2000 µg). After21 days (meaning after each culturing phase) one 226 culturing vessel per system was exchanged. Vessels and specimens were left in the culturing system for the 227 complete culturing phase (21 days) and no exchange took place during a culturing phase. One culturing vessel 228 containing all three species was left in the system from the beginning until the end of the experiment (from phase 229 0 to phase 3) for 84 days. Data of these specimens are not available due to time constraints caused by the outbreak 230 of the COVID-19 pandemic. 231 Temperature and salinity were kept stable at 15.0 °C and 30.2 units (trace metals) and at 14.9 °C and 30.4 units 232 (control) over the complete culturing period. As the system was mostly closed, evaporation had a minor effect. 233 Demineralized water was added when necessary to keep the salinity stable. The exchanges of culturing vessels 234 between phases inferred a partial water exchange of approximately 10 % (= 1.5 l) every three weeks, which ensured 235 a repetitive renewal of water with adequate quality. 236 237

Collection of water samples 239
Water samples for determining the heavy metal concentrations were taken frequently from the supply tanks (see 240 ml with blue cap and ring, boro 3.3). Water samples to be analysed for mercury concentrations had to be treated 243 differently due to analytical constraints as detailed below. The water was filtered through a 0.2 µm PES filter 244 (CHROMAFIL Xtra disposable filters, membrane material: polyether sulfone pore) for heavy metal samples and 245 through a 0.2 µm quartz filter for Hg samples (HPLC syringe filters, 30 mm glass fibre syringe filters/ nylon). 246 Filters were rinsed with the sample before the sample was taken. Every water sample was immediately acidified 247 with concentrated ultrapure HCl to a pH of approximately 2 to avoid changes in the trace metal concentrations due 248 to adsorption to the sample bottle walls or the formation of precipitates. 249 250

Preparation of water samples before analysis 251
For Mn, Zn, Ni, Pb, Cu, and Cd concentration analyses, the water samples were pre-concentrated offline by using 252 a SeaFAST system (ESI, USA). Twelve ml of each sample were used to fill a 10 mL sample loop and concentrated 253 by a factor of 25 into 1.5M HNO3. All samples were spiked with indium as an internal standard for monitoring 254 and correcting for instrumental drift. Both MilliQ water and bottle blanks of acidified MilliQ water (pH ~ 2) stored 255 in the same bottles until the samples were passed through the pre-concentration system. Additionally, procedural 256 blanks which were filtered as the samples were also pre-concentrated and measured. A variety of international 257 https://doi.org/10.5194/bg-2021-158 Preprint. Discussion started: 12 August 2021 c Author(s) 2021. CC BY 4.0 License.
(Open Ocean Seawater NASS-6, River Water SLRS-6, Estuarine Seawater SLEW-3, all distributed by NRC-258 CNRC Canada) and in-house (South Atlantic surface water, South Atlantic Gyre water) reference materials were 259 pre-concentrated like the samples. All samples were subsequently analysed by ICP-MS (inductively coupled 260 plasma mass spectrometry). 261 For the metals that cannot be preconcentrated by the SeaFAST system as they are not retained on the Nobias resin 262 (Cr, Ag and Sn) samples were diluted 1/25 and directly introduced into the ICP-MS. The dilution was performed 263 with indium-spiked nitric acid (2%) and to match the matrix of these samples blanks and standards with added 264 NaCl were prepared. 265 All trace metals except mercury were measured using an Agilent 7500ce quadrupole ICP-MS and raw intensities 266 calibrated with mixed standards, made from single element solutions, covering a wide concentration range. 267 Additionally, a dilution series (dilution factors: 1, 1/10, 1/100 and 1/1000) of SLRS-6 of river water reference 268 material (NRC Canada; Yeghicheyan et al., 2019) was measured for quality control. Mean values and relative 269 standard deviations (RSD) derived from the reference materials are summarised in the appendix (Table A2). 270 Prior to the measurements of Hg concentrations, all samples were treated with BrCl solution at least 24 hours 271 before the analysis to guarantee the oxidation and release of mercury species that are possibly present in a different 272 oxidation states or phases. The BrCl was removed again from the sample by adding hydroxylamine hydrochloride 273 at least one hour prior to analysis before the Hg was reduced to the volatile Hg 0 species with acidic SnCl2 (20 % 274 w v -1 ) during the measuring process. All preparations of the water samples took place in a Clean Lab within a 275 trace metal clean atmosphere and all vials were acid cleaned prior to use. Mercury concentrations were determined 276 using a Total Mercury Manual System (Brooks Rand Model III). The reduced volatile Hg 0 is nitrogen-purged onto 277 a gold-coated trap and released from again by heating before it is measured via cold vapour atomic fluorescence 278 (CVAFS) under a continuous argon carrier stream. Quality control of the Hg measurements was carried out by 279 measuring mixed standards, made from single element solutions and confirmed with replicate measurements 280 throughout each analysis. The measurement uncertainty was smaller than 4.5 % RSD for all analyses. 281 The calcium concentration of culture seawater was analysed using a VARIAN 720-ES ICP-OES (inductively 282 coupled plasma optical emission spectrometer). Yttrium was added as an internal spike and samples were diluted 283 1/10. IAPSO seawater standard (ORIL) was measured after every 15 samples for further quality control which 284 revealed a measurement uncertainty < 0.35 (RSD %) for the elements analysed (mean Ca concentration IAPSO 285 this study = 419.6 ± 0.15 mg l -1 ; reference Ca concentration IAPSO Batch 161 = 423 mg l -1 ). 286 287

