# Increase in organic carbon stocks in subsoil layers after two decades of organic amendment application cannot be explained by the estimated contribution of multiple C inputs

Florent Levavasseur, David Montagne, Yvan Capowiez, Claire Chenu, Frédéric Rees, Ophélie Sauzet, Sabine Houot

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## To cite this version:

Florent Levavasseur, David Montagne, Yvan Capowiez, Claire Chenu, Frédéric Rees, et al.. Increase in organic carbon stocks in subsoil layers after two decades of organic amendment application cannot be explained by the estimated contribution of multiple C inputs. Geoderma, 2025, 463, pp.117546. 10.1016/j.geoderma.2025.117546. hal-05326221

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Submitted on 3 Nov 2025

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# Increase in organic carbon stocks in subsoil layers after two decades of organic amendment application cannot be explained by the estimated contribution of multiple C inputs

Florent Levavasseur<sup>a, *</sup>, David Montagne<sup>a</sup>, Yvan Capowiez<sup>b, *</sup>, Claire Chenu<sup>a, #</sup>, Frédéric Rees<sup>a</sup>, Ophélie Sauzet<sup>c, #</sup>, Sabine Houot<sup>a</sup>

## ARTICLE INFO

Handling Editor: Annette Cowie

**Keywords:**

Soil organic carbon

Subsoil

Organic waste

Organic amendment

## ABSTRACT

Soil organic carbon (SOC) is important for both soil health and climate change mitigation. Organic amendment (OA) applications are known to increase SOC stocks. However, most existing studies have focused on surface soil layers, whereas the subsoil is an important SOC stock. In this study, we quantified SOC stock accrual over 0–90 cm in the QualiAgro long-term experiment after 24 years of OA application. While surface SOC stocks increased only with OA application, they increased significantly in the subsoil both with and without OA application. Compared to the control, OA application increased subsoil SOC stocks: from 5 % to 10 % and from 3 % to 13 % of the total SOC stocks accrual over the whole soil profile occurred in the 35–50 cm and 50–90 cm soil layers, respectively. C inputs to the subsoil layers were estimated: belowground crop C inputs using crop yields and allometric coefficients, dissolved organic carbon (DOC) leaching by direct measurement (lysimeter), illuviation and bioturbation by image analysis of soil thin sections. Potential C inputs did not fully explain the subsoil SOC stocks accrual. The contribution of belowground crop C ranged from 32 % to 54 % of the SOC stocks accrual. DOC leaching showed a limited contribution (1 to 5 %). The contribution of bioturbation and illuviation was very limited (≤3%). Our study suggests discrepancies in our current understanding of the mechanisms influencing subsoil SOC, and highlights the importance of considering the subsoil when monitoring the effect of OA application on SOC stocks.

## 1. Introduction

Soil organic carbon (SOC) is considered as a good indicator of soil health (Liptzin et al., 2022). SOC is also one of the most important C stocks on Earth, and increasing or at least maintaining this stock is crucial for mitigating climate change (Minasny et al., 2017). While most research in the past has focused on SOC stocks in the surface soil (around 0–30 cm), the assessment of SOC stocks in subsoil layers has recently gained interest. In their meta-analysis, Balesdent et al. (2018) found that soils at a depth of 30–100 cm below the surface contain, on average, 47 % of the topmost meter SOC stocks. This SOC stored in the subsoil could also be more stable compared to surface SOC stocks due to the interaction with the mineral phase and to the partial separation of SOM, microorganisms and extracellular enzyme activity (Rumpel and Kögel-

Knabner, 2011). This reinforces the interest in storing C in the subsoil for climate change mitigation. According to Rumpel and Kögel-Knabner (2011), the main sources of SOC in the subsoil are plant roots and exudates, leaching of dissolved organic carbon (DOC), and bioturbation by soil macrofauna. In clay-illuviated soils, Torres-Sallan et al. (2017) also highlighted that illuviation enables durable subsoil storage of carbon deposited in the upper profile. Illuviation transports SOC in microaggregates and silt plus clay fractions in subsoil, and this SOC fraction has a higher residence time than the other SOC fractions (Balesdent, 1996).

The application of organic amendment (OA) (e.g., manure, compost, sewage sludge) is a well-known practice to increase SOC stocks in the surface soil layers, depending on the type of OA, on the application rate and on the soil and climatic conditions (Levavasseur et al., 2020).

<footnote><sup>a</sup> Université Paris-Saclay, INRAE, AgroParisTech, UMR ECOSYS, 91120 Palaiseau, France</footnote>

<footnote><sup>b</sup> INRAE, UMR1114, EMMAH, INRAE/Université d'Avignon, Site Agroparc, 84914 Avignon cedex 09, France</footnote>

<footnote><sup>c</sup> University of Applied Sciences and Arts of Western Switzerland (HES-SO), HEPIA inTNP, Soils and Substrates Group, Geneva, Switzerland</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: florent.levavasseur@inrae.fr (F. Levavasseur).</footnote>
Although much debated in the literature (Derrien et al., 2023), the chemical recalcitrance of OA can explain the variability in SOC stocks accrual between different OA for a given set of pedoclimatic conditions (Derrien et al., 2023). In the subsoil layers, only few studies have focused on the effects of OA application on SOC stocks. In a recent analysis of four long-term experiments (LTE) on luvisols with several decades of farmyard manure application, Skadell et al. (2023) found an increase in SOC stocks of 6.4, 0.7 and 0.1 t C ha⁻¹ in the surface (0–30 cm), upper subsoil (30–50 cm) and lower subsoil (50–100 cm), respectively. In the same study, it was found that 19 % of the total increase in SOC stocks due to different practices (e.g., mineral fertilization, manure application, irrigation) was found in the upper subsoil and 3 % in the lower subsoil, while most of the increase (78 %) occurred in the surface soil. In a tropical context, Rambaut et al. (2022) also showed significant increases in SOC stocks in the 30–60 cm soil layer after 15 years of repeated manure or compost application. Finally, in addition to the limited number of studies focusing on subsoil SOC stocks accrual, very few studies have attempted to quantify the potential sources of this accrual.

The objectives of this study were i) to quantify the SOC stocks accrual in surface and subsoil layers in an LTE with repeated OA applications, and ii) to quantify the main mechanisms by which SOC stocks increased in subsoil layers (belowground crop C input, DOC leaching, illuviation and bioturbation). Our first hypothesis was that OA applications resulted in a significant SOC stocks accrual in subsoil layers, depending on the type of OA, especially its recalcitrance. Our second hypothesis was that this SOC stock accrual could be explained by the different known C inputs to the subsoil, i.e., belowground crop C inputs, DOC leaching, bioturbation and illuviation. We used the QualiAgro LTE, located in northern France, where different OA have been applied regularly since 1998.

## 2. Material and methods

### 2.1. The QualiAgro long-term experiment

#### 2.1.1. General characteristics

The 6 ha field of the QualiAgro LTE is located in northern France, 20 km west of Paris (48°53'27'' N, 1°58'20'' E). In this study, we focused on the northern part of the experiment, called N + from 1998 to 2013 and PRO since 2014 (Fig. S1, Supplementary Material). The field has been cultivated with arable crops for decades before the start of the experiment. The experiment started in 1998 with the aim of comparing the agronomic and environmental effects of the application of urban composts with farmyard manure and mineral fertilization only. The climate is temperate oceanic in the Köppen classification, with mean annual temperature, mean annual precipitation and mean evapotranspiration for the period 2003–2021 equal to 10.9 °C, 613 mm and 709 mm, respectively (on-site weather station since 2003). The soil is classified as a Haplic Luvisol (IUSS Working Group WRB, 2015) and developed on Quaternary loess deposits. The soil depth is about 150 cm without any rocky elements. In our study, we focused on the upper 90 cm because this corresponds to the lower limit of the fourth soil layer and to the depth of the existing lysimeter plates. Four distinct soil layers are observed down to 90 cm (Paradelo et al., 2019), the depth considered in our study. The 0–28 cm soil layer is the plowed layer. The 28–35 cm soil layer corresponds to the plow pan or to the former bottom of a deeper plowed layer. The 35–50 cm soil layer is an eluviated horizon. The 50–90 cm soil layer is an argic horizon, with redoximorphic features. The surface layer has a silt-loam texture, while the subsoil layers are more clayey (Table 1). The soil is decarbonated to a depth of 150 cm. The pH at the beginning of the experiment was neutral in all soil layers. At the beginning of the experiment in 1998, the carbon content showed a classical trend along the soil profile, decreasing from 10.6 g kg⁻¹ in the 0–28 cm soil layer to 2.4 g kg⁻¹ in the 50–90 cm soil layer.

