# A wet chemical extraction protocol for measuring biogenic silica in sediments of marginal seas and open ocean

Dongdong Zhu, Su Mei Liu, Aude Leynaert, Paul Tréguer, Morgane Gallinari, Heting Zhou, Jill N Sutton

► To cite this version:

Dongdong Zhu, Su Mei Liu, Aude Leynaert, Paul Tréguer, Morgane Gallinari, et al.. A wet chemical extraction protocol for measuring biogenic silica in sediments of marginal seas and open ocean. MethodsX, 2025, 10.1016/j.mex.2025.103723Reference:MEX103723. hal-05379919

HAL Id: hal-05379919

https://hal.science/hal-05379919v1

Submitted on 24 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Journal Pre-proof

A wet chemical extraction protocol for measuring biogenic silica in sediments of marginal seas and open ocean

Dongdong Zhu, Su Mei Liu, Aude Leynaert, Paul Tréguer, Morgane Gallinari, Heting Zhou, Jill N. Sutton

PII: S2215-0161(25)00567-9

DOI: https://doi.org/10.1016/j.mex.2025.103723

Reference: MEX 103723

To appear in: *MethodsX*

Received date: 23 September 2025

Accepted date: 13 November 2025

Please cite this article as: Dongdong Zhu, Su Mei Liu, Aude Leynaert, Paul Tréguer, Morgane Gallinari, Heting Zhou, Jill N. Sutton, A wet chemical extraction protocol for measuring biogenic silica in sediments of marginal seas and open ocean, *MethodsX* (2025), doi: https://doi.org/10.1016/j.mex.2025.103723

This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability. This version will undergo additional copyediting, typesetting and review before it is published in its final form. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article. Please note that Elsevier's sharing policy for the Published Journal Article applies to this version, see: https://www.elsevier.com/about/policies-and-standards/sharing#4-published-journal-article. Please also note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2025 Published by Elsevier B.V.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

<figure><img src="image_2.png" /></figure>
**Article information**

**Article title**

A wet chemical extraction protocol for measuring biogenic silica in sediments of marginal seas and open ocean

**Authors**

Dongdong Zhu<sup>a,b</sup>, Su Mei Liu<sup>a,*</sup>, Aude Leynaert<sup>b</sup>, Paul Tréguer<sup>b</sup>, Morgane Gallinari<sup>b</sup>, Heting Zhou<sup>a</sup>, Jill N. Sutton<sup>b,*</sup>

**Affiliations**

<sup>a</sup>Frontiers Science Center for Deep Ocean Multi-spheres and Earth System, Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China.

<sup>b</sup>University of Brest, Centre national de la recherche scientifique, Institut de recherche pour le développement, Ifremer, Institut Universitaire Européen de la Mer, Plouzané 29280, France.

**Corresponding authors' email address and Twitter handle**

* Corresponding authors.

E-mail address: sumeiliu@ouc.edu.cn (Su Mei Liu), jill.sutton@univ-brest.fr (Jill N. Sutton).

**Keywords**

Analytical method; opal; marine sediments; lithogenic silica; silicon cycle;

**Related research article**

D. Zhu, J.N. Sutton, A. Leynaert, P.J. Tréguer, J. Schoelynck, M. Gallinari, Y. Ma, S.M. Liu, Revisiting the biogenic silica burial flux determinations: A case study for the East China seas. Front. Mar. Sci. 9 (2023) 1058730. https://doi.org/10.3389/fmars.2022.1058730

**Abstract**

This study describes a wet chemical extraction protocol for measuring the biogenic silica (bSi) in sediments from diverse marine environments. The protocol lists the reagents, materials, equipment, and sample preparation procedures, and provides a detailed explanation of the methods for examining the alkaline-leachable silicon (Si), and calculating bSi content. Although, the protocol was primarily developed for measuring bSi in sediments from the Chinese marginal seas, it was also validated using sediments from the Chesapeake Bay, the Atlantic Ocean, and the Southern Ocean. The protocol can be used to quantify bSi in recently deposited and aged sediments from the Holocene period. The protocol contributes to the ongoing efforts to minimize the methodological bias that exist in bSi quantification and the bSi burial flux evaluation, thereby assisting in our understanding of Si cycling in the modern ocean.

* This protocol provides a step-by-step wet chemical extraction procedures and the measurement of dissolved Si in an alkaline solution using spectrophotometer.

* This protocol is easy to set up and reproduce, and determines bSi content with high precision.

* The protocol can be used to determine bSi in sediments of marginal seas and the open ocean.
## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Specifications table