Foraminiferal samples 288
After every culturing phase, the culturing vessels were taken out of the system and foraminiferal specimens where 289 collected from their cavities within one day. The individuals were cleaned with tap water and ethanol before they 290 were mounted in cell slides to mechanically remove salt scale and organic coatings with a paintbrush. 291 In order to check, whether the foraminifera had grown during the experiment, the total number of chambers was 292 counted before and after the experiment for every specimen ( Imager 2) if new chambers without color were added, hence whether the particular specimen had grown or not 295 (Fig. 2e). Only individuals clearly showing new chambers were analysed by Laser ablation ICP -MS. 296 Prior to the laser ablation analyses, the foraminifera were transferred into individual acid-leached, 500 µl micro-297 centrifuge tubes and thoroughly cleaned, applying a procedure adapted from Martin and Lea (2002). The 298 specimens were rinsed three times with MilliQ water and introduced into the ultrasonic bath for a few seconds at 299 the lowest power setting after each rinse. Afterwards, clay and adhering particles were removed by rinsing the 300 sample with Ethanol twice, which was followed by three MilliQ rinses again with minimal ultrasonic treatment. 301 Oxidative cleaning was applied using 250 µl of a 0.1M NaOH and 0.3 % H2O2 mixture added to each sample and 302 the vials were kept for 20 min in a 90 °C water bath. Afterwards, the samples were rinsed with MilliQ three times 303 to remove the remaining chemicals. The reductive step of the cleaning procedure by Martin and Lea (2002) was 304 not applied. This step is necessary to remove metal oxides, which of course could also influence the trace metal 305 concentration within the foraminiferal shell carbonate but these are usually considered to be added during early 306 deposition (e.g., Boyle, 1983) and therefore unlikely to occur during culture experiments. For Laser Ablation 307 Inductively Coupled Plasma Mass Spectroscopy (LA-ICP-MS) measurements, all cleaned specimens were fixed 308 on a double-sided adhesive tape (PLANO). 309 Micro-analytical analyses with LA-ICP-MS were performed at the Institute of Geosciences, Kiel University, using 310 a 193nm ArF excimer GeoLasPro HD system (Coherent) with a large volume ablation cell (Zurich-type 311 LDHCLAC, Fricker et al., 2011) and helium as the carrier gas with 14 mL min -1 H2 added prior to passing the 312 ablation cell. For the foraminiferal samples, the pulse rate was adjusted to 4 to 5 Hz with a fluence between 2 and 313 3.5 J cm -2 . The spot size was set to 44 or 60 µm depending on the size of the foraminiferal chamber. All chambers 314 of a foraminifer that were built up in the culturing medium were analysed, starting from the earliest, inner chamber 315 adjacent to the calcein-stained chamber. The laser was manually stopped once it broke through the foraminiferal 316 shell. The ablated material was analysed by an ICP-MS/MS instrument (8900, Agilent Scientific Instruments) in 317 no gas mode. The NIST SRM 612 glass (Jochum et al., 2011) was used for calibration and monitoring of instrument 318 drift while NIST SRM 614 was measured for quality control. Glasses were ablated with a pulse rate of 10 pulses 319 per second, an energy density of 10 J cm -2 and a crater size of 60 µm. Carbonate matrix reference materials coral 320 JCp-1, giant clam JCt-1, limestone ECRM752-1 and synthetic spiked carbonate MACS-3 (Inoue et al., 2004;321 Jochum et al., 2019) in the form of nano-particle pellets were analysed (Garbe-Schönberg and Müller, 2014). 322 MACS-3 was used for calibrating the mercury content in the samples as Hg is not present in the NIST SRM 323 glasses. All results for the reference materials are displayed in the appendix (Table A3). Trace element-to-calcium 324 ratios were quantified using the following isotopes: 26 43 Ca. If more than one isotope was measured for an element, the 326 average concentration of these was used after data processing. Uncertainty (in % RSD) was better than 5 % for all 327 TE/Ca ratios. The lowest RSD % based on the NIST SRM 612 glass was 2.1 % for Mn/Ca and the highest 5.0 % 328 for Ag/Ca. Uncertainties of all used standards and reference materials are expressed are summarized in Table A3. 329 Each acquisition interval lasted for 90 seconds, started and ended with measuring 20 s of gas blank, used as the 330 background baseline to subtract from sample intensities during the data reduction process. Furthermore, the 331 background monitoring ensured that the system was flushed properly after a sample. In cases when foraminiferal 332 test walls were very fragile causing the test to break very quickly and, hence, the length of the sample data 333 acquisition interval was less than 15 seconds, these profiles were excluded from further consideration. determination of element/Ca ratios were performed after the method of (Rosenthal et al., 1999). High values of 337 25 Mg, 27 Al or 55 Mn at the beginning of an ablation profile were related to contamination on the surface of the 338 foraminiferal shell or remains of organic matter (e.g., Eggins et al., 2003) and these parts of the profiles were 339 excluded from further data processing. The detection limit was defined by 3.3*SD of the gas blank in counts per 340 seconds for every element in the raw data. Only values above this limit were used for further analyses. After 341 processing the data with Iolite, an outlier detection of the TE/Ca ratios of the samples was performed. If trace 342 metal values from a spot deviated more than ±2SD from the average of the samples from the corresponding 343 culturing phase, values were defined as outliers and discarded. The number of rejected points is indicated in the 344 supplementary material (Table S1). 345 All statistical tests were carried out using the statistical program PAST (Hammer, 2001). As the concentration of 346 heavy metals in seawater was varying during individual phases in the metal system (Table A1 and     were added during the complete culturing period (phase 0 to 3) and closed symbols represent the metal system. 389 In this system, phase 0 is the control phase without any extra added metals and for phase 1 to 3, the heavy metal 390 concentration in the culturing medium was elevated. Note that the standard error is comparably high in phase 3 391 because the heavy metal concentration in this phase varied more strongly, which is shown in the appendix (Table  392 https://doi.org/10.5194/bg-2021-158 Preprint. Discussion started: 12 August 2021 c Author(s) 2021. CC BY 4.0 License. A1, Fig. B1). Therefore, this error is derived from the real values in the seawater and not from analytical 393 uncertainties. Note that the Cr/Ca values from the control system in phase 0 and 1 are not given as these values 394 were below the detection limit. 395