<caption>**Table 1**</caption>

Mean (and standard deviation) initial soil characteristics in 1998 (n = 20 for the 0–28 cm soil layer, n = 5 for the subsoil layers). C<sub>org</sub> and C/N values of the 28–35 cm soil layer were not determined in 1998 and the C<sub>org</sub> and C/N values of the 28–35 cm soil layer in the CON treatment in 2004 was used as a proxy.

<table><thead><tr><th></th><th>Soil layer<br>(cm)<br>0–28</th><th>28–35</th><th>35–50</th><th>50–90</th></tr></thead><tbody><tr><td>Clay (&lt;2 µm) (g/kg)</td><td>146 (7)</td><td>196<br>(34)</td><td>216<br>(18)</td><td>309<br>(14)</td></tr><tr><td>Light silt (2–20 µm) (g/kg)</td><td>269 (8)</td><td>283 (6)</td><td>279<br>(11)</td><td>244 (5)</td></tr><tr><td>Coarse silt (20–50 µm) (g/kg)</td><td>516 (8)</td><td>468<br>(27)</td><td>443<br>(13)</td><td>400 (7)</td></tr><tr><td>Light sand (50–200 µm) (g/<br>kg)</td><td>61 (5)</td><td>38 (8)</td><td>45 (4)</td><td>37 (2)</td></tr><tr><td>Coarse sand (0.2–2 mm) (g/<br>kg)</td><td>8 (1)</td><td>5 (2)</td><td>17 (5)</td><td>10 (2)</td></tr><tr><td>CaCO<sub>3</sub> (g/kg)</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1</td><td>&lt; 1</td></tr><tr><td>pH (H<sub>2</sub>O)</td><td>7.1 (0.2)</td><td>7.6<br>(0.5)</td><td>7.2<br>(0.1)</td><td>7.3<br>(0.2)</td></tr><tr><td>C<sub>org</sub> (g/kg)</td><td>10.6 (0.6)</td><td>9.4<br>(0.4)</td><td>3.8<br>(0.3)</td><td>2.4<br>(0.3)</td></tr><tr><td>C/N</td><td>9.5 (0.4)</td><td>9.5<br>(0.0)</td><td>8.0<br>(0.4)</td><td>6.4<br>(0.4)</td></tr></tbody></table>

The soil was plowed to a depth of 28 cm every year in autumn or winter. From 1998 to 2013, the crop rotation was grain maize (Zea mays) – winter wheat (Triticum aestivum), with the exception of winter barley (Hordeum vulgare) in 2007. Maize residues were returned to the soil, while wheat straw was harvested. Pesticides were used to control weeds and pests. Since 2014, organic farming practices were introduced. The crop succession changed slightly, with grain maize followed by one to several years of cereals: rye (Secale cereale), spring barley or winter wheat. Also, since 2014, all crop residues have been returned to the soil and the use of pesticide has been stopped.

#### 2.1.2. Description of the treatments and of the organic amendments applied

The experimental design is a randomized block design, with four replicates per treatment. Each individual plot has a length of 45 m and a width of 10 m. The five treatments include a control with no OA (CON) and four OA treatments with different OA: a compost of green waste and sewage sludge (GWS), a compost of green waste and the home-sorted organic fraction of municipal solid waste (BIO), a compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging (MSW) and dairy farmyard manure (FYM). From 1998 to 2013, OA was applied every two years at the end of the summer preceding the maize at a rate of 4 t C ha⁻¹. This rate and frequency of application resulted in approximately twice the amount of OA usually applied by farmers. Since 2014, OA was still applied every two years, but at a reduced rate of 2 t C ha⁻¹. OA were applied with a manure spreader. From 1998 to 2013, mineral N fertilizers (urea ammonium nitrate) were applied to achieve optimal yield in both the control and OA treatments. The rate of mineral N fertilizer was identical for wheat regardless of the treatment, while for maize it was adapted per treatment depending on the soil N supply and crop N requirements. Since 2014, no mineral N fertilizer has been applied, while an organic fertilizer (blood and bone meal) is applied when needed to achieve optimal yield in both the control and OA treatments. The rate of organic N fertilizer was adapted per treatment depending on the soil N supply and crop N requirements. The C input with this fertilizer was neglected in this study due to the limited quantity of C applied (a maximum of 0.5 t C ha⁻¹ yr⁻¹ in the CON treatment) and its rapid mineralization.

The MSW compost and FYM had higher organic C content (NF ISO 10694 method) and C:N<sub>org</sub> than the GWS and BIO composts (Table 2). However, the estimated stability of the C in FYM and MSW was significantly lower than in the GWS and BIO, as indicated by their lower
<caption>**Table 2**</caption>

Mean (and standard deviation) of the organic amendment characteristics and of the input flows per application (n = 9 and n = 4 for 1998–2013 and 2015–2021, respectively). Letters indicate significant differences (p-value < 0.05). DM: dry matter, I<sub>ROC</sub>: Indicator of potential residual carbon in soils (g C 100 g<sup>-1</sup> C of OA), FM: fresh matter, OA: organic amendment.

<table><thead><tr><th>Period</th><th>OA</th><th>DM<br>(%<br>FM)</th><th>C<br>(g kg<sup>-1</sup><br>DM)</th><th>C:<br>N<sub>org</sub></th><th>I<sub>ROC</sub><br>(% C)</th><th>FM (t<br>ha<sup>-1</sup>)</th><th>C (t<br>ha<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="5">1998–2013</td><td>GWS</td><td>64<br>(8)</td><td>265 (45)</td><td>12.9</td><td>77<br>(9) a</td><td>26.2<br>(5.3)</td><td>4.36<br>(1.04) a</td></tr><tr><td>BIO</td><td>71<br>(9)</td><td>211 (47)</td><td>12.6</td><td>75<br>(6) a</td><td>27.7<br>(8.1)</td><td>3.92<br>(0.72) a</td></tr><tr><td>MSW</td><td>67<br>(12)</td><td>310 (45)</td><td>18.5</td><td>48<br>(13) c</td><td>18.3<br>(4.1)</td><td>3.74<br>(1.01)</td></tr><tr><td>FYM</td><td>39<br>(9)</td><td>324 (68)</td><td>15.5</td><td>65<br>(8) b</td><td>34.6<br>(11.0)</td><td>4.06<br>(0.82) a</td></tr><tr><td>2015–2021</td><td>GWS</td><td>64<br>(2)</td><td>249 (29)</td><td>12.6</td><td>71<br>(1) ab</td><td>11.9<br>(1.4)</td><td>1.93<br>(0.34) c</td></tr><tr><td></td><td>BIO</td><td>79<br>(6)</td><td>308 (27)</td><td>12.3</td><td>66<br>(7) b</td><td>11.9<br>(2.9)</td><td>2.87<br>(0.76)</td></tr><tr><td></td><td>MSW</td><td>76<br>(4)</td><td>325 (16)</td><td>21.3</td><td>39<br>(7) c</td><td>9.0<br>(1.4)</td><td>2.23<br>(0.44) c</td></tr><tr><td></td><td>FYM</td><td>42<br>(5)</td><td>358 (36)</td><td>18.1</td><td>52<br>(8) c</td><td>14.1<br>(2.0)</td><td>2.11<br>(0.22) c</td></tr></tbody></table>

indicator of potential residual carbon (I<sub>ROC</sub>) (Lashermes et al., 2009). There was no significant difference in the OA C input between treatments.