<table><tr><td>Subject area</td><td>Earth and Planetary Sciences</td></tr><tr><td>More specific subject area</td><td>Geochemistry, biogeochemistry, marine sciences</td></tr><tr><td>Name of your protocol</td><td>A wet chemical extraction protocol for measuring biogenic silica in marine sediments<br/>While we provide the source of the materials used, the reagents and equipment listed in this box can be purchased from any supplier.</td></tr><tr><td>Reagents/tools</td><td><strong>Reagents:</strong><br/>Sodium metasilicate (Na₂SiO₃·9H₂O): Sigma-Aldrich S4392.<br/>Sodium hydroxide (NaOH): Sigma-Aldrich S5881.<br/>Sodium carbonate (Na₂CO₃): Sigma-Aldrich 223530.<br/>Hydrochloric acid (HCl): Sigma-Aldrich 258148.<br/>Hydrogen peroxide (H₂O₂): Nanjing Reagent C0404510123.<br/>Sulfuric acid (H₂SO₄): Sigma-Aldrich 258105.<br/>Ammonium heptamolybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O): Aladdin A597691.<br/>Oxalic acid (C₂H₂O₄): Aladdin O107180.<br/>Metol (C₇H₉NO·0.5H₂SO₄): Aladdin M110716.<br/>Sodium sulfite (Na₂SO₃): Aladdin S112300.<br/><br/><strong>Equipment:</strong><br/>Analytical balance (±0.1 mg): Cubis® II 224S-2CCN, Sartorius AG, Otto-Brenner-Straße 20, 37079 Göttingen, Germany.<br/>Electric balance (±10 mg): YP502N, Shanghai Precision Instrument Co., Ltd, Shanghai, China.<br/>Water Purification System: Elix® Reference water purification system, Merck Millipore, EMD Millipore Corporation, Billerica, MA, U.S.A.<br/>Freeze drier: Alpha 1-2 LSCbasic, Martin Christ, Gefriertrocknungsanlagen, An der Unteren Söse 50, 37520 Osterode am Harz, Germany.<br/>Centrifugal machine: Multifuge 3SR+, Thermofisher Scientific, Thermo Electron LED GmbH, D-37520, Osterode am Harz, Deutschland.</td></tr></table>
<table><tr><td></td><td>Water bath: Julabo SW22, JULABO GmbH, Gerhard-Juchheim-Strasse 1, 77960 Seelbach, Germany.<br/>Mixer: DLAB MX-S, Dragon Lab, Beijing, China.<br/>Magnetic stirrer: B11-1, Shanghai Sile Instruments Co., Ltd., Shanghai, China.<br/>Pipette: Eppendorf Reference®2, Eppendorf SE Barkhausenweg 1, D-22339, Hamburg, Germany.<br/>Ultraviolet-Visible Spectrophotometer: EU-2600A, Shanghai Onlab Instrument Co., Ltd., Shanghai, China.<br/>Microscope: Zeiss Axio Observer A1, Carl Zeiss AG, Carl-Zeiss-Straße 22, 73447 Oberkochen, Germany.<br/>Desiccator: Shuniu, Sichuan Shubo group Co., Ltd.<br/>Auto-analyzer: AA3 HR auto-analyzer, SEAL Analytical.<br/>ICP-OES: iCAP6300, Thermo Scientific.<br/>Centrifuge tube: 50 mL fluorinated ethylene propylene (FEP) centrifuge tube, Nalgene no. 3114-0050, Caps no. DS3131-0024; 50 mL polypropylene copolymer centrifuge tube, Nalgene no. 3119-0050, Caps no. DS3132-0024; 15 mL polypropylene (PP) Conical Centrifuge Tube, Axygen®, Corning.<br/>Lab spatula: Merck HS15909.</td></tr><tr><td>Experimental design</td><td>The types of biogenic silica (bSi) present in freeze-dried sediments were identified under a microscope to assist in selecting appropriate alkaline solution. After gentle homogenization using an agate mortar and lab spatula, the sediments were weighed into a conical centrifuge tube and filled with alkaline solution. The tube was then soaked in a preheated water bath to speed up the dissolution of bSi. Subsampling of the alkaline solution was conducted at one-hour intervals after centrifugation. The alkaline-leachable silicon content was measured using a UV spectrophotometer, and the bSi content was evaluated based on the time-course dissolution of silica.</td></tr><tr><td>Trial registration</td><td>N/A</td></tr><tr><td>Ethics</td><td>N/A</td></tr><tr><td>Value of the Protocol</td><td>This protocol provides a detailed description of wet chemical extraction procedures and subsequent measurement of alkaline-leachable silicon concentrations.<br/>It is easy to reproduce and provides a standardized, high-precision method for determining biogenic silica content.<br/>It can be used to determine biogenic silica content in sediments ranging from marginal seas to the open ocean.</td></tr></table>

## Background

Biogenic silica (bSi), primarily derived from siliceous microfossils such as diatoms, radiolarians, and sponge spicules, constitutes a critical component of marine biogeochemical cycles and climate regulation [1,2]. As the primary repository of dissolved silicon (Si) in oceanic systems, bSi serves as a proxy for reconstructing past productivity, assessing modern carbon sequestration mechanisms, and understanding the intricate coupling of Si and carbon cycles in marginal seas and open ocean environments [3,4].

Accurate quantification of bSi in marine sediments is indispensable for elucidating historical changes in marine productivity, evaluating anthropogenic impacts on nutrient dynamics, and predicting future responses of marine ecosystems to climatic perturbations [2,5]. However, conventional wet chemical extraction methods—often relying on standardized alkaline solutions (e.g., 0.1–0.5 M Na₂CO₃/NaOH) and fixed digestion times—face significant challenges when applied to heterogeneous marine sediments. These include incomplete dissolution of resistant bSi
phases (e.g., radiolarians and sponge spicules), co-dissolution of lithogenic silicates, and matrix interference from organic matter or authigenic minerals, leading to systematic biases in bSi burial flux estimates [5,6].

Existing protocols, such as the widely adopted 5-hour DeMaster (1981) method [7], lack flexibility to address sediment-specific complexities. For instance, sediments rich in radiolarians or sponge spicules require prolonged extraction (>8 hours) and stronger alkalis (e.g., 0.5 M NaOH) to maximize bSi recovery, while diatom-dominated samples risk overestimation due to aluminosilicate dissolution under aggressive conditions [5-7]. Additionally, traditional homogenization techniques (e.g., powdering) accelerate lithogenic silicate dissolution, further compromising accuracy [5]. These limitations are exacerbated in marginal seas (e.g., river-influenced East China Sea) and open-ocean settings (e.g., Southern Ocean siliceous oozes), where sediment composition varies widely.

To address these gaps, we present an optimized wet chemical extraction protocol that integrates sediment-specific alkaline solution selection, kinetic dissolution modeling, and rigorous quality control. Key innovations include: microscopy-guided alkaline solution selection, extended extraction kinetics, standardized dissolved Si quantification, and dual bSi calculation methods. Validated across diverse Holocene sediments—from eutrophic estuaries (Chesapeake Bay) to oligotrophic open oceans—this protocol achieves 70% higher bSi recovery in radiolarian-rich sediments compared to conventional Na₂CO₃ extractions. Its reproducibility (triplicate RSD <5%) and adaptability to low-bSi open-ocean sediments (<0.5% SiO₂) address critical gaps in global bSi burial flux assessments. By harmonizing methodological rigor with practical accessibility, this protocol advances the fidelity of marine Si cycle studies and supports emerging research in climate-biogeochemistry interactions.

# Description of protocol

## Sample preparation

Sample preparation includes drying the sediments, micrography, sediment homogenization, and weighing.

### 1. Freeze drying

Approximately 200 g of wet sediments are stored at -20 °C for 12 hours, and then dried in a freeze dryer for 48-72 hours until dry. Freeze-drying is recommended, as oven drying can potentially cause fragmentation of the biosiliceous structure [8].