396
In phases 1 and 0 the concentration in both systems were nearly equal for most elements. Only Cr and Sn had 397 slightly elevated concentrations in the metal system, whereas Cu and Mn concentration were higher in the control 398 system in phase 0 (Fig. 3). This also holds true for Mn in phase 2, when all other metals showed higher 399 concentrations in the metal system than in the control system. In phase 3, the concentration of all heavy metals 400 were elevated in the metal system as compared to the control system. The variation of the metal concentration was 401 in both systems highest in phase 3 for all elements but Cu, which showed the highest variation in phase 0 (Fig. 3). 402 The control system generally displayed a smaller degree of variation than the metal system. 403 The target concentration of the metals was not accomplished for most metals in phase 1 and 2, the only exception 404 is Ag in phase 1 ( Table 3). The factors between the target and measured concentration was highest (> 50) for Ni, 405 Cu and Zn in phase 1 and gets smaller in phase 2 and 3. Generally, all elements but Mn were concentrated higher 406 in phases 1 and 2 than expected. In phase 3 Cr, Mn, Cu, Ag and Sn reached concentrations closer (factor 0.4 -0.8) 407 to the target concentration and Ni, Zn, Cd, Hg and Pb were concentrated higher (factor 3.1 -9.9) than expected. 408 Furthermore, the factor between individual phases (Table 3) was small for the transition from phase 0 to 1 (factor 409 < 1.4) for all elements but Cd (factor 2.6) and Hg (factor 7.5). Same patterns can be seen between phase 1 and 2, 410 while the difference between phase 2 and 3 was more distinct (factor > 4) for Ni, Zn, Ag, Cd, Pb and Hg. Mn, Cu 411 and Sn showed little variation between phase 2 and 3 (factor < 1.7). Generally, the factor between each phase 412 should have been approximately 10, which was not achieved in most cases. Exceptions were Ag, Cd and Pb, which 413 had factors >15 between phase 2 and 3. Furthermore, Hg showed concentrations that were higher by a factor 414 around 10 between all phases (phase 0-1 = 7.5, phase 1-2 = 8.5, phase 2-3 = 9.3). 415 416  Pearson's correlation coefficient R 2 and its significance (p) are given for the calculation with all phases and when 422 removing phase 3 from the calculations. It´s also indicated whether the regression line is forced through the origin 423 or not. In cases when a regression did not show significant correlation, the DTE range separately calculated from 424 the individual phases is given. In cases when the regression was significant, the DTE values represent the slope of 425 the regression line. Ph = Phase. Values in Table S1   Measurable incorporation into the foraminiferal calcite was found for all the trace metals analysed but the degree 430 of incorporation varied profoundly within and between species (Fig. 4 and Table 4). In both systems, the trace 431 metal concentration in E. excavatum was higher than in the other species (A. aomoriensis and A. batava) for Cr, 432