## 2.2. SOC stock calculation

### 2.2.1. Soil sampling and SOC content analysis

In 1998, soil was sampled from the 0–28 cm layer in each individual plot. Soil from the 35–50 cm and 50–90 cm soil layers was also sampled in five plots throughout the experiment (plots 201, 202, 203, 302, 303, Fig. S1, Supplementary Material). The 28–35 cm soil layer was not sampled in 1998, but was sampled in 2004. The SOC content of the 28–35 cm soil layer in the CON treatment in 2004 was used as a proxy for the initial SOC content of the 28–35 cm soil layer in 1998.

Soil was sampled in each plot in early September 2009 (0–28 cm) and spring 2010 (28–35, 35–50, and 50–90 cm), as well as in early September 2021 (0–28 and 28–35 cm) and spring 2022 (35–50 and 50–90 cm). September surface soil sampling was always done prior to OA application. Sampling in the subsoil layers was done in the spring because the soil was too dry in September to allow sampling. It was assumed that the soil sampled in the deeper layers was not affected by the most recent OA application. Total carbon was determined by dry combustion (NF ISO 10694 method), inorganic carbon by calcimetry (NF ISO 10693 method), and total organic carbon was calculated as the difference between total and inorganic carbon.

### 2.2.2. Determination of soil bulk density

Soil bulk density was determined using the cylinder method (NF X31-501 method). For the 0–28 cm soil layer, the mean (n = 20) value over the experiment, equal to 1.26, was used for all the plots in 1998. In 2009 and 2021, bulk density of the 0–28 cm soil layer was determined for each individual plot (four replicates per plot) and these individual values per plot were used to calculate the SOC stocks. The bulk density of the 28–35 cm soil layer was determined for each plot in 2004 and 2011 (four replicates per plot). No effect of treatment was observed (data not shown) and the mean value of 1.49 g cm<sup>-3</sup> was used for all plots and years. The bulk density of the 35–50 and 50–90 cm soil layers was determined in five pits dug in 2004 across the experiment. A mean bulk density of 1.46 g cm<sup>-3</sup> and 1.50 g cm<sup>-3</sup> was used for all plots and years

in the 35–50 and 50–90 cm soil layers, respectively.

### 2.2.3. SOC stocks at fixed depth

In the absence of stony elements over 0–90 cm, SOC stocks were simply calculated at a fixed depth for each soil layer (0–28, 28–35, 35–50, and 50–90 cm) as follows:

$$SOC_{stock\ FD\ i} = \sum_{i} \frac{SOC_{content\ i}}{1000} \times BD_{i} \times z_{i} \times 10000 \quad (1)$$

With SOC<sub>stock FD i</sub> the SOC stock with the fixed depth method of the soil layer i (t C ha<sup>-1</sup>), SOC<sub>content i</sub> the SOC content of the soil layer i (g kg<sup>-1</sup>), BD<sub>i</sub> the bulk density of the soil layer i (g cm<sup>-3</sup>), z<sub>i</sub> the thickness of the soil layer i (m).

### 2.2.4. SOC stocks with equivalent soil mass

Due to changes in soil bulk density over time and differences between treatments, SOC stocks were also calculated for an equivalent soil mass (ESM) following von Haden et al. (2020). We considered a cumulative mineral soil mass that served as a conserved reference between sampling dates and treatments (cumulative mineral soil mass in 1998 at 28, 35, 50, and 90 cm in our case). For each plot and sampling date, cumulative SOC stocks were then interpolated to the reference masses using a cubic-spline interpolation model. Non-cumulative SOC stocks were finally calculated by subtracting the cumulative SOC stocks between adjacent depth intervals (von Haden et al., 2020).

### 2.2.5. SOC stock accrual

Absolute SOC stocks accrual per soil layer was calculated for all treatments as the difference between SOC stocks in 2022 and SOC stocks in 1998. SOC stock accrual relative to CON was calculated for OA treatments as the difference between SOC stocks in OA treatments in 2022 and SOC stocks in CON in 2022.

## 2.3. Measurement of DOC flows

In 2004, a pair of fiberglass wick lysimeters (0.0625 m<sup>2</sup> each) was installed in a single plot of each of the treatments GWS, MSW, and CON at a depth of 45 cm (plots 201, 202 and 303, Fig. S1, Supplementary Material). In 2007, additional pairs of lysimeters were installed at the same depth for the treatments BIO and FYM (plots 203 and 302). In 2011, additional pairs of lysimeters were installed on the same plots (201, 202, 203, 302, and 303) at a depth of 100 cm (0.125 m<sup>2</sup> each). Drained water was regularly pumped from each lysimeter and the volume measured (mainly from December to March), while water samples were collected for DOC analysis. The collection areas of the 45 cm and 100 cm lysimeters were made of a 0.0625 m<sup>2</sup> and of a 0.125 m<sup>2</sup> stainless steel plates, respectively. DOC flows between two sampling dates were computed as follows:

$$fDOC_i = \frac{10^{-6} \times DOC_i \times V_i}{S \times 10^{-4}} \quad (2)$$

With fDOC<sub>i</sub>, the flow (kg C ha<sup>-1</sup>) of DOC during the period i, DOC<sub>i</sub> the DOC content (mg L<sup>-1</sup>) of the drained water during the period i, V<sub>i</sub>, the volume (L) of drained water during the period i, and S the surface area of the lysimeter plate (m<sup>2</sup>).

## 2.4. Estimation of belowground crop C input with allometric coefficients

Plant allometric relationships were used to estimate the contribution of roots to the total possible C inputs over the monitoring period. Crop yield and total aboveground plant biomass were measured each year in each individual plot. Using a shoot:root ratio specific to each crop (Clivot et al., 2023, Clivot et al. (2019)), we estimated each year the belowground C input to the soil from roots and extra-root material (rhizodeposition) using the following equation:
$$C_{BG_{p,0-i}} = \frac{DM_{AG,p}}{SRR_p} \times 0.4 \times 1.65 \times (1 - \beta_p^i) \quad (3)$$

With $C_{BG_{p,0-i}}$ the belowground C input of the considered crop p to soil from roots and extra-root material up to a considered soil depth i, $DM_{AG,p}$ the total aboveground biomass of the considered crop p (t ha⁻¹ dry matter), $SRR_p$ the shoot:root ratio of the considered crop p, 0.4 the C content of belowground crop biomass (t t⁻¹) (Boiffin et al., 1986), 1.65 a coefficient to estimate total belowground C input when assuming that extra-root material represents 65 % of standing root biomass at harvest (Bolinder et al., 2007) and, β a parameter describing root distribution with soil depth of the considered crop p (Clivot et al., 2023).

We then computed each year the belowground C input of the considered crop p to soil from roots and extra-root material for subsoil layers:

$$C_{BG_{p,28-35}} = C_{BG_{p,0-35}} - C_{BG_{p,0-28}} \quad (4)$$

$$C_{BG_{p,35-50}} = C_{BG_{p,0-50}} - C_{BG_{p,0-35}} \quad (5)$$

$$C_{BG_{p,50-90}} = C_{BG_{p,0-90}} - C_{BG_{p,0-50}} \quad (6)$$

Both mean and standard deviation values per treatment were computed (4 plots/replicates per treatment).