### 2. Micrography

Approximately 5 mg of dried sediment was reacted with 1 mL of 1.0 M HCl and 1 mL of 30% H₂O₂ for two hours in a 15 mL centrifuge tube. The aim was to remove the organic matter and calcareous skeletons, leaving the bSi and lithogenic silicate in the residual. The residual was then washed three times with 5 mL of Milli-Q water, then mixed with an additional 1 mL of Milli-Q water using an electrical mixer (1000 rpm, 10 seconds). The well-mixed samples were transferred onto a glass microscope slide using a pipette. Different types of bSi, i.e., phytolith, diatoms, sponge spicules, radiolarians, and silicoflagellates, were identified using an optical microscope.

Samples containing mainly diatoms, phytoliths and silicoflagellates (90% of the total bSi abundance) require wet chemical extraction (8 hours) using 0.1 M Na₂CO₃ solution. Samples containing an important abundance of sponge spicules and radiolarians (>10% of the bSi abundance), or with a bSi content larger than 10%, require digestion (10-12 hours) using a 0.5 M NaOH solution. The bSi content of the sediment can be estimated approximately in accordance with Figure 1 in order to select the appropriate alkaline solution.
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Biogenic silica content (SiO₂%) in surface sediments of the global ocean. This figure was created using previously published data [5,9,10]. The white area represents data that are not available. The figure was plotted using Ocean Data View (ODV) program [11].</figcaption></figure>

**3. Homogenization of sediments and weighing**

The freeze-dried sediments were gently homogenized using an agate mortar and lab spatula. This procedure aims to improve the reproducibility of bSi measurements by mixing the sediments, while minimize the destruction of the physical structures of biosiliceous skeletons and lithogenic silicates. Powdering the sediment can cause the destruction of bSi and lithogenic silicates, thus enhancing their dissolution when exposed to alkaline solution [12]. To reduce the interference of lithogenic silica dissolution on bSi quantification, it is not recommended to powder sediments containing high levels of lithogenic silicates. The well-homogenized sediments were stored in a desiccator at room temperature.

The sediments were weighed using an analytical balance. A 50 mL fluorinated ethylene propylene (FEP) or polypropylene copolymer centrifuge tube was placed on the balance, and 30–150 mg of homogenized freeze-dried sediment was added to the centrifuge tube. Weighing the samples in a centrifuge tube can minimize the loss of sediment material. The mass of sediment (e.g., 30–150 mg) is designed for an effective extraction of bSi and for the analysis of dissolved Si concentration (see Table 1). Here, the maxim concentration of Si in alkaline solution is approximately 1,000 µmol L⁻¹, which is much lower than the Si concentration (9,000 µmol L⁻¹) proposed by Mortlock and Froelich [13].
<table><caption>**Table 1.** An example showing sediment mass, biogenic silica content, and the Si concentration in an alkaline solution and a working solution. The Si concentration in the alkaline solution was designed to be lower than 1,000 µmol L⁻¹, and the Si concentration in the working solution (after dilution by a factor of 75) was lower than 15 µmol L⁻¹. Therefore, a working standard solution ranging from 0 to 20 µmol L⁻¹ is suitable for quantifying the dissolved Si concentration in leachate.</caption><thead><tr><th>Mass of sediments [mg]</th><th>Biogenic silica [%SiO<sub>2</sub>]</th><th>Si in sediment [µmol]</th><th>Volume of alkaline solution [mL]</th><th>Si concentration in alkaline solution [µmol L<sup>-1</sup>]</th><th>Estimated Si concentration in working solution [µmol L<sup>-1</sup>]</th></tr></thead><tbody><tr><td>30</td><td>5.0</td><td>2.5</td><td>40</td><td>62.5</td><td>0.83</td></tr><tr><td>30</td><td>10.0</td><td>5.0</td><td>40</td><td>125.0</td><td>1.67</td></tr><tr><td>30</td><td>20.0</td><td>10.0</td><td>40</td><td>250.0</td><td>3.33</td></tr><tr><td>30</td><td>30.0</td><td>15.0</td><td>40</td><td>375.0</td><td>5.00</td></tr><tr><td>30</td><td>50.0</td><td>25.0</td><td>40</td><td>625.0</td><td>8.33</td></tr><tr><td>30</td><td>80.0</td><td>40.0</td><td>40</td><td>1000.0</td><td>13.33</td></tr><tr><td>150</td><td>0.1</td><td>0.3</td><td>40</td><td>6.3</td><td>0.08</td></tr><tr><td>150</td><td>0.5</td><td>1.3</td><td>40</td><td>31.3</td><td>0.42</td></tr><tr><td>150</td><td>1.0</td><td>2.5</td><td>40</td><td>62.5</td><td>0.83</td></tr><tr><td>150</td><td>3.0</td><td>7.5</td><td>40</td><td>187.5</td><td>2.50</td></tr><tr><td>150</td><td>5.0</td><td>12.5</td><td>40</td><td>312.5</td><td>4.17</td></tr><tr><td>150</td><td>10.0</td><td>25.0</td><td>40</td><td>625.0</td><td>8.33</td></tr></tbody></table>

## Wet chemical extraction

Figure 2 shows the wet chemical extraction process. Here, 40 mL of an alkaline solution (0.1 M Na₂CO₃ or 0.5 M NaOH, depending on the bSi content and types) was added to a centrifuge tube containing pre-weighed sediments. The solution and sediments were then mixed using an electric mixer (1000 rpm, 30 seconds). After sealing the tubes tightly with caps, they were placed in a water bath that had been pre-heated to 85 °C and shaken at a frequency of 100 rpm. At each one-hour time interval, the tube was immediately centrifuged at 4000 rpm for 5 min, after which 200 µL of leachate was taken using a pipette for dissolved Si analysis. Depending on the concentration of dissolved Si in the leachate, this volume can be adjusted between 100 and 500 µL. The leachate was stored in a 15 mL polypropylene conical centrifuge tube at 4 °C before measurement. In addition, the sediment and alkaline solution were mixed well using an electric mixer (1000 rpm, 30 seconds) after subsampling, after which the tubes were placed in the hot shaking water bath again. The period of extraction is suggested to be 8 hours [14], which is three hours longer than the traditional 5-hour exaction proposed by DeMaster [7]. Moreover, it is recommended that sediments containing important amounts of sponge spicules and radiolarian skeletons be extracted for 10–12 hours.

