**2. Materials and Methods**

For the cleaning test, *Globigerinoides ruber albus* (NCBI:txid2606480) sensu stricto specimens were used, from the 45 cm interval of unconsolidated sediments from the North Aegean Sea core M22-67 (245 m water depth; 38◦21.87′ N, 25◦56.96′ E) with a radiocarbon date (AMS <sup>14</sup>C) of 14.5 ka before present. The core consisted mainly of fine-grained hemipelagic muds and clays and represents a sedimentary archive of the last 85 kyr. The predominant clay minerals in the area are [30] and have been during the study interval [31] illite and smectite. The carbonate content of the sample was measured to be ~42% and since it was not from a sapropel or sapropelic layer its organic content is estimated to be less than 0.6% [32]. *G. ruber albus* s.s. was chosen for species under investigation because of its high abundance in the sample and its importance in paleoclimatic studies. It is likely that foraminiferal tests from different settings, and possibly different foraminifera species of different size, will respond differently to cleaning.

The sample was oven-dried overnight at 50 ◦C and was weighed unprocessed 24 g. Subsequently, it was divided into six aliquots (~4.10 g each) that were transferred into different 50 mL glass beakers and underwent treatment for 20 min at room temperature before wet sieving over a 63 µm mesh, using six processing methods: (1) addition of Calgon® (sodium hexametaphosphate, (NaPO3)6); (2) 2.5% hydrogen peroxide (H2O2); (3) 2.5% hydrogen peroxide and subsequent treatment with Calgon; (4) simultaneous treatment with 2.5% hydrogen peroxide and Calgon; (5) 4% hydrogen peroxide (H2O2); and (6) distilled water without chemical additions (see Table 1 for procedures). Hydrogen peroxide tends to acidify the solution by oxidizing the organic residuals, while Calgon is an alkaline dispersant

that neutralizes the charge of clay particles. Both reagents have traditionally been used in sediment or rock processing methods and in the present study they are applied in a specific order that aims to best use their effects.

Beaker 1 received treatment with Calgon by filling up the beaker with 5% Calgon solution (50 g Na6P6O<sup>18</sup> diluted in 950 mL distilled water) as proposed by [23]. Beaker 2 received treatment with 30% hydrogen peroxide by adding 4 mL of the reagent and filling up the beaker to 50 mL with distilled water, producing a 2.5% hydrogen peroxide solution. Beaker 3 received a "two step treatment". The sample was initially treated with 2.5% hydrogen peroxide solution, like beaker 2, and after washed through a 63-µm sieve the remaining coarse fraction was transferred back to the beaker and treated with 5% Calgon solution, similar to beaker 1 (HyPerCal treatment). Beaker 4 received simultaneous treatment with hydrogen peroxide and Calgon by adding 4 mL of 30% hydrogen peroxide in 46 mL of 5% Calgon solution. Beaker 5 received treatment with 4% hydrogen peroxide solution by diluting 4 mL of 49.5% hydrogen peroxide in 46 mL distilled water, and beaker 6 only received treatment with distilled water. All beakers were gently agitated periodically sonicated every 2 min for 4 s, since a 4 s sonication step has been found to provide a greater detritus cleaning effect and minimize test breakage [23].

After their treatment the sample aliquots were thoroughly washed with tap water over a 63 µm wire mesh sieve and left overnight in the oven to dry at 50 ◦C. They were subsequently dry-sieved and the first random 15 non-fragmented *G. ruber albus* s.s. specimens from the 300–355 µm sieve fraction were picked from each aliquot for further analyses. In order to minimize the effect of specimen size (i.e., size of apertural openings, chamber size) in the cleaning efficiency tests from a narrow size fraction were used. This particular sieve fraction was chosen because of its frequent use in paleoceanographic studies. For assessing the effect that each treatment had on the surface ultrastructure of the foraminifera specimens, 5 specimens from each sample were mounted and gold-coated for SEM imaging. The samples were examined with a JEOL JSM-6390 instrument at a 1100× magnification, a working distance of 2.1 mm and an accelerating voltage of 20 kV in the Department of Geology and Geoscience of the National Kapodistrian University of Athens. In order to evaluate the extent of detritus removal from the interior of the specimens and quantify weight loss from each chemical treatment method, 5 additional specimens from each sample were weighed and subsequently scanned using X-ray computed tomography. The tests were initially weighed as a group of five individuals to obtain their average mass and subsequently in three groups of two individuals in order to record the weight variation in each sample. After weighing the tests were oriented and photographed (Figure 1) using a *Leica* M165 C stereo microscope with an integrated camera at the Department of Geology and Geoscience of the National Kapodistrian University of Athens. The weight analysis took place using a Sartorius microbalance (1 mg precision) also at the University of Athens.

The specimens were subsequently tomographically scanned using Synchrotron X-ray radiation at the Diamond Manchester Imaging Branchline (I13-2) at Diamond Light Source. The tests were transferred into quartz capillaries of 1 mm inner diameter (similar to [29]) that were subsequently attached to magnetic cryo-cap holders and mounted on to a goniometer. The data was acquired with partially coherent, pink X-ray beam which has broader energy spectrum centered around 27 keV. For each of the sessions exposure time of 0.5 s were used, at a 0.09 degree rotation step size producing an acquisition of 2000 projections with 2560 × 2160 pixel resolution using a pco.edge 5.5 camera at a 4× magnification, which resulted in an effective pixel size of 0.8125 µm. The reconstruction of the acquisition data and their downsampling to 8bit tomographic images were performed with Savu package [33]. Links to the raw tomographic data are given the Appendix A below. The images were subsequently analyzed in Avizo software, where the test and sedimentary infilled areas were segmented and discriminated as described in Section 3.3 of [24].


**Table 1.**Table summarizing the different cleaning methods followed.

**Figure 1.** Images of the analyzed specimens after their treatment with the different cleaning protocols.