## 2.5. C transfer from illuviation and bioturbation

Sauzet et al. (2023, 2016) used image analysis on large soil thin sections of around 140 mm width × 60 mm height to quantify the cumulative intensity of illuviation and bioturbation during soil development in the two plots 303 (CON) and 203 (FYM) of the QualiAgro LTE. Briefly, the total mass of clay coatings and papules (i.e., fragments of clay coatings reworked by bioturbation) produced during soil development per unit surface area of soil ($M_{ill}$ in kg m⁻²) was quantified:

$$M_{ill} = \sum_{i=1}^{n} (V_{pap,i} + V_{coat,i}) \times \rho_{coat} \times z_i \quad (7)$$

with $V_{pap,i}$ and $V_{coat,i}$ the volumetric proportions of papules and coatings, respectively, measured by image analysis on three soil sections from each soil layer i, expressed as a percentage of the volume of the soil layer (Sauzet et al., 2017), $z_i$ the thickness of the i-th sampled layer (m), and n the number of 10-cm thick soil layers from 30 to 90 cm depth. The bulk density of the clay coatings, $\rho_{coat}$, measured with the kerosene method on 21 manually sorted replicates of clay coatings was 1.750 kg m⁻³ (Sauzet et al., 2017).

The total mass of earthworms' casts between 30 and 90 cm depth per unit area of soil ($M_{tur}$ in kg m⁻²) was estimated:

$$M_{cast} = \sum_{i=1}^{n} V_{cast,i} \times \rho_{cast} \times z_i \quad (8)$$

with $V_{cast,i}$ the volumetric proportion of earthworm casts in percent of the total volume of the soil layer i, $\rho_{cast}$ the bulk density (kg m⁻³) of earthworms casts, $z_i$ the thickness of the soil layer i, and n the number of 10-cm thick soil layers from 30 to 90 cm depth.

The volumetric proportion of earthworm casts was estimated as the ratio between the proportion of illuvial features fragmented by biological activity, the so-called papules ($V_{pap,i}$) and the total proportion of illuvial features ($V_{pap,i}$ plus $V_{coat,i}$) on thin sections (Sauzet et al., 2023):

$$V_{cast,i} = \frac{V_{pap,i}}{V_{pap,i} + V_{coat,i}} \quad (9)$$

The bulk density of earthworm casts was set to 1.5 kg m⁻³, the mean of the bulk densities measured by Le Bayon et al. (2021) and Le Mer et al. (2022) for *Lumbricus terrestris* casts. In the QualiAgro LTE, *L. terrestris*

accounted for most of the earthworm biomass and was the only anecic earthworm species observed (Sauzet et al., 2023). By dividing the total masses of illuviated clay features and earthworm casts by the estimated duration of the illuviation process in pedogenesis, the mean yearly transfer of illuviated and bioturbated materials in the 35–50 and 50–90 cm soil layers was estimated. According to Finke and Hutson (2008) and to Quénard et al. (2011), the total duration of illuviation in soils developed in Quaternary loess deposits ranged from 1,000 to 14,000 years, depending on climate and parent material conditions, i.e. a mean duration of 7,500 years. We considered these three durations of illuviation (1,000, 7,500, and 14,000 years) to obtain a range of possible mean yearly transfers of illuviated and bioturbated materials.

Finally, the mean yearly illuviation and bioturbation C transfers during the period 1998–2022 were obtained by multiplying the mean yearly transfer of illuviated and bioturbated material during the soil development by C contents assumed to be representative of the illuvial clay features and earthworm casts produced during the period considered. For illuviation, the C content of the clay-sized fraction of the topsoil of each treatment sampled in 2019 (Kpemoua et al., 2024) was used as a proxy for the C content of illuvial clay features. For bioturbation, the C content of earthworm casts was assumed to be 45 % higher than that of bulk topsoil (Van Groenigen et al., 2019). The topsoil C content in 2009 was chosen as a proxy for the mean topsoil C content over the study period.

## 2.6. Statistical analyses

We tested the significance of the effect of the treatment and/or of the year on the soil C content, soil bulk density, soil C stocks, soil C stocks accrual and belowground crop C inputs. We could not test the significance of the differences in DOC leaching, because only two pseudoreplicates per treatment were available (two lysimeters on the same plot for each treatment). No statistical analyses could be performed for illuviation and bioturbation because estimates were only made for two treatments, with no replicates. We tested the significance of the OA type on the IROC value and on the OA C input. We used Kruskal-Wallis tests followed by posthoc tests using the criterium Fisher's least significance difference. All tests were performed in R 4.3.2, using “kruskal” function of the “agricolae” package.

# 3. Results

## 3.1. SOC content evolution

In 2009–2010, the SOC content had significantly increased in all soil layers after OA application compared to 1998 (Table 2, Fig. S2, Supplementary Material). Significantly higher SOC content was also measured in the 50–90 cm soil layer for CON. SOC content was significantly higher than CON for all OA treatments in the 0–28 and 28–35 cm soil layer (mean SOC content in the 0–28 cm soil layer of 14.1 and 10.6 g kg⁻¹ for the four OA treatments and CON, respectively), but only for FYM in the 35–50 cm soil layer (4.9 g kg⁻¹ and 4.3 g kg⁻¹ for FYM and CON, respectively). SOC content in the 50–90 cm soil layer was not significantly different in the OA treatments compared to CON. SOC content was also significantly higher in the BIO, GWS and FYM treatments (14.8, 14.8 and 14.4 g kg⁻¹, respectively) compared to the MSW treatment (12.4 g kg⁻¹) in the 0–28 cm soil layer, as well as in the 28–35 cm soil layer.

In 2021–2022, a further significant increase in SOC content was observed compared to 2009–2010 in the 0–28 cm soil layer for BIO and GWS, and in the 28–35 cm, 35–50, and 50–90 cm soil layers for all OA treatments (Table 2, Fig. S2, Supplementary Material). SOC content also increased in CON in the 35–50 and 50–90 cm soil layers, so that SOC content was significantly higher than CON only for BIO and GWS in the 35–50 cm soil layer (7.9, 7.6 and 5.7 g kg⁻¹ for BIO, GWS and CON, respectively) and was not significantly higher than CON in OA
treatments in the 50–90 cm soil layer.

## Tableau 1

Mean SOC content (and standard deviation) (g kg⁻¹) per soil layer (0–28, 28–35, 35–50, and 50–90 cm) for each treatment and sampling year in the QualiAgro LTE. Letters indicate significant differences (p-value < 0.05) and can only be compared between treatments and dates for the same soil layer. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure, CON: control.

<table><thead><tr><th>Period</th><th>Layer (cm)</th><th>Treatment BIO</th><th>GWS</th><th>FYM</th><th>MSW</th><th>CON</th></tr></thead><tbody><tr><td rowspan="4">1998</td><td>0–28</td><td>10.6 (0.6) e</td><td></td><td></td><td></td><td></td></tr><tr><td>28–35</td><td>9.4 (0.4) e</td><td></td><td></td><td></td><td></td></tr><tr><td>35–50</td><td>3.8 (0.3) g</td><td></td><td></td><td></td><td></td></tr><tr><td>50–90</td><td>2.4 (0.3) e</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">2009–2010</td><td>0–28</td><td>14.8 (0.6) c</td><td>14.8 (0.8) c</td><td>14.3 (0.4) c</td><td>12.4 (0.3) d</td><td>10.6 (0.3) e</td></tr><tr><td>28–35</td><td>11.9 (1.2) c</td><td>12.0 (1.2) c</td><td>12.0 (0.4) c</td><td>10.7 (0.6) d</td><td>9.4 (0.6) e</td></tr><tr><td>35–50</td><td>4.5 (0.2) ef</td><td>4.5 (0.3) ef</td><td>4.9 (0.3) de</td><td>4.5 (0.2) f</td><td>4.3 (0.2) fg</td></tr><tr><td>50–90</td><td>3.0 (0.2) cd</td><td>3.0 (0.2) cd</td><td>3.2 (0.2) bc</td><td>2.8 (0.2) de</td><td>3.0 (0.2) cd</td></tr><tr><td>2021–2022</td><td>0–28</td><td>16.8 (0.2) ab</td><td>17.4 (0.3) a</td><td>14.9 (0.4) bc</td><td>14.2 (0.7) cd</td><td>10.3 (0.3) e</td></tr><tr><td rowspan="3">2009–2010</td><td>28–35</td><td>16.4 (0.5) a</td><td>16.8 (0.8) a</td><td>13.7 (0.6) b</td><td>13.6 (0.8) b</td><td>9.9 (0.4) de</td></tr><tr><td>35–50</td><td>7.9 (0.7) a</td><td>7.6 (1.6) ab</td><td>6.5 (0.3) bc</td><td>6.6 (0.6) abc</td><td>5.7 (0.6) cd</td></tr><tr><td>50–90</td><td>3.9 (0.2) a</td><td>3.6 (0.4) a</td><td>3.8 (0.3) a</td><td>4.0 (0.5) a</td><td>3.5 (0.4) ab</td></tr></tbody></table>