Incorporation of trace metals into the foraminiferal shell 417
Mn, Ni, Cu, Hg and Sn. This trend is also visible but less pronounced in the Cu values of the control system. Calculations were performed with and without phase 3 (Fig. 4, Fig. B2 and Table 4)  System as only data from one newly formed chamber are available. All values can be found in Table 4.An enlarged 462 graph based on the calculations without phase 3 is provided in the appendix (Fig. B2). be detected. Furthermore, calculations were performed with and without phase 3 (Fig. 4 , Fig. B2 and Table 4). The element concentrations within the culturing medium of each culturing phase were comparably stable for most 491 elements in the control system. In the metal system, the variations were higher, which is due to the punctual input 492 of the stock solution for reaching the next phase concentration (Table A1,  When taking into account that the amount of the stock solution added to the culturing medium of the metal system 499 at the beginning of each culturing phase was elevated 1 to 1.5 order of magnitudes between phases, the measured 500 metal concentrations are smaller than expected for phases 0, 1 and 2. This in combination with the varying metal 501 concentration within one phase suggests that several processes are affecting the concentration in such a complex 502 culturing system. One possible mechanism is sorption of the metals onto surfaces (e.g., tubing, culturing vessels, 503 plates, organic matter or the foraminiferal test itself), which could have lowered the metal concentration in the 504 culturing medium. Therefore, sorption could have contributed to the overall budget of the metals. On the other 505 hand, Cu appears to have been released from components of the culturing system even though the system was 506 cleaned before and was operated with seawater for 14 days before the experiments begun. For instance, the 507 concentration of Cu was high in phase 0, where no metals were added suggesting release from system parts. In 508 phase 1, the Cu concentration decreased meaning the contamination derived from the system was removed by a 509 process similar to that observed for the other metals after additions were made. Similar effects have been reported whereas recent studies constrained them as a reaction to stressful environments, not necessarily created by high 520 heavy metal concentrations (Frontalini and Coccioni, 2008;Polovodova and Schönfeld, 2008). The lack of 521 malformations in our experiments suggests that the foraminifera were neither poisoned by elevated trace metal 522 concentrations nor stressed too much by strongly varying environmental parameters, maintaining a normal 523 metabolism and growth. Reproduction was generally very rare, which may indicate that the conditions were not 524 ideal. In field studies foraminiferal reproduction has been linked to short periods of elevated food supply (e.g., Lee 525 et al., 1969;Gooday, 1988;Schönfeld and Numberger, 2007). The regular feeding of foraminifera in our 526 experiment twice a week at constant rates therefore probably did not provide supply levels that trigger 527 reproduction. 528 https://doi.org/10.5194/bg-2021-158 Preprint. Discussion started: 12 August 2021 c Author(s) 2021. CC BY 4.0 License.
Calcein was used for staining the foraminiferal test before they were placed into the culturing system. Calcein 529 binds to Ca and is incorporated into the mineralised calcium carbonate . It is conceivable 530 that the trace metal incorporation could also be affected by calcein. However, no evidence has been found by a 531 variety of studies (e.g., Hintz et al., 2006;De Nooijer et al., 2007;Dissard et al., 2009). Furthermore, calcein was 532 only used prior to the experiment to mark the last chamber that was grown outside the culturing system. Therefore, 533 the incorporation of the metals measured in subsequent chambers was not affected by the calcein application. 534 is getting too high and an imminent intoxication is probable, which may be managed by controlling the ion uptake 569 via TMT. Therefore, it may well be possible that the highest concentration of the metals in our study was close to 570 the tipping point of the biological mechanism taking over and protecting the organism. 571 Besides biologically controlled factors, also physicochemical properties play an important role when it comes to 572 the uptake of ions. One chemical factor is the aqueous speciation and solubility of the metals. Metals with a free 573 ion form with a charge of 2+ are more similar to Ca 2+ , which makes incorporation more likely (Railsback, 1999). 574 Nearly all metals in this study were added as dissolved chlorides and therefore had a charge of 2+. The only 575 exceptions were Ag, which was added as AgNO3 with a charge of 1+ and Cr, which was added as CrCl3*6 H20. 576 The charge of the cation as such does not seem to make a major difference as Ag was incorporated into all three 577 species and Cr into E. excavatum with a significant positive correlation to concentrations in the culturing medium. 578 Furthermore, it is possible that the oxidative state of the elements is changing due to their pH dependency, which 579 will be discussed for every element separately. Furthermore, other ions with a charge of 1+ are also known to be 580 around 8.0 ± 0.1 (measured twice a week), speciation changes between phases due to varying pH values can be 587 excluded. However, it is possible that some metals were not available in a form that could be readily incorporated 588 in the calcite such as the free ion or carbonate species. Cr is not available in an optimal speciation to substitute Ca 589 as a pH of 8 would favour Cr 3+ or Cr 4+ as well as oxides and hydroxides (Elderfield, 1970 (Table A2), which also corroborates the assumption that these interferences can 597 be neglected. Similar pH processes could also have effected Cu as a pH around 8, like in this experiments, favours 598 copper carbonates over free Cu 2+ ions (e.g., Escudero Maret, 2016). Therefore, these elements should preferentially be taken up into the foraminiferal cell, where 620 they were used for further processes. This in turn could lead to the consumption of these metals before they can 621 be incorporated into the foraminiferal tests. All of these ions have a similar ionic radius (Cu = 0.73 Å, Mn = 0.67 622 Å, Zn = 0.74 Å) in six-fold coordination (Rimstidt et al., 1998), which would also suggest, that their behaviour is 623 comparable. The ionic radii are much smaller than that of Ca, but are rather similar to Mg (0.72 Å, Rimstidt et al.,624 1998) (Fig. 5). 625

