*3.2. Synchrotron X-ray Absorption and Weight Analysis*

– Tomographic slices of the scanned specimens that underwent different treatment are shown in Figure 3 and the results of the tomographic analysis together with the weight measurements are summarized in Table 2. The visual inspection of the tomographs clearly shows that the HyPerCal treatment of Method 3 is the most effective way to eliminate contamination from the internal tests cavities of foraminifera. As it can be seen in Figure 3k–o even the smallest chambers or the secondary apertures and pores are free from detrital material. The two-step treatment with H2O<sup>2</sup> and subsequently with Na6O18P<sup>6</sup> shows also reduced contamination in the smaller chambers but there is still sedimentary material attached to the interior of the larger chamber walls. Na6O18P<sup>6</sup> alone is less effective in removing contamination, especially in the smaller chambers, while treatment only with water leaves the test infilling in an aggregated form. Treatment with H2O<sup>2</sup> has left the tests with considerable amounts of detritus and the cohesion of this remaining detrital mass seems to increase with H2O<sup>2</sup> concentration.

– – – – – – **Figure 3.** X-ray tomographic images of the interior of the specimens after their treatment with the different cleaning methods: (**a**–**e**) tomographs of specimens after treatment with Na6O18P<sup>6</sup> , (**f**–**j**) tomographs of specimens after treatment with 2.5% H2O<sup>2</sup> solution, (**k**–**o**) tomographs after treatment first with 2.5% H2O<sup>2</sup> and subsequently with Na6O18P<sup>6</sup> solution (HyPerCal), (**p**–**t**) tomographs after treatment simultaneously with 2.5% H2O<sup>2</sup> and Na6O18P<sup>6</sup> solution, (**u**–**y**) tomographs after treatment with 4% H2O<sup>2</sup> , and (**z**–**ad**) tomographs after treatment with only with distilled water.

**Table 2.** Table showing the results of the X-ray tomographic and weight analyses. The degree of contamination is given as a percentage of cell's volume. Furthermore, the difference in the measured weights in regard to the average shell weight of the least contaminated tests.


**№ μ** ( ( ( Apart from the visual inspection, the X-ray analysis allowed the determination of the total foraminifera cell volume and that of the area in the interior of the test, which is occupied by sedimentary infill. Thus the degree of contamination of each test is presented in Table 2 as the percentage of detritus within the cell's volume. It can be seen that the HyPerCal treatment of the sample with H2O<sup>2</sup> and Na6O18P<sup>6</sup> in two steps has almost completely removed the sediment infill (0%, Table 2) from the test's interior, as this is also evident in Figure 3k–o. The simultaneous treatment with H2O<sup>2</sup> and Na6O18P<sup>6</sup> within the same solution had adequate results since detrital contamination was reduced to only 5% by volume. Treatment with Na6O18P<sup>6</sup> or water had a similar effect on detrital removal by reducing

(

contamination to 14% and 12% respectively, while treatment with H2O<sup>2</sup> (of different concentrations) had the minimum efficiency in specimen cleaning. (

Shell weight is found to be a function of the degree of contamination as shown in Figure 4. It can be seen that samples treated with hydrogen peroxide solution group in the right corner of the graph, while samples treated with aqueous solutions (i.e., water or Calgon) group in its middle. The heaviest tests were the ones that were treated with 2.5% hydrogen peroxide (Method 2). Their average shell weight was 44% greater than that measured for the least contaminated tests. Although treatment with 4% hydrogen peroxide produced consistently lower shell weights its effect on contamination removal was the lowest, suggesting possibly calcite dissolution by the unbuffered solution [34]. Treatment with water produced weights increased by 35% compared to the actual/uncontaminated ones. From the single-constituent solutions Calgon was the one to have the greatest effect on shell weight but also with the greatest variability (12%) in the extent of sediment detrital removal. The simultaneous treatment of the sample with Calgon and hydrogen peroxide is found to be an effective method for specimen cleaning since contamination was found consistently reduced to 5%. Finally, the most effective method that almost completely removed contamination (0% ± 1%) was the HyPerCal treatment.

**Figure 4.** Plot of *G. ruber albus* s.s. shell weights after treatment with the different cleaning methods against their contamination as per volume percentage.