## 3.2. SOC stocks

In 1998, at the beginning of the experiment, SOC stocks (with ESM) were equal to 37.1, 9.8, 8.4 and 14.4 t C ha⁻¹ in the 0–28, 28–35, 35–50

and 50–90 cm soil layers, respectively, which represented 53, 14, 12 and 21 % of the total 0–90 cm SOC stocks (69.6 t C ha⁻¹), respectively (Fig. 1).

In 2009–2010, SOC stocks had significantly increased compared to 1998 in all treatments in the 0–28 (except for CON), 28–35, and 35–50 cm (except for BIO) soil layers, while they had significantly increased only in the BIO, GWS and FYM treatments in the 50–90 cm soil layer. SOC stocks were also higher compared than CON in all the OA treatments for the surface layer, in the BIO, GWS and FYM treatments for the 28–35 cm soil layer, and in the BIO, GWS and FYM treatments for the 50–90 cm soil layer.

In 2021–2022, further significant increases in SOC stocks were observed in all soil layers for most OA treatments compared to 2009–2010. SOC stocks also increased significantly in CON in the 35–50 and 50–90 cm soil layers, but did not change significantly in the 0–28 and 28–35 cm soil layers. Finally, SOC stocks in 2021–2022 were higher for all OA treatments compared to CON in the 0–28 cm soil layer (59.7, 61.8, 52.6, 50.3, and 36.0 t C ha⁻¹ for BIO, GWS, FYM, MSW and CON, respectively) and in the 28–35 cm soil layer (17.6, 18.1, 15.0, 14.6, and 11.4 t C ha⁻¹ for BIO, GWS, FYM, MSW and CON, respectively). In the subsoil layers, SOC stocks were significantly higher compared to CON only in BIO and GWS for the 35–50 cm soil layer (17.8, 17.2 and 13.9 t C ha⁻¹ for BIO, GWS and CON, respectively) and in BIO only for the 50–90 cm soil layer (23.0 and 20.4 t C ha⁻¹ for BIO and CON, respectively).

## 3.3. SOC stocks accrual

Between 1998 and 2022, the accrual in SOC stocks in the surface soil layer was 22.7, 24.7, 15.5 and 13.2 t C ha⁻¹ for BIO, GWS, FYM and MSW, respectively (Fig. 2a), which represented 39 % (MSW) to 51 % (GWS) of the total SOC stocks accrual over the whole soil profile (Fig. 2b). The contribution of each of the 28–35, 35–50 and 50–90 cm soil layers to the total SOC stocks accrual ranged from 14 to 26 %. During the same period, the SOC stocks decreased by 1 t C ha⁻¹ for CON in the 0–28 cm soil layer, while SOC stocks increased in the 35–50 and 50–90 cm soil layers (5.5 and 6.0 t C ha⁻¹, respectively).

In 2022, relative to CON, OA induced a SOC stock accrual in the surface soil layer of 23.7, 25.8, 16.6 and 14.3 t C ha⁻¹ for BIO, GWS, FYM and MSW, respectively (Fig. 2c), which represented 65 % (MSW

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Mean SOC stocks with the equivalent soil mass method per soil layer, treatment and year in the QualiAgro LTE. Error bars represent standard deviation and letters indicate significant differences (p-value < 0.05). The lowercase letters were used for comparisons for the same soil layer between the different dates and treatments, while the uppercase letters were used for the different dates and treatments for the whole soil profile. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure, CON: control.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Mean absolute SOC stocks accrual (a) and SOC stocks accrual relative to control (c) in 2022 with the equivalent soil mass method per soil layer and treatment in the QualiAgro LTE and proportion of the total (0–90 cm) absolute (b) or relative (d) SOC stocks accrual per soil layer. Absolute SOC stocks accrual is calculated as the difference in SOC stocks for each treatment between 2022 and 1998 (a,b), while relative SOC stocks accrual is the difference in SOC stocks in 2022 between the OA treatments and the control (c,d). Error bars represent standard deviation. Letters indicate significant differences (p-value < 0.05) and can only be compared between treatments for the same soil layer. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure, CON: control.</figcaption></figure>

and BIO) to 73 % (FYM) of the total SOC stock accrual (Fig. 2d). The contribution of each of the 28–35, 35–50, and 50–90 cm soil layers to SOC stock accrual (relative to CON) varied from 3 % (GWS 50–90 cm) to 13 % (MSW 50–90 cm).

3.4. *Estimation of C input by DOC leaching*

On average, 237 and 209 mm yr⁻¹ of water were collected in the lysimeters at 45 and 100 cm, respectively, with slight differences observed between treatments (Fig. S5, Supplementary Material). The mean DOC content of drained water (mean of all water samples collected at both depths) was 11 mg L⁻¹ (standard deviation of 16 mg L⁻¹). Cumulative DOC flows at 45 cm ranged from 226 kg C ha⁻¹ for CON to 380 kg C ha⁻¹ for GWS from 2004 to 2019, and from 362 kg C ha⁻¹ for

FYM to 440 kg C ha⁻¹ for BIO from 2007 to 2019 (Fig. 3). Cumulative DOC flows at 1 m from 2011 to 2019 ranged from 63 kg C ha⁻¹ for CON to 136 kg C ha⁻¹ for BIO. Mean yearly DOC leaching at 45 cm over the 2004–2019 period was 26, 26, and 16 kg C ha⁻¹ yr⁻¹ for GWS, MSW and CON, respectively. Mean yearly DOC leaching at 45 cm over the 2007–2019 period was 38 and 32 kg C ha⁻¹ yr⁻¹ for BIO and FYM, respectively. Mean yearly DOC leaching at 1 m over the 2011–2019 period was 18, 16, 10, 10, and 9 kg C ha⁻¹ yr⁻¹ for BIO, GWS, FYM, MSW and CON, respectively. We calculated the difference between the mean yearly DOC leaching at 45 cm and at 1 m (whatever the period) and we divided it by the corresponding soil depth equal to 55 cm to obtain a mean yearly DOC deposition rate equal to 0.36, 0.19, 0.39, 0.27 and 0.13 kg C ha⁻¹ yr⁻¹ cm⁻¹ for BIO, GWS, FYM, MSW, and CON, respectively.

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Cumulative DOC leaching in the lysimeters of the QualiAgro LTE at 45 cm and 1 m. Measurement at 45 cm started in 2004 for GWS, MSW and CON and in 2007 for BIO and FYM. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure. CON: control.</figcaption></figure>
### 3.5. Belowground crop C input

The cumulative production of aboveground biomass of wheat, barley, or rye over the period 1998–2022 was significantly higher in all OA treatments than in CON; for maize, aboveground biomass production was only significantly higher in BIO and FYM (Fig. S6, Supplementary Material). Total cumulative aboveground biomass (all crops) was equal to 310, 313, 318, 306 and 278 t DM ha⁻¹ in the BIO, GWS, FYM, MSW, and CON treatments, respectively. Using allometric coefficients (see section 2.4), we logically estimated significantly higher total below-ground C inputs in all soil layers for all OA treatments compared to CON (Fig. 4). For example, the mean belowground C inputs in the 35–50 cm soil layer were 0.14, 0.14, 0.14, 0.14 and 0.12 t C ha⁻¹ yr⁻¹ in the BIO, GWS, FYM, MSW and CON treatments, respectively.