Mn shows a positive correlation between its concentration in seawater and the foraminiferal test in the two 626
Ammonia species when the calculations include phase 3. This indicates that this element serves as a well-behaved that the presence of toxic metals such as Cd, Ni or Hg can inhibit the uptake of essential metals like Mn if these 635 metals are present in low concentrations (e.g., Huntsman, 1998a, 1998b). It is possible that this 636 mechanism is more pronounced in E. excavatum than in the Ammonia species. Zn was clearly incorporated above 637 control level into all three species, but it´s behaviour is influenced by more factors than the concentration of Zn in 638 https://doi.org/10.5194/bg-2021-158 Preprint. Discussion started: 12 August 2021 c Author(s) 2021. CC BY 4.0 License. the culturing medium. This can be inferred by the fact that there was no significant correlation recognised between 639 Zn concentration in calcite and seawater (Fig. 4, Table 4). further removing mechanism. The ionic radii of Pb 2+ in calcite-coordination is 1.19 Å, which is remarkably higher 664 than those of Hg 2+ (1.02 Å) and Cd 2+ (0.95 Å), which are comparable to Ca (Fig. 5). This similarity should also 665 favour the incorporation of Cd and Hg into calcite, which holds only partly true, as Cd shows no trends, but Hg 666 correlates in A. batava and in E. excavatum if phase 3 is not integrated into the calculations. This indicated that 667 the incorporation of Cd is not straight-forward and is indeed depending on more complex factors than seawater 668 concentration of Cd. Nevertheless, Cd is incorporated well above control level in all three species. Because the 669 ionic radius of Pb is bigger than that of Ca a smaller degree of Ca 2+ substitution following the ionic radius to charge 670 ratio theory after Rimstidt et al. (1998) is expected. This is not the case as Pb emerged as a well-behaved proxy. 671 All three species incorporated Pb with a significant positive trend indicating that the main controlling factor is the 672 seawater concentration of Pb (Fig. 4, Table 4). The importance of other metals like Sn, Cr, Ag and Ni is not fully understood yet but some of them are believed 696 to have certain biological functions in the cells of animals or plants (Horovitz, 1988;Mertz, 1993;Lukaski, 1999; compared to other studies as no literature is available, but the general trend, the ionic radius and the DTE values 708 are comparable to other elements in this study e.g., to Pb. Ag and Ni both display a well-behaved proxy for the 709 estimation of seawater concentration of these ions (Fig. 4, Table 4). furthermore, the differences between the phases are also very low (Fig. 3, Fig. B1 and Table 3). It may be that the 717 concentration of Cr needs to be further elevated and the concentration range needs to be extended before the 718 foraminifera are able to incorporate Cr with significant differences between concentrations. We may speculate that 719 the same could apply for Sn. Besides, Remmelzwaal et al. (2019) suggested, that Cr in foraminiferal shells is 720 mainly a result of post-depositional overprinting. Diagenetic processes are very unlikely to play a role in our 721 experiments, which would explain, why we do not recognise a correlation between the concentration of Cr in the 722 culturing medium and in foraminiferal calcite. 723 724