<figure><img src="image_5.png" /></figure>
**Figure 2.** Schematic diagram shows the alkaline extraction procedure.

## Measurement of dissolved silicon concentration

The dissolved Si concentration was determined using a molybdenum blue photometric method with an Ultraviolet-Visible (UV) spectrophotometer [5]. Prior to measuring the dissolved Si, the reagents listed in Table 2 were prepared. The alkaline leachate (0.5 M NaOH, 200 µL) was neutralized using an HCl solution (100 µL of 1.0 M HCl), and the Si working standards were prepared according to Table 3. The solution matrix of the dissolved Si working standard is similar to that of the alkaline leachate solution. The neutralized solution was then diluted with Milli-Q water to 10 mL. Then, 2 mL of acidified ammonium molybdate solution was added to the sample and mixed. After 20 minutes, 3 mL of chromogenic reagent (reagent E in Table 2) was added to the tube. The mixture solution (15 mL) was thoroughly mixed using an electric mixer (1000 rpm, 10 seconds). After three hours, the standards and samples were analyzed using the UV spectrophotometer (λ = 810 nm, 1 cm cuvette). Note that the UV spectrophotometer should be switched on 30 minutes prior to measurement, and that the standards should be analyzed before the sediment leachate samples. A coefficient of determination (R²) for the standard curve greater than 0.9995 is preferred. Furthermore, the concentration of dissolved Si in the reagent blank solutions needs to be evaluated [10].

The Si concentration of leachate was calculated based on the linear relationship between the Si concentration (unit: µmol L⁻¹) and absorbance value (unit: 1) of Si working standards. We compared the dissolved Si measurements obtained using the UV spectrophotometer, a nutrient auto-analyzer, and an ICP-OES. The results obtained using these methods are comparable (see Figure 5 in the Protocol Validation section). This protocol was chosen because it uses a UV spectrophotometer to determine the dissolved Si concentration, which is cost-effective and can measure up to 400 liquid samples (approximately 50 sediment samples) in one day. Note that the Si concentrations in leachate can be converted into SiO₂% (see Eq. 1). As shown in Eq. 1, C, V and M represent the Si concentration, the volume of leachate and the mass of the sediment, respectively.

$$SiO_2\% = 100 \times [10^{-6} \times C (\mu mol \ L^{-1}) \times V (L) \times 60 (g \ mol^{-1})/(M (g))] \quad Eq. 1$$

<table><caption>**Table 2.** Preparation of reagents for dissolved silicon colorimetric reaction.</caption><thead><tr><th>Reagents</th><th>Operating instructions</th><th>Storage condition</th></tr></thead><tbody><tr><td>A: Saturated oxalic acid (10% C<sub>2</sub>H<sub>2</sub>O<sub>4</sub>)</td><td>Dissolve 100 g of C<sub>2</sub>H<sub>2</sub>O<sub>4</sub> in 1 L of Milli-Q water.</td><td>Store at 4 °C in a refrigerator, and it will remain stable for one year.</td></tr><tr><td>B: Sulfuric acid solution (H<sub>2</sub>SO<sub>4</sub> : H<sub>2</sub>O = 1:2)</td><td>Mix 250 mL of concentrated H<sub>2</sub>SO<sub>4</sub> with 500 mL of Milli-Q water.</td><td>Store at room temperature, and it will remain stable for one year.</td></tr><tr><td>C: Acidified ammonium molybdate</td><td>Dissolve 8.0 g of ammonium molybdate in 976 mL of Milli-Q water in a brown plastic bottle. Then, add 24 mL of concentrated HCl solution.</td><td>Store at 4 °C in a refrigerator under dark conditions, and it will remain stable for one month.</td></tr><tr><td>D: Metol-Sodium sulfite solution</td><td>Dissolve 20 g of C<sub>7</sub>H<sub>9</sub>NO · 0.5H<sub>2</sub>SO<sub>4</sub> in 800 mL of Milli-Q water, then add 12 g of Na<sub>2</sub>SO<sub>3</sub>. Once the sodium sulfite has fully dissolved, add a further 200 mL of Milli-Q water.</td><td>Store at 4 °C in a refrigerator under dark conditions, and it</td></tr></tbody></table>
## Article Template

E: Chromogenic reagent

60 mL A solution + 90 mL B solution + 100 mL D solution + 50 mL Milli-Q water

will remain stable for one month.

Prepare the solution before use.

Silicon stock solution (20.0 mmol L⁻¹)

Dissolve 5.684 g of Na₂SiO₃·9H₂O in 500 mL of Milli-Q water in a 1 L plastic volumetric flask. Once the Na₂SiO₃·9H₂O has fully dissolved, top up with Milli-Q water to reach the 1 L mark.

Store at 4 °C in a refrigerator under dark conditions, and it will remain stable for six months.

Silicon standard solution (100 µmol L⁻¹)

Transfer 0.5 mL of the silicon stock solution to a 100 mL plastic volumetric flask, and top up with Milli-Q water until the 100 mL mark is reached.

Prepare the standard solution before use.

<table><caption>**Table 3.** An example of a dissolved Si working standard. In this study, a solution matrix similar to that of the standards and sediment leachate (0.5 M NaOH) was created, with a final sample mixture solution volume of 10 mL prior to the chromogenic reaction. This volume can be adjusted to 10 mL, 25 mL or 50 mL according to the volume of colorimetric tube and the concentration of dissolved silicon in a specific study. Those who are unfamiliar with chemical analysis are advised to analyses the standard tens of times.</caption><thead><tr><th>Number</th><th>Concentration of Si [µmol L<sup>-1</sup>]</th><th>Volume of Si standard solution [µL]</th><th>Volume of alkaline solution (0.5 M NaOH) [µL]</th><th>Volume of acid (1.0 M HCl) used for neutral reaction [µL]</th><th>Volume after addition of Milli-Q water [mL]</th><th>Volume of acidified ammonium molybdate [mL]</th><th>Volume of Chromogenic reagent [mL]</th></tr></thead><tbody><tr><td>1</td><td>0</td><td>0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>2</td><td>0.25</td><td>37.5</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>3</td><td>0.50</td><td>75.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>4</td><td>0.75</td><td>112.5</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>5</td><td>1.00</td><td>150.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>6</td><td>3.00</td><td>450.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>7</td><td>5.00</td><td>750.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>8</td><td>10.00</td><td>1500.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr><tr><td>9</td><td>15.00</td><td>2250.0</td><td>200</td><td>100</td><td>10</td><td>2</td><td>3</td></tr></tbody></table>

## Calculation of biogenic silica content

This section describes two commonly used methods for calculating the biogenic silica content: A) calculating the bSi content using a first-order dissolution kinetic model (see Eq. 2, and Figure 3A) [15,16]; and B) a linear regression calculation based on the Si vs. time curve (see Figure 2B) [7,14,17].