### 3.6. Illuviated C transfer

In the 35–50 cm soil layer, the mean volumetric proportion of clay coatings and papules was equal to 1.3 % and 1.7 % of the soil volume in the plot 303 (CON) and 203 (FYM), respectively (Table 3). In the 50–90 cm soil layer, the proportion was higher and equaled 6.4 % and 6.9 % in the plot 303 (CON) and 203 (FYM), respectively. Using the C content of the clay fraction, the illuviated C transfer in the 35–50 cm soil layer was estimated to be less than 0.002 t C ha⁻¹ yr⁻¹ in both plots, regardless of the illuviation duration considered. In the 50–90 cm soil layer, the illuviated C transfer was estimated to be 0.001 t C ha⁻¹ yr⁻¹ whatever the plot for an illuviation duration of 14,000 years, and 0.014 and 0.017 t C ha⁻¹ yr⁻¹ for plots 303 and 203, respectively, for an illuviation duration of 1,000 years. The mean illuviated C transfer for a mean illuviation duration of 7,500 years (regardless of plot) was equal to 0.000 t C ha⁻¹ yr⁻¹ and 0.002 t C ha⁻¹ yr⁻¹ in the 35–50 cm and 50–90 cm soil layers, respectively.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Mean yearly belowground C input per treatment and per soil layer in the QualiAgro LTE. Error bars represent standard deviation due to yield variability among the 4 replicates per treatment. Letters indicate significant differences (p-value < 0.05) and can only be compared between treatments for the same soil layer. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure. CON: control.</figcaption></figure>

<caption>**Table 3**</caption>

Proportion of clay coatings plus papules, cumulative mass of particles illuviated during soil development, yearly mass of illuviated particles averaged over different durations (1,000, 7,500 or 14,000 years), estimated C content of illuviated particles between 1998 and 2022, and yearly C transfer between 1998 and 2022 as a function of the duration of illuviation for the plot 303 (CON, control) and 203 (FYM, farmyard manure) in the QualiAgro LTE.

<table><thead><tr><th>Soil layer (cm)</th><th>35-50</th><th>35-50</th><th>50-90</th><th>50-90</th></tr></thead><tbody><tr><td>Plot (treatment)</td><td>303<br>(CON)</td><td>203<br>(FYM)</td><td>303<br>(CON)</td><td>203<br>(FYM)</td></tr><tr><td>Mean volumetric proportion of clay coatings and papules (%)</td><td>1.3</td><td>1.7</td><td>6.4</td><td>6.9</td></tr><tr><td>Cumulative mass of illuviated particles (t ha<sup>-1</sup>)</td><td>34</td><td>45</td><td>445</td><td>485</td></tr><tr><td>Yearly mass of particles illuviated for a duration of:</td><td></td><td></td><td></td><td></td></tr><tr><td>1,000 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.034</td><td>0.045</td><td>0.445</td><td>0.485</td></tr><tr><td>7,500 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.005</td><td>0.006</td><td>0.059</td><td>0.065</td></tr><tr><td>14,000 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.002</td><td>0.003</td><td>0.032</td><td>0.035</td></tr><tr><td>C content of the clay fraction between 1998 and 2022 (g kg<sup>-1</sup>)</td><td>30.5</td><td>35.0</td><td>30.5</td><td>35.0</td></tr><tr><td>Yearly illuviated C transfer as a function of the duration of illuviation:</td><td></td><td></td><td></td><td></td></tr><tr><td>1,000 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.001</td><td>0.002</td><td>0.014</td><td>0.017</td></tr><tr><td>7,500 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.000</td><td>0.000</td><td>0.002</td><td>0.002</td></tr><tr><td>14,000 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.000</td><td>0.000</td><td>0.001</td><td>0.001</td></tr></tbody></table>

### 3.7. C transfer from bioturbation

In the 35–50 cm soil layer, the cumulative volumetric proportion of bioturbated soil was equal to 63 % in both plots 303 (CON) and 203 (FYM) (Table 4). In the 50–90 cm soil layer, the proportion was lower and equal to 41 % and 39 % in the plot 303 (CON) and 203 (FYM), respectively. Depending on the duration during which bioturbation occurred and on the estimated C content of the casts during the period 1998–2022, the estimated C transfer from bioturbation ranged from 0.001 t C ha⁻¹ yr⁻¹ (plot 303, CON, 14,000 years) to 0.029 t C ha⁻¹ yr⁻¹ (plot 203, FYM, 1,000 years) in the 35–50 cm soil layer and from 0.003 t C ha⁻¹ yr⁻¹ (plot 303, CON, 14,000 years) to 0.048 t C ha⁻¹ yr⁻¹ in the 50–90 cm soil layer (plot 203, FYM, 1,000 years).

<caption>**Table 4**</caption>

Proportion of the soil bioturbated during soil development, cumulative mass of bioturbated soil in the 35–50 and 50–90 cm soil layers, yearly transfer of bioturbated soil averaged over different durations for illuviation (1,000, 7,500 or 14,000 years), C content in topsoil and earthworm casts between 1998 and 2022, and yearly C transfer by bioturbation as a function of the different duration for the plot 303 (CON, control) and 203 (FYM, farmyard manure) in the QualiAgro LTE.

<table><thead><tr><th>Soil layer (cm)</th><th>35-50</th><th>35-50</th><th>50-90</th><th>50-90</th></tr></thead><tbody><tr><td>Plot (treatment)</td><td>303<br>(CON)</td><td>203<br>(FYM)</td><td>303<br>(CON)</td><td>203<br>(FYM)</td></tr><tr><td>Cumulative bioturbated volumetric proportion (%)</td><td>63</td><td>63</td><td>41</td><td>39</td></tr><tr><td>Cumulative mass of bioturbated soil (t ha<sup>-1</sup>)</td><td>1418</td><td>1418</td><td>2475</td><td>2325</td></tr><tr><td>Yearly mass of bioturbated soil for a duration of:</td><td></td><td></td><td></td><td></td></tr><tr><td>1,000 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>1.42</td><td>1.42</td><td>2.48</td><td>2.33</td></tr><tr><td>7,500 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.19</td><td>0.19</td><td>0.33</td><td>0.31</td></tr><tr><td>14,000 years (t ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.10</td><td>0.10</td><td>0.18</td><td>0.17</td></tr><tr><td>C content of topsoil in 2009 (g kg<sup>-1</sup>)</td><td>10.2</td><td>14.2</td><td>10.2</td><td>14.2</td></tr><tr><td>C content of earthworm casts (g kg<sup>-1</sup>)</td><td>14.8</td><td>20.7</td><td>14.8</td><td>20.7</td></tr><tr><td>Yearly bioturbated C transfer as a function of the duration of illuviation:</td><td></td><td></td><td></td><td></td></tr><tr><td>1,000 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.021</td><td>0.029</td><td>0.037</td><td>0.048</td></tr><tr><td>7,500 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.003</td><td>0.004</td><td>0.005</td><td>0.006</td></tr><tr><td>14,000 years (t C ha<sup>-1</sup> yr<sup>-1</sup>)</td><td>0.001</td><td>0.002</td><td>0.003</td><td>0.003</td></tr></tbody></table>
## 3.8. C balance

We compared the amplitude of SOC stocks accrual (difference between 2022 and 1998) in the 35–50 cm and 50–90 cm soil layers with the estimated C inputs in these layers: crop belowground C input, DOC deposition, illuviated C transfer (average of both plots 203 and 303, assuming 7,500 years of illuviation duration, i.e., the mean assumption of duration) and bioturbated C transfer (average of both plots 203 and 303, assuming 7,500 years of bioturbation duration) (Fig. 5). Crop belowground C inputs (roots and rhizodeposits) represented from 35 % of SOC stocks accrual for BIO to 54 % for CON in the 35–50 cm soil layer, and from 32 % for MSW to 43 % for CON in the 50–90 cm soil layer. DOC deposition represented only from 1 % (BIO, GWS, MSW, CON) to 2 % (FYM) of SOC stocks accrual in the 35–50 cm soil layer, and from 2 % (CON) to 5 % (FYM) in the 50–90 cm soil layer. Both illuviation and bioturbation transfers represented a very low C input compared to SOC stocks accrual (≤1% and ≤ 3 % whatever the treatment and the soil layer for illuviation and bioturbation, respectively).