Interspecies variability 725
The three different species cultured in this study clearly incorporate the same metal in different ways, which is 726 most visible in the overall higher TE/Ca values of E. excavatum compared to species from the genus Ammonia 727 (Fig. 4, Table 4). Koho et al. (2017) suggested that these differences in the incorporation result from different 728 microhabitats used by different foraminiferal species. This might be true in nature. In our experiments, however, 729 the sediment in the cavities was only a few mm thick and no redox horizon was recognised when recovering the 730 foraminifera after the experiment. Therefore, all foraminifera were living in the same microhabitat. 731 Another possible reason for the difference between E. excavatum and Ammonia species is their nutrition strategy. 732 As discussed above, DTE values were markedly higher in symbiont-bearing species. Ammonia species do not 733 harbour endosymbionts (Jauffrais et al., 2016), whereas at least five intertidal Elphidium species were husbanding 734 diatom chloroplasts, including E. excavatum (e.g., Pillet et al., 2011;Cesborn et al., 2017). However, an earlier 735 study could not corroborate the assumption that Elphidium species living at greater water depth in the Baltic Sea 736 may contain endosymbiontic zooxantellae (Schönfeld and Numberger, 2007). In our experiment, dead 737 Nannochloropsis were fed, which is certainly not the preferred food source for E. excavatum (Pillet et al., 2011). 738 This could lead to a slower growth and E. excavatum built on average only 1 chamber during the individual 739 culturing period of 21 days while Ammonia species built more than four chambers. Furthermore, E. excavatum did 740 not reproduce, even though the culturing period is close to the generation time of this species (Haake, 1962). When 741 growth is slower it could be possible that a higher amount of a metal is incorporated into the shell, which would 742 lead to higher TE/Ca values in this species. Another possibility is the timing of chamber formation. As E. 743 excavatum formed on average one new chamber, it is possible that this chamber was formed during the high peak 744 in the metal concentration during the beginning of the culturing phases (Fig. B1, Table A1). This could in turn 745 lead to a higher uptake of the metals and higher DTE values. Both Ammonia species on the other hand, formed 746 more chambers, which makes it most likely that not only the first high concentration influences their overall DTE 747 value. Unfortunately, it is not possible to constrain exactly when the specimens formed their new chambers. 748 749 During the past years, many studies were performed to assess the pollution level of seawater. The range of heavy 755 metal concentrations in the culturing medium of this study are compared to the metal concentrations in polluted 756 environments measured over the past 40 years in different regions all over the world (Table 5) European rivers (Byrd and Andreae, 1982;Kannan et al., 1998;Thomas et al., 2002). Furthermore, the maximum 764 metal concentration as recommended by the EPA is the lower boundary of the concentration range from this study 765 (Prothro, 1993). A lower concentration than the EPA value is also covered by our study during the control phase 766 or in the control system. This enables us to assess metal levels at the very beginning of the harmful pollution phase 767 in environments, which could be used as an early-warning system for the ecological status of an area (Sagar et al., 768 2021). Furthermore, it allows to assess the effectiveness of contamination reducing measures. This advantage will 769 be important in the future for the possibility to intervene or to apply more promising measures within an adequate 770 time frame. In some regions of the world, seawater heavy metal concentrations are higher than in this study. 771