**Method A:** In Eq. 2, [bSiO₂%] represents the bSi content, y and t are the concentrations of Si (SiO₂%) in leachate and the extraction time (hours), k and b represent the apparent rate constants in the alkaline solution that summarize the impact of biogenic and lithogenic silica on the dissolved Si concentration respectively [16]. This calculation was
conducted using Origin 2021b software in the current study, but it can also be performed using other programs (i.e., Solver in Excel, MATLAB or Python).

$$y = [bSiO_2\%] * (1 - e^{-kt}) + bt \quad (Eq. 2)$$

**Method B:** A linear regression was calculated based on the last 3–4 data points due to their linearly dissolving characteristics (see Figure 4B), and the intercept was considered as bSi content. This method is the most commonly applied in previous studies [5,7,18]. However, it should be stated that this method is not recommended, as the selection of data points for linear extrapolation is highly subjective.

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Examples of bSi content (SiO₂%) calculated using two methods. A, non-linear fitting using a first-order dissolution kinetic model (see Eq. 2); B, linear regression of the linearly dissolving phases (e.g., the last four data points). The figures were plotted based on the alkaline extraction of sample C12 from the East China Sea, performed in triplicate. The bSi content was found to be 1.71 ± 0.04%, and 1.89 ± 0.02% using methods A and B, respectively.</figcaption></figure>

## Protocol validation

### 1. Materials

The protocol was validated using eight sediment samples that were obtained from marginal seas and the open ocean. Additionally, this study determined two samples from a previous international intercomparison campaign [19]: sample Still Pond and R64 (see Figure 4). Sample F104 was obtained off the coast of New Zealand. Samples B10 and C12 were obtained from the Bohai Sea and the East China Sea, respectively [5]. Samples Still Pond and R64 were obtained from the Chesapeake Bay [19]. Sample MTB2 was obtained from the Congo deep-sea fan (>4000 m water depth) during the Western Atlantic Climate Study (WACS) cruise [20]. Samples 2714CQ and 3585CQ were obtained from the siliceous ooze in the Southern Ocean. Following the protocol, all the samples were freeze-dried and gently homogenized using an agate mortar and lab spatula. The samples were then stored in a desiccator at room temperature before analysis.
<figure><img src="image_7.png" /><figcaption>**Figure 4.** Location of eight sediment samples used for protocol validation. The samples are surface sediment (0 – 2 cm) obtained from marginal seas (e.g., B10, C12, F104, Still Pond, and R64) and the open ocean (MTB2, 3585CQ, and 2714CQ). The water depth at the sites where these samples were collected ranges from 10 meters to 5,000 meters.</figcaption></figure>

**2. Microscopy and selection of alkaline solution**

Following microscopic observation, the predominant types of biosiliceous structure were identified (see Table 4). Sample B10 contains mainly diatoms, while samples F104 and C12 contain a mixture of diatoms, radiolarians and sponge spicules. Samples Still Pond, R64 and MTB2 mainly comprise diatoms and sponge spicules, and samples 2714CQ and 3585CQ mainly contain diatoms and radiolarians. According to the protocol, the alkaline solution recommended for each sample is listed in Table 4. Notably, this study conducted alkaline extractions using both 0.1 M Na₂CO₃ and 0.5 M NaOH in order to demonstrate the difference in bSi yield between the two extraction methods.

<table><caption>**Table 4.** Types of biogenic silica in sediment samples and the proposed alkaline solution. “Y” and “N” represent the observation or non-observation of biosiliceous structures, respectively.</caption><thead><tr><td>Sample code</td><td>Diatoms</td><td>Radiolarians</td><td>Sponge spicules</td><td>Alkaline solution</td></tr></thead><tbody><tr><td>F104</td><td>Y</td><td>Y</td><td>Y</td><td>0.5 M NaOH</td></tr><tr><td>B10</td><td>Y</td><td>N</td><td>N</td><td>0.1 M Na₂CO₃</td></tr><tr><td>C12</td><td>Y</td><td>Y</td><td>Y</td><td>0.5 M NaOH</td></tr><tr><td>Still Pond</td><td>Y</td><td>N</td><td>Y</td><td>0.5 M NaOH</td></tr><tr><td>R64</td><td>Y</td><td>N</td><td>Y</td><td>0.5 M NaOH</td></tr><tr><td>MTB2</td><td>Y</td><td>N</td><td>Y</td><td>0.5 M NaOH</td></tr><tr><td>2714CQ</td><td>Y</td><td>Y</td><td>N</td><td>0.5 M NaOH</td></tr><tr><td>3585CQ</td><td>Y</td><td>Y</td><td>N</td><td>0.5 M NaOH</td></tr></tbody></table>

**3. Dissolved Si measurement**

Dissolved Si was measured using a molybdenum blue photometric method with a UV-spectrophotometer [5]. A Si working standard (Figure 5A) was determined in accordance with the procedures outlined in Table 2 and Table 3. A linear relationship (R² = 1) was found between the measured absorbance and Si concentration, demonstrating the reliability of the measurement. Additionally, measurements of dissolved Si obtained using an ICP-OES and a nutrient auto-analyser were compared with those obtained using a UV spectrophotometer (Figure 5B). The results suggest that there is no obvious difference in the Si concentrations examined among the three techniques (Si concentration ranges from 0 to 80 µL⁻¹). Therefore, reliable data can be obtained by measuring the dissolved Si of the alkaline leachate using the molybdenum blue photometric method with a UV spectrophotometer.
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Si working standard curve (A) and comparison of dissolved Si concentrations determined using different methods (B). The absorbance of the Si working standards and corresponding dissolved Si concentration were determined using an UV-spectrophotometer (A). The Si concentrations in plot B were determined using an ICP-OES, a nutrient auto-analyser and a UV spectrophotometer.</figcaption></figure>