#### **4. Discussion**

*δ δ* We performed a systematic experiment with chemical treatments commonly utilized to disaggregate marine sediment and which are known to not significantly affect foraminiferal based proxies, such as species abundance, shell fragmentation, δ <sup>18</sup>O, δ <sup>13</sup>C, and Mg/Ca. The chemical agents used in solutions were hydrogen peroxide (H2O2) in two different concentrations, 5% Calgon (sodium hexametaphosphate, Na6P6O18), a swap and a combination of them. We find that the most effective way for preparing foraminifera samples for their subsequent micropaleontological or geochemical analyses is the initial cleaning of the sedimentary material with H2O<sup>2</sup> followed by treatment of the sieved sample residual with Na6O18P<sup>6</sup> solution and we refer to this procedure as HyPerCal. In the present experiment the samples were treated for 20 min in every solution and were sonicated every 2 min for 4 s in order to minimize shell breakage [23] but duration of treatment may vary depending on the cohesion of the sedimentary mass. After cleaning, single-species specimens from a certain sieve fraction were picked, weighed, and subsequently inspected using light microscope, SEM and SµCT. The analyses showed that the different procedures had a variable effect in contamination removal (Figure 4) from the surface and the interior of the examined specimens and that the HyPerCal treatment left the specimens free of (surface or internal) sedimentary residuals, shiny and translucent (Figure 1c).

Sodium hexametaphosphate is a common dispersing agent in research of marine sediments and is more effective than water in removing clay clumps from tests interiors [35], while foraminifera shell weight loss has been previously reported with [21] and without [23] the use of it during cleaning. Our tomographic analysis supports previous studies and confirms that weight differences are the result of sediment contamination removal. The initial treatment with H2O<sup>2</sup> promotes the degradation of organic matter, which is the major binding agent in benthic sediments [36] and thus minimizing the adhesive forces within the medium. Cohesive forces are at molecular scale the result of the attractive interactions in vacuum between contiguous particles of the same medium, while the adhesive forces are defined as the additional binding forces between particles, due to the presence of a second, interparticle medium [37]. The dispersing action of Na6O18P6, as a second treatment step, helps to neutralize the attraction electrostatic forces between (clay) particles [38] and is thus reducing particle cohesion. The use of only one of these two reagents alone (Na6O18P6, H2O2) in specimen cleaning did not produce satisfying results both under the SEM and SµCT analyses. The use of both reagents in the same solution, compared to HyPerCal, produced fairly satisfactory results by reducing contamination to only 5%. The high efficiency, however, of the HyPerCal treatment can also stem from the fact that during a two-step treatment the sample processed and sonicated twice as much or from the fact that Na6O18P<sup>6</sup> is only applied on the coarse fraction of sample, free of a substantial amount of material.

Due to the highly reactive nature of the used reagents, there is number of studies that accuse them for foraminifera specimen dissolution [21,34,39]. The release of CO<sup>2</sup> during organic matter oxidation by the unbuffered H2O<sup>2</sup> increases ambient pH and raises dissolution concerns, while Na6O18P<sup>6</sup> is known to react with calcite [40]. Nevertheless, both of our imaging analyses do not reveal signs of foraminifera calcite dissolution. Dissolution can be assessed by the preservation state of four ultrastructural test features such as pores, interpore space, spines, and ridges [25]. As dissolution proceeds, pores get widened, the interpore areas is etched, ridges and spines become denuded. However, such features are not observed on the well decontaminated ultrastructural surface of most of the tests that were cleaned with HyPerCal (Figure 2k–o) that have thus undergone treatment with both reagents. Further evidence of negligible dissolution can be found by the examination of the SµC-tomographs that show intact chamber walls and well defined initial juvenile chambers (Figure 3k–o), since dissolution first attacks the high-Mg calcite parts of the test. The first signs of dissolution apparent in CT images is that chamber walls become blurred and paler in color, especially in the middle, while the smallest inner chambers start to vanish [41]. Such alterations are not here observed possibly also due to the low organic content of the oligotrophic in nature Mediterranean Sea.

The effectiveness of sediment cleaning procedures is a function sediment matrix mineralogy, grain size and degree of consolidation. The present sediment core material consists of fine-grained (hemipelagic) sediment and originates from the Southeastern Mediterranean basin, which is known for the fine particle size of its clay minerals [42]. The chemical treatment tested here has proved appropriate for removal of the fine material that are usually found in sedimentary basins and should remain so for recent sediment, where the depth of burial is not considered important to initiate diagenetic alteration of the clay minerals [43]. The efficiency of the HyPerCal procedure in the cleaning of calcitic microfossils makes it complementary for foraminifera shell weight studies since it was shown to bring the measured weight closer to that of an "original" shell. Furthermore, it paves the way for its use in modern analytical techniques that require some degree of automatization, such as image recognition software that are unable to recognize a lot of foraminifera images, whose umbilical aperture is not fully cleaned and is infilled with remaining nannofossil ooze [28]. On the other hand, it has proved

beneficial for the upcoming practice of microfossil X-ray tomography, since CT image analysis software cannot easily discriminate between contaminated areas and areas referring to the foraminifera tests unless (subjective) manual labor intensive segmentation is employed [24].