Application of TE/Ca values in the foraminiferal shell 750
Examples are Suva, Fiji (Arikibe and Prasad, 2020), the Gulf of Kutch, Arabian Sea (Chakraborty et al., 2014) or 772 the East London and Port Elizabeth harbours, U.K. (Fatoki and Mathabatha, 2001) (Table 5). These areas seem to 773 be extremely polluted, which would make it necessary to apply a higher metal concentration to the cultured 774 foraminifera if a reconstruction covering these values should be made. However, this study clearly indicates a 775 reduced uptake of metals of interest, when the concentration of these metals in the seawater is exceeding a certain 776 threshold value (here between phase 2 and 3). This will make it generally difficult to model extreme high pollution 777 levels. Indeed, it is possible to distinguish between a heavy and a moderate pollution level. Overall, the 778 concentration of heavy metals in seawater should be decreasing all over the world due to a rigorous legislation for 779 reduction of the heavy metal input into the environment, and due to various emission reducing measures that are 780 applied already. This means that the concentration range of metals covered by this study is adequate for future 781 research and monitoring of polluted systems. 782 783

Conclusion 784
The aim of this study was to assess the incorporation of heavy metals into the foraminiferal calcite as a function 785 of their concentration in the seawater the foraminifera calcified in. Culturing experiments with different 786 foraminiferal species (A. aomoriensis, A. batava and E. excavatum) that were exposed to a mixture of ten different 787 metals (Cr, Mn, Ni, Cu, Zn, Ag, Cd, Sn, Hg and Pb) at varying concentrations (Table 3, Fig. 3, Fig. B1) were 788 carried out to gain further insights into the uptake of heavy metals. Laser ablation ICP-MS analysis of the newly 789 formed calcite revealed species-specific differences in the incorporation of heavy metals. Nevertheless, all metals 790 used in this study were incorporated into the foraminiferal calcite of all three species (Fig. 4, Table 4). Some 791 elements showed a behaviour inferring that the uptake of these metals mainly depends on its concentration in     Figure B1: TE/Ca values in the culturing medium of the metal system in µmol mol -1 or nmol mol -1 divided by 837 individual culturing phases. In this system, phase 0 is the control phase without any extra added metals and for 838 phase 1 to 3, the heavy metal concentration in the culturing medium was elevated. The data the figure is based on 839 can be found in Table A1. can be found in Table 4. 852

Supplements 853
Table S1-S3: TE/CaCalcite values from Ammonia aomoriensis (Table S1), Ammonia batava (Table S2) and 854 Elphidium excavatum (Table S3). Values represent single laser ablation spots on foraminiferal chambers that were 855 formed during the individual culturing period in the control and the metal system. Only values above the detection 856 limits of the individual element are presented. Furthermore, outliers are also excluded. These values are the basis 857 for the calculation of the mean TE/Ca values in Table 4 and Fig. 4. The sample ID indicates the species (AA = A. 858 aomoriensis, AB = A. batava, E = E. excavatum), the culturing phase, the system (R = metal system, L = control 859 system), the individual and the chamber that was ablated, starting from the innermost chamber going to the 860 youngest one. 861

Data availability 862
All data generated or analysed during this study are included in this published article and its supplementary 863 information files. 864

Author contribution 865
This study was initiated by JS and EH. SS collected the samples, cultured the foraminifera, processed the samples 866 in the laboratory and acquired, analysed and interpreted the water and foraminiferal data. JS helped with the 867 sampling logistics, design and implementation of the culturing experiments. EH advised and helped with the 868 processing and analysis of the water samples and EH and DGS advised and helped with the measurements of the 869 foraminiferal samples. SS wrote the manuscript with all the authors contributing to the discussion and data 870 interpretation, and editing of the work. 871

Competing interests 872
The authors declare that they have no conflict of interest. 873

Acknowledgements 874
We are indebted to Tal