**4. Alkaline extraction and biogenic silica content calculation**

Figure 6 shows the sediment extraction using both 0.1 M Na₂CO₃ and 0.5 M NaOH. Triplicate alkaline extractions were performed in order to test the reproducibility of the protocol. The silica content extracted using 0.5 M NaOH was higher than that extracted using 0.1 M Na₂CO₃ (Figure 6), indicating that 0.5 M NaOH is capable of extracting more resistant biogenic silica and lithogenic silicate than the 0.1 M Na₂CO₃ solution. Of all the samples, the silica content extracted using alkaline solution was lowest in sample F104 (approximately 0.5%, see Figure 6A), and the highest in sample 3585CQ (approximately 60%, see Figure 6H). The alkaline-extractable silica content of sample B10, C12 and Still Pond was lower than 5% (Figure 6B–D), and lower than 10% for samples R64 and MTB2 (Figure 6E–F). Notably, the silica extracted using 0.5 M NaOH was 70% higher than that extracted using 0.1 M Na₂CO₃ (29.37% vs. 17.21%), highlighting the importance of selecting the appropriate alkaline solution based on micrography.
<figure><img src="image_9.png" /><figcaption>**Figure 6.** Silica (SiO₂%, dry weight/dwt%) extracted using a 0.1 M Na₂CO₃, and a 0.5 M NaOH solution. The error bars represent triplicate alkaline extractions. A) sample F104 from offshore New Zealand, B) sample B10 from the Bohai Sea, C) sample C12 from the East China Sea, D and E) sample Still Pond from the Chesapeake Bay, F) sample MTB2 from the Congo deep-sea fan, G and H) sample 2714CQ and 3585CQ from the Southern Ocean. The types of bSi in each sample were shown in Table 4. Note that the scales on the two axes are different.</figcaption></figure>

The bSi contents were analysed using the two methods described in this protocol (Table 5). Based on a two-way analysis of variance (ANOVA), no significant difference was observed between the two methods (0.1 M Na₂CO₃ extraction: *p* = 0.154, 0.5 M NaOH extraction: *p* = 0.585). However, the bSi yield from the 0.5 M NaOH extraction was significantly higher than that from the 0.1 M Na₂CO₃ extraction (*p* = 0.01; Table 5). The “estimated bSi contents” shown in Table 5 were calculated using the recommended alkaline solution for the eight samples (see Table 4 for sample information). This value is considered as an appropriate sediment bSi content due to the rigorous analytical procedure
and the quality control measures during sample preparation, alkaline extraction, dissolved Si measurement and the mathematical calculation.

Furthermore, this study determined the bSi content of Still Pond and R-64 (samples from the inter-laboratory comparison campaign) in accordance with our proposed protocol. These bSi values were then compared with the data presented in Conley (1998) [19] and other study [5] (see Figure 7). Our results (Table 5) are consistent with the inter-laboratory comparison data (Still Pond: 2.82 ± 1.17%, R-64: 6.49 ± 2.06%) [19], but are smaller than the bSi content determined by extracting powdered sediments with a 2.0 M Na₂CO₃ solution [13] or a measurement that did not display replicate extractions [22]. As presented in Conley (1998) [19], a significant discrepancy in the bSi values occurs when different alkaline extraction techniques are employed (i.e., sample preparation, alkaline solution and concentration, and mineral correction): 41% for Still Pond and 32% for R-64. Powdering of sediment destructs the structure of silicate minerals and enhances the dissolution of lithogenic silicates, thus overestimating the bSi content. Therefore, this protocol utilizes an agate mortar and lab spatula to homogenize freeze-dried sediments instead of grinding them into powder. Figure 7 also shows that the bSi values determined in this study are higher than those obtained by alkaline extraction for 5 hours [23]. This may be due to the incomplete extraction of resistant bSi (e.g., sponge spicules) presented in the samples [5]. This emphasizes the necessity of knowing the types of bSi present in sediments prior to selecting an appropriate alkaline extraction method (e.g., alkaline solution, and extraction time).

<table><caption>**Table 5.** Evaluation of the biogenic silica content (SiO₂%). The errors in values marked with “a” were calculated using modelling, while errors in other values were calculated based on triplicate extractions. N/A indicates that data are unavailable because the sample did not undergo Na₂CO₃ extraction.</caption><thead><tr><th rowspan="2">Sample</th><th colspan="2">0.1 M Na₂CO₃ extraction</th><th colspan="2">0.5 M NaOH extraction</th><th rowspan="2">Estimated bSi<br>content</th></tr><tr><th>Calculation<br>method A</th><th>Calculation<br>method B</th><th>Calculation<br>method A</th><th>Calculation<br>method B</th></tr></thead><tbody><tr><td>F104</td><td>0.19 ± 0.07</td><td>0.04 ± 0.01</td><td>0.19 ± 0.02<sup>a</sup></td><td>0.18</td><td>0.19 ± 0.02</td></tr><tr><td>B10</td><td>0.48 ± 0.05</td><td>0.38 ± 0.02</td><td>1.31 ± 0.10<sup>a</sup></td><td>1.39</td><td>0.48 ± 0.05</td></tr><tr><td>C12</td><td>1.28 ± 0.01</td><td>1.29 ± 0.08</td><td>1.71 ± 0.04</td><td>1.89 ± 0.02</td><td>1.71 ± 0.04</td></tr><tr><td>Still Pond</td><td>2.19 ± 0.02</td><td>1.93 ± 0.05</td><td>2.78 ± 0.17<sup>a</sup></td><td>2.82</td><td>2.78 ± 0.17</td></tr><tr><td>R64</td><td>5.19 ± 0.14</td><td>5.30 ± 0.09</td><td>6.43 ± 0.13<sup>a</sup></td><td>6.73</td><td>6.43 ± 0.13</td></tr><tr><td>MTB2</td><td>3.99 ± 0.07</td><td>3.47 ± 0.11</td><td>4.52 ± 0.01<sup>a</sup></td><td>4.70</td><td>4.52 ± 0.01</td></tr><tr><td>2714CQ</td><td>13.60 ± 0.47</td><td>12.02 ± 0.35</td><td>22.59 ± 0.65<sup>a</sup></td><td>22.35</td><td>22.59 ± 0.65</td></tr><tr><td>3585CQ</td><td>N/A</td><td>N/A</td><td>60.62 ± 0.79<sup>a</sup></td><td>60.40</td><td>60.62 ± 0.79</td></tr></tbody></table>