# 4. Discussion

## 4.1. Uncertainty in SOC stocks evaluation

Twelve years after the start of the experiment, we showed a significant increase in SOC content in all QualiAgro LTE treatments in the 35–50 and 50–90 cm soil layers (except for CON in the 35–50 cm soil layer and MSW in the 50–90 cm soil layer). This increase was even greater after 24 years. Considering the changes in bulk density and calculating equivalent soil mass SOC stocks, we showed a significant increase in SOC stocks in the 35–50 cm and 50–90 cm soil layers for all treatments, including CON. The results were similar with the fixed depth method (Fig. S4, Supplementary Material), indicating a limited effect of the evolution of bulk density in the topsoil (Fig. S3, Supplementary Material). Some uncertainties may have affected our estimated SOC accrual in the subsoil. SOC content data were not available for all plots in the subsoil layers in 1998, but only for four plots. However, the field was initially considered to be homogenous, as indicated by the low standard deviation of the SOC content in 1998. Bulk density was also not measured in the subsoil layers after 1998, while some changes in the subsoil could be expected with repeated applications of OA (Menichetti et al., 2015).

In the control treatment, the proportion of SOC stocks in the surface layer (0–28 cm) relative to the 0–90 cm SOC stocks ranged from 53 % in

1998 to 44 % in 2022, in agreement with Balesdent et al. (2018). Given this agreement with Balesdent et al. (2018) and the consistent and gradual evolution of SOC content from 1998 to 2009–2010 and from 2009 to 2010 to 2021–2022, we considered the uncertainty in our estimation of SOC stocks and SOC stocks accrual to be limited.

## 4.2. Unexplained SOC stocks accrual in subsoils in the control treatment without organic amendment application

Since 1998, SOC stocks in the 0–28 cm soil layer in the control treatment without OA application decreased, though not significantly (−0.05 t C ha⁻¹ yr⁻¹). This slight decrease in surface SOC stocks in QualiAgro was well explained by the cropping system characteristics, corresponding crop C inputs, and the soil and climate conditions in a modeling study (Levavasseur et al., 2020). However, we observed a significant increase in SOC stocks in the 28–35, 35–50, and 50–90 cm soil layers of this control treatment (0.06, 0.23, and 0.25 t C ha⁻¹ yr⁻¹, respectively). Yu et al. (2022) also showed an increased allocation of new organic carbon to subsoil layers with high nitrogen application. They suggested an increased vertical transport of SOC. Kätterer et al. (2014) and Skadell et al. (2023) also showed an increase in SOC stocks in subsoils (below the plough layer) with mineral N (or NPK) fertilization compared to the unfertilized control. They assumed an increased contribution of belowground C inputs associated with increased net primary production. In our case, the increase in SOC stocks in the subsoil could not be explained by the estimated C inputs to the subsoil due to crop belowground C inputs, DOC, illuviated and bioturbated C. Despite the uncertainty associated with these C transfers (section 4.4), this increase in SOC stocks in the subsoil associated with a decrease in SOC stocks in the topsoil thus remains largely unexplained. Moreover, even if the detailed cropping systems were not known before the start of the experiment, the field has been cultivated with arable crops and fertilized with mineral fertilizers for a long time. There were no radical changes in the cropping system (such as a shift from no fertilization to mineral fertilization) that could explain the observed changes in SOC stocks, as suggested by the studies cited above (Kätterer et al., 2014; Skadell et al., 2023; Yu et al., 2022). However, the study area has experienced a strong increase in crop net primary production over the last century (mainly due to increased use of mineral fertilizers), resulting in a SOC stocks accrual in the topsoil (Le Noë et al., 2019). We could hypothesize that while topsoil SOC stocks have already reached a new equilibrium following this increase in crop net primary production, subsoil SOC stocks are still increasing due to slower processes in the subsoil.

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Mean yearly SOC stocks accrual (equivalent soil mass method) and C inputs in the 35–50 and 50–90 cm soil layers in the QualiAgro LTE. BIO: compost of green waste and the home-sorted organic fraction of municipal solid waste, GWS: compost of green waste and sewage sludge, MSW: compost of mechanically separated organic fractions from residual waste after selective collection of dry and clean packaging, FYM: dairy farmyard manure. CON: control.</figcaption></figure>
### 4.3. Significant SOC stocks accrual in subsoils with OA application

Since a significant increase in subsoil SOC stocks was observed after 22 years in the control treatment without OA application, we discussed the effect of OA application based on the differences between SOC stocks in the amended treatments and SOC stocks in the control treatment. In 2022, 24 years after the start of the experiment, the SOC stocks were significantly higher in the plow pan (28–35 cm) for all OA treatments. They were significantly higher for the GWS and BIO treatments in the 35–50 cm soil layer and for the BIO treatment in the 50–90 cm soil layer. Interestingly, the higher SOC stocks with BIO and GWS were also observed in the topsoil and were associated with higher recalcitrance of these OA (Levavasseur et al., 2021). Considering only the SOC stocks in the surface soil (0–28 cm), 34 % (MSW) to 55 % (GWS) of the cumulative applied OA C was stored in the soil. When considering the 0–90 cm soil layer, this proportion reached 51 and 79 % for MSW and GWS, respectively. This highlighted the importance of considering the subsoil when estimating the OA C retention coefficient for greenhouse gas emission inventories, whereas it is usually not considered (Maillard and Angers, 2014).

In this study, we showed higher effects of OA application on subsoil SOC stocks than previously reported. Skadell et al. (2023) showed a significant effect of FYM application on SOC stocks of four different LTE in the 30–50 cm soil layer, but no effect below 50 cm. Menichetti et al. (2015) showed no effect of OA application below 35 cm in the Ultuna LTE after 53 years with the same C rate as in QualiAgro. At the Lanna agricultural research station, Kätterer et al. (2014) showed a significant effect of compost application only down to 40 cm after 13 years with the same C rate as in QualiAgro. This higher effect in QualiAgro could be related to the different climate, soil and cropping system, but this difference between LTE deserves further investigation.

Regarding the contribution of each soil layer to the total SOC stocks accrual in 0–90 cm (relative to CON), we showed a contribution ranging from 65 % (MSW) to 73 % (FYM) for the plowed layer, while Skadell et al. (2023) found a contribution of FYM of 89 % for the 0–30 cm soil layer. In our case, even if most of the remaining contribution was observed in the 28–35 cm soil layer (15 to 18 %), the contribution of the subsoil layers was higher (5–10 % and 3 to 13 % for the 35–50 cm and 50–90 cm soil layers, respectively) compared to that reported by Skadell et al. (2023) (9 and 2 % for the 30–50 cm and 50–100 cm soil layers, respectively).