<figure><img src="image_10.png" /><figcaption>**Figure 7.** Comparison of the biogenic silica content (SiO₂%) of sample Still Pond and R-64 determined in this study and the previously published data [5, 13, 19, 22, 23]. The blue and grey dashed lines represent the mean bSi value of sample R-64 and Still Pond, respectively.</figcaption></figure>
**Limitations**

The samples used in this study are Holocene sediments, which are less than 11,700 years old. Biosiliceous structures in sediments that are older than the Holocene (i.e., the Miocene or Paleocene, which are 2–24 million years old) may undergo alteration and transformation (e.g., transformation from amorphous silica/opal-A to crystalized silica/opal-CT). These structures are therefore more difficult to fully extract using the alkaline concentration presented in this protocol. Future research involving Miocene – Paleocene (or even older) sediments should use strong alkalis (e.g., 2.0 M NaOH or KOH) to leach these physically altered biosiliceous structures for 9–12 hours [21], in accordance with our protocol. Moreover, the diagenesis and alternation of bSi will be enhanced under a high geothermal gradient [24]. Therefore, measuring bSi in sediments with a high geothermal gradient may require strong alkaline extraction or an extended extraction time.

**CRediT author statement**

**Dongdong Zhu:** Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Visualization and Funding acquisition. **Su Mei Liu:** Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration and Funding acquisition.

**Aude Leynaert:** Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration and Funding acquisition. **Paul Tréguer:** Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration and Funding acquisition. **Morgane Gallinari:** Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration and Funding acquisition. **Heting Zhou:** Investigation, Resources, Writing - Review & Editing. **Jill N. Sutton:** Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration and Funding acquisition.

**Acknowledgments**

We would like to thank Prof. Daniel Conley, Philippe Pondaven, Olivier Ragueneau, and David DeMaster for their valuable contributions to the initial discussions surrounding this work. Two samples, Still Pond and R64, were provided by Prof. Daniel Conley. The sample MTB2 from Conge deep-sea fan was provided by Prof. Olivier Ragueneau.

We would also like to thank Dr. Brivaëla Moriceau, Dr. Gaspard Delebecq, and Mrs. Manon Le Goff at the CNRS and all those who assisted with laboratory experiments.

This work was supported by the China Postdoctoral Science Foundation under Grant Number 2024M753048, the Qingdao Postdoctoral Grant (QDBSH20240202136), the National Natural Science Foundation of China (42176040, U1806211), the Taishan Scholars Programme of Shandong Province, and was supported by the French National Research Agency (18-CEO1-0011-01) and the ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir" embedded in France 2030..

**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

**Supplementary material and/or additional information [OPTIONAL]**

Not applicable.

**Data availability**

Data will be made available on request.
## References

[1] P. Tréguer, C. Bowler, B. Moriceau, S. Dutkiewicz, M. Gehlen, O. Aumont, L. Bittner, R. Dugdale, Z. Finkel, D. Ludicone, O. Jahn, L. Guidi, M. Lasbleiz, K. Leblanc, M. Levy, P. Pondaven. Influence of diatom diversity on the ocean biological carbon pump. Nature Geosci 11, 27–37 (2018). https://doi.org/10.1038/s41561-017-0028-x.

[2] P. Tréguer, J.N. Sutton, M. Brzezinski, M.A. Charette, T. Devries, S. Dutkiewicz, C. Ehlert, J. Hawkings, A. Leynaert, S.M. Liu, N. Llopis Monferrer, M. López-Acosta, M. Maldonado, S. Rahman, L. Ran, O. Rouxel. Reviews and syntheses: The biogeochemical cycle of silicon in the modern ocean. Biogeosciences 18, 1269-1289 (2021). https://bg.copernicus.org/articles/18/1269/2021.

[3] O. Ragueneau, P. Tréguer, A. Leynaert, R.F. Anderson, M.A. Brzezinski, D.J. DeMaster, R.C. Dugdale, J. Dymond, G. Fischer, R. François, C. Heinze, E. Maier-Reimer, V. Martin-Jézéquel, D.M. Nelson, B. Quéguiner. A review of the Si cycle in the modern ocean: recent progress and missing gaps in the application of biogenic opal as a paleoproductivity proxy. Glob. Planet. Chang. 26, 317-365 (2000). https://doi.org/10.1016/S0921-8181(00)00052-7.

[4] S. Westacott, N. Planavsky, M. Zhao, P.M. Hull. Revisiting the sedimentary record of the rise of diatoms, Proc. Natl. Acad. Sci. U.S.A. 118 (27) e2103517118 (2021). https://doi.org/10.1073/pnas.2103517118.

[5] D. Zhu, J.N. Sutton, A. Leynaert, P. Tréguer, J. Schoelnick, M. Gallinari, Y. Ma, S.M. Liu. Revisiting the biogenic silica burial flux determinations: A case study for the East China seas. Front. Mar. Sci. 9, 1058730 (2023). https://doi.org/10.3389/fmars.2022.1058730.

[6] D. Zhu, J.N. Sutton, A. Leynaert, P. Tréguer, S.M. Liu. The global marine silicon cycle and its major challenges. Earth Science Frontiers 29(5), 47-58 (2022). https://doi.org/10.13745/j.esf.sf.2021.8.6.

[7] D.J. DeMaster. The supply and accumulation of silica in the marine environment. Geochim. Cosmochim. Acta 45, 1715–1732 (1981). https://doi.org/10.1016/0016-7037(81)90006-5.

[8] R.J. Flower. Diatom preservation: experiments and observations on dissolution and breakage in modern and fossil material. Hydrobiologia 269, 473-484 (1993). https://doi.org/10.1007/BF00028045.

[9] C.T. Hayes, K.M. Costa, R.F. Anderson, E. Calvo, Z. Chase, L.L. Demina, J.C. Dutay, C.R. German, L.E. Heimbürger-Boavida, S.L. Jaccard, A. Jacobel, K.E. Kohfeld, M.D. Kravchishina, J. Lippold, F. Mekik, L. Missiaen, F.J. Pavia, A. Paytan, R. Pedrosa-Pamies, M.V. Petrova, S. Rahman, L.F. Robinson, M. Roy-Barman, A. Sanchez-Vidal, A. Shiller, A. Tagliabue, A.C. Tessin, M. van Hulten, J. Zhang. Global ocean sediment composition and burial flux in the deep sea. Glob. Biogeochem. Cycles, 35, e2020GB006769 (2021). https://doi.org/10.1029/2020GB006769.

[10] D. Zhu, S.M. Liu, A. Leynaert, P. Tréguer, J. Ren, J. Schoelnick, Y. Ma, J.N. Sutton. Muddy sediments are an important potential source of silicon in coastal and continental margin zones. Mar. Chem. 258, 104350 (2024). https://doi.org/10.1016/j.marchem.2024.104350.

[11] R. Schlitzer. Ocean Data View, odv.awi.de (2023). https://odv.awi.de/.

[12] P.J. Müller, R. Schneider. An automated leaching method for the determination of opal in sediments and particulate matter. Deep-Sea Res. 40, 425-444 (1993). https://doi.org/10.1016/0967-0637(93)90140-X.

[13] S.M. Liu, X.W. Ye, J. Zhang, Y.F. Zhao. Problems with biogenic silica measurement in marginal seas. Mar. Geol. 192, 383-392 (2002). https://doi.org/10.1016/S0025-3227(02)00531-5.

[14] R.A. Mortlock, P.N. Froelich. A simple method for the rapid determination of biogenic opal in pelagic marine sediments. Deep Sea Research, 36, 1425-1426 (1989). https://doi.org/10.1016/0198-0149(89)90092-7.

[15] E. Koning, G.J. Brummer, W. Van Raaphorst, J. Van Bennekom, W. Helder, J. Van Iperen. Settling, dissolution and burial of biogenic silica in the sediments off Somalia (northwestern Indian Ocean). Deep-Sea Research II 44, 1341-1360 (1997). https://doi.org/10.1016/S0967-0645(97)00018-0.

[16] D. Rickert, D., M. Schlüter, K. Wallmann. Dissolution kinetics of biogenic silica from the water column to the sediments. Geochim. Cosmochim. Acta. 66, 439–455 (2002). https://doi.org/10.1016/S0016-7037(01)00757-8.

[17] A. Landén, O. Holby, P.O.J. Hall. Determination of biogenic silica in marine sediments—selection of pretreatment method and effect of sample size. Vatten 52, 85-92 (1996).

[18] D.J. Conley, C.L. Schelske. Potential role of sponge spicules in influencing the silicon biogeochemistry of Florida lakes. Canadian Journal of Fisheries and Aquatic Sciences. 50(2), 296-302 (1993). https://doi.org/10.1139/f93-034.
[19] D.J. Conley. An interlaboratory comparison for the measurement of biogenic silica in sediments. Mar. Chem. 63, 39-48 (1998). https://doi.org/10.1016/S0304-4203(98)00049-8.

[20] C. Rabouille, K. Olu, F. Baudin, A. Khripounoff, B. Dennielou, S. Arnaud-Haond, N. Babonneau, C. Bayle, J. Beckler, S. Bessette, B. Bombled, S. Bourgeois, C. Brandily, J.C. Caprais, C. Cathalot, K. Charlier, R. Corvaisier, C. Croguennec, P. Cruaud, C. Decker, L. Droz, N. Gayet, A. Godfroy, S. Hourdez, J. Le Bruchec, J. Saout, M. Le Saout, F. Lesongeur, P. Martinez, L. Mejanelle, P. Michalopoulos, O. Mouchel, P. Noel, L. Pastor, M. Picot, P. Pignet, L. Pozzato, A.M. Pruski, M. Rabiller, M. Raimonet, O. Ragueneau, J.L. Reyss, P. Rodier, B. Ruesch, L. Ruffine, F. Savignac, C. Senyarich, J. Schnyder, A. Sen, E. Stetten, M.Y. Sun, M. Taillefert, S. Teixeira, N. Tisnerat-Laborde, L. Toffin, J. Tourolle, F. Toussaint, G. Vétion, J.M. Jouanneau, M. Bez. The Congolobe project, a multidisciplinary study of Congo deep-sea fan lobe complex: overview of methods, strategies, observations and sampling. Deep-Sea Res. II Top. Stud. Oceanogr. 142, 7-24 (2017). https://doi.org/10.1016/j.dsr2.2016.05.006.

[21] A.O. Lyle, M.W. Lyle. Determination of biogenic opal in pelagic marine sediments: A simple method revisited. Proc. Ocean Drill. Program 199, 1215-1222 (2002). https://doi.org/10.2973/odp.proc.ir.199.106.2002.

[22] L. Barão, F. Vandevenne, W. Clymans, P. Frings, O. Ragueneau, P. Meire, D.J. Conley, E. Struyf. Alkaline-extractable silicon from land to ocean: a challenge for biogenic silicon determination. Limnol. Oceanogr. Methods 13, 329-344 (2015). https://doi.org/10.1002/lom3.10028.

[23] H. Vogel, C. Meyer-Jacob, L. Thöle, J.A. Lippold, S.L. Jaccard. Quantification of biogenic silica by means of Fourier transform infrared spectroscopy (FTIRS) in marine sediments. Limnol. Oceanogr. Methods 14, 828-838 (2016). https://doi.org/10.1002/lom3.10129.

[24] S. Varkouhi, N.J. Tosca, J.A. Cartwright. Temperature-time relationships and their implications for thermal history and modelling of silica diagenesis in deep-sea sediments. Marine Geology 439, 106541 (2021). https://doi.org/10.1016/j.margeo.2021.106541.