### 4.4. Sources of SOC stocks accrual in subsoil layers

In our study, we attempted to estimate the potential C inputs contributing to SOC stocks accrual in subsoil. The amount of roots and rhizodeposits we estimated was by far the most important source of C to the subsoil. However, it was not sufficient to explain either the amount of SOC stocks accrual or the difference between treatments (depending on the difference in crop yields). However, this estimate was highly uncertain. Roots and rhizodeposits were not measured in the field, but estimated from the aboveground biomass and allometric coefficients, and were assumed to be distributed within each soil horizon in a pre-determined asymptotic way. It should be noted that the coefficients used to estimate total root biomass production were sometimes derived from studies that did not sample roots over the entire soil profile. In the study by Bolinder et al. (2007), on which our coefficients are mostly based, root sampling depth varied from 18 to 180 cm, with 75 % of the studies considering a root depth of less than 50 cm. Thus, our estimation of root biomass production may have been underestimated. Similarly, rhizodeposited C was assumed to represent 65 % of the C remaining in the root biomass at harvest, while less conservative estimates of up to 100 % have been suggested (Bolinder et al., 2007). These coefficients are known to be associated with large uncertainties, and the use of the same allometric coefficients or depth-distribution regardless of pedoclimatic context and agronomic practices is often questioned (Rees et al., 2025;

Taghizadeh-Toosi et al., 2016). Considering a margin of error of more or less 50 % around both shoot:root ratio and rhizodeposition (conservative assumption with respect to Bolinder et al., (2007)), the maximum contribution of roots and rhizodepositon could be more than doubled and reach the amount of SOC stocks accrual quantified in our study (from 0.03 to 0.32 t C ha⁻¹ yr⁻¹ depending on the treatment and considered soil layer, compared to 0.13 t C ha⁻¹ yr⁻¹ on average with our assumption).

In addition, repeated OA applications could have altered both chemical properties (e.g., nutrient form and availability) and physical properties of the soil, which are known to influence root growth (Bai et al., 2021; Simões-Mota et al., 2022). An improvement in soil physical properties was observed in the QualiAgro LTE (Paradelo et al., 2019), such as reduced bulk density (Fig. S3, Supplementary Material), although its effect on root growth was not investigated.

The application of OA in our study tended to increase DOC leaching (not statistically tested), both at 45 cm and at 1 m. Didelot et al. (2024) also reported an increase in DOC flows (at 40 cm) after digestate application in a similar context, while a global meta-analysis by Ren et al. (2024) indicated that manure application increased DOC leaching in the surface layer. However, DOC leaching remained limited in all cases. The DOC leaching reported here (from 8 to 18 kg C ha⁻¹ yr⁻¹ at 90 cm) was similar to that of Didelot et al. (2024) (from 1 to 14 kg C ha⁻¹ yr⁻¹ at 1 m) in a similar soil (luvisol) and climate context (oceanic) in an arable crop field. DOC leaching measured at 45 cm was also limited, ranging from 15 to 36 kg C ha⁻¹ yr⁻¹, again similar to Didelot et al. (2024) at 40 cm (11 to 51 kg C kg C ha⁻¹ yr⁻¹). Considering the small amplitude of C inputs by DOC leaching, we concluded that DOC is not the main contributor to the observed SOC stocks accrual in the subsoil layers.

Although recognized as potential contributors to SOC stocks accrual in subsoil (Rumpel and Kögel-Knabner, 2011; Torres-Sallan et al., 2017), C transfers to the subsoil due to bioturbation or illuviation have never been estimated to our knowledge. In the studied soil subjected to illuviation, these C transfers were limited and could not explain the observed SOC stocks accrual in the subsoil and the differences between treatments. However, this result is highly dependent on the estimate of the average annual mass of bioturbated or illuviated soil with long-term estimates over 1,000 to 14,000 years, as used here, which may differ from current estimates. For example, Sauzet et al., (2016) demonstrated that illuviation is sensitive to changes in land use and management that are not well captured by long-term estimates that are primarily representative of forest soil development. More specifically, if the application of OA tended to increase C transfers through bioturbation and illuviation, thanks to a higher C content of the casts and of the clay fraction, it could also modify the annual mass of bioturbated or illuviated soil. Indeed, Capowiez et al. (2009) and Ricci et al. (2015) showed that OA application induced an increase in the abundance and/or biomass of earthworms in the QualiAgro LTE (although not always significant), which could increase the intensity of bioturbation. However, to reach a significant contribution to SOC stocks accrual in the subsoil (e.g., around 100 kg C ha⁻¹ yr⁻¹), the current intensity of bioturbation or illuviation would have to be 2 to 100 times higher than the long-term estimates based on a duration of soil development as short as 1,000 years, which would be surprising.

Finally, despite our attempt to quantify all subsoil C inputs recognized as potential contributors to SOC stocks accrual (Rumpel and Kögel-Knabner, 2011; Torres-Sallan et al., 2017), the sum of all estimated subsoil C inputs was lower than the calculated SOC stocks accrual. This indicates that we did not estimate some C inputs well, or that we missed some others. As discussed above, the contribution of bioturbation and illuviation may have been underestimated due to the assumed duration of these processes. Another contribution that we did not estimate was a direct contribution of OA or crop residues through macropore transport in soil cracks or macropores, as suggested by Kätterer et al. (2014). However, soil cracks were not common in this
field experiment (due to low clay content). In addition to C inputs into the subsoil, we did not consider C flows out of the subsoil (except for DOC). Even if it was shown that SOC has a longer residence time in the subsoil, part of it is mineralized each year due to significant microbial activity in the subsoil (Rumpel and Kögel-Knabner, 2011). This would require even higher incoming C inputs to the subsoil. Nevertheless, our study provides a unique estimation of subsoil C stocks accrual and potentially contributing C inputs in an arable crop field with OA application.

## 5. Conclusion

We studied the SOC stocks accrual in the soil profile of the QualiAgro LTE after 24 years of repeated application of OA. We showed a significant increase in SOC content and SOC stocks in the surface soil layers (0–28 and 28–35 cm) and in subsoil layers (35–50 and 50–90 cm) in the OA treatments and in the control (except in the 0–28 cm soil layer). Compared to the control, OA application increased SOC stocks in all soil layers up to 90 cm (significantly only for GWS and BIO in the 35–50 cm layer and only for BIO in the 50–90 cm soil layer). From 5 % to 10 % and from 3 % to 13 % of the total SOC stocks accrual over the whole soil profile occurred in the 35–50 cm and 50–90 cm soil layers, respectively. The OA that increased the most SOC stocks in the subsoil layers also increased the most SOC stocks in the surface soil and were characterized by a higher recalcitrance (GWS and BIO composts). The estimated contribution of belowground crop C inputs to SOC stocks accrual in the subsoil was high, but limited to 54 % of the observed SOC stock accrual when using conventional allometric coefficients. DOC leaching remained limited and was not an important contributor to SOC stock accrual. Although we estimated a very limited contribution of illuviation and bioturbation to SOC stocks accrual in the subsoil, large uncertainties were associated with their estimation. Our study highlights the importance of considering the subsoil when estimating the effect of OA application on subsoil SOC stocks accrual, and suggests that more studies should be conducted to understand the source of SOC stock accrual in the subsoil.

### CRediT authorship contribution statement

**Florent Levavasseur:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. **David Montagne:** Writing – review & editing, Validation, Methodology, Formal analysis, Conceptualization. **Yvan Capowiez:** Writing – review & editing, Validation. **Claire Chenu:** Writing – review & editing, Validation. **Frédéric Rees:** Writing – review & editing, Validation. **Ophélie Sauzet:** Writing – review & editing, Validation. **Sabine Houot:** Writing – review & editing, Validation, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

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.

### Acknowledgment

We thank the technical staff in charge of managing the QualiAgro experiment management, Camille Resseguier and Grigorios Androdimis. The QualiAgro experiment is part of the SOERE-PRO (network of long-term experiments dedicated to the study of the impact of the recycling of organic waste products) and is integrated as a service of the “Investment in the Future” infrastructure AnaEE-France, overseen by the French National Research Agency (ANR-11-INBS-0001). The QualiAgro experiment was founded and is still supported by INRAE and Veolia.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.geoderma.2025.117546.

### Data availability

Data will be made available on request.

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