Soil organic carbon mobility in equatorial podzols: soil column experiments

. Transfer of organic carbon from topsoil horizons to deeper horizons and to the water table is still little documented, in particular in equatorial environments despite the high primary productivity of the evergreen forest. Due to its complexing capacity, organic carbon also plays a key role in the transfer of metals in the soil profile and therefore in 10 pedogenesis and for metal mobility. Here we focus on equatorial podzols, which are known to play an important role in carbon cycling. We carried out soil column experiments using soil material and percolating solution sampled in an Amazonian podzol area in order to better constrain the conditions of transfer of organic carbon at depth. The dissolved organic matter (DOM) produced in the topsoil was not able to percolate through the clayey, kaolinitic material from the deep horizons and was retained in it. When it previously percolated through the Bh material, there was production of fulvic-like, 15 protein-like compounds and small carboxylic acids able to percolate through the clayey material and increasing the mobility of Al, Fe and Si. Podzolic processes in the Bh can therefore produce a DOM likely to be transferred to the deep water table, playing a role in the carbon balances at the profile scale, and owing to its complexing capacity, playing a role in deep horizon pedogenesis and weathering. The order of magnitude of carbon concentration in the solution percolating at depth was around 1.5-2.5 mg L -1 . Our findings reveal a fundamental mechanism that favors the formation of very thick kaolinitic 20 saprolites.

or respiration experiments (Fontaine et al., 2007;Lucas et al., 2020). Despite this, little is known about the fluxes and characteristics of organic matter capable of migrating in depth.
In this context, we were interested in equatorial podzols which are known to play a significant role in carbon cycling. In Amazonia, these soils store a large carbon pool, estimated around 13.6 PgC (Montes et al., 2011), and Pereira et al. (2016) specified that they contain in average 105.9 kgC m -2 , of which 83.2 are in the deep Bh. Doupoux et al. (2017) modelled their 35 genesis and dynamics by considering both total C fluxes and 14 C fluxes. They noticed, however, that dissolved organic carbon (DOC) fluxes at depth were not enough well known to constrain the model unambiguously. The DOC fluxes exported and reaching the deep water table were generally approximated by the analysis either of groundwater taken from boreholes, of spring water baseflow at the outlet of an elementary watershed of known characteristics or by tracer-aided modelling at the scale of a larger catchment (Birkel et al., 2020). Such data are scarce, and Table 1 summarizes those we 40 found relating to soil systems from tropical or equatorial environments. They show that the solutions which percolate at depth in the soils of tropical or equatorial environments have significant DOC contents, varying from 0.3 to 2.3 mg L -1 .
Regarding podzols, data relating to springs give information on the solutions that flows laterally in the eluvial horizons but there is very little detailed data on solutions from horizons below the Bh. Analyzer, Thermo Fisher Scientific). Kaolinite and gibbsite determination were performed by Thermogravimetry-Differential Thermal Analysis (TG-DTA) using a Shimadzu DTG-60H-Simultaneous DTA-TG. Fe-oxydes were calculated as Fe 2 O 3 after total Fe determination by ICP-AES on aqua regia digestion extracts.

45
The columns used were 60 cm long with an internal diameter of 3 cm. To represent the E horizon we used a quartz sand (Q) (pure Fontainebleau sand, commercial, particle size <350 µm). To represent the other horizons, we sampled soil material in 85 Amazonian podzol profiles: two sandy Bh material (Bh1, SOC 4.5% and Bh2, SOC 1.7%) and a kaolinitic material (K) from a horizon underlying a Bh (Table 2). All materials were passed through a 2-mm sieve and carefully packed in column to avoid large voids. These materials were introduced into the columns in layers of 5 cm for kaolinitic material and Bh material, and of 10 cm for sand. A 0.1 mm nylon mesh was inserted between each layer avoid mixing of the phases, at the top of the column to damp the fall of the drops, and at the base of the column to avoid suffosion of the material.
Six column experiments were conducted according to the arranged percolating device shown in Fig. 1. Two columns 95 were packed with a Q-Bh arrangement (Q-Bh1 and Q-Bh2) to observe the DOM transfer through and from the Bh. Three columns (Q-Bh1-K(a), Q-Bh1-K(b) and Q-Bh2-K) were packed with a Q-Bh-K arrangement to observe the adsorption of the DOM issued from the Bh by the kaolinitic clay material. One column (Q-K) was packed with a Q-K arrangement to observe the direct adsorption of the DOM circulating in the E horizons by the kaolinitic clay material.
The input solution was a pH 4.1 black water taken from a spring located towards the center of a podzolic area (S 0°6'42'', 100 W 66°54'09''), corresponding to the water of the perched water-table circulating in the E horizons. It was kept around 4°C until experiment. The input solution was injected at the top of the column by a peristaltic pump to obtain a downward flow of 0.05 ml mn -1 for 3 weeks to obtain around 1.3 L of percolate. In parallel, the peristaltic pump was used in suction mode at the output to homogenize the speed and have the same residence time in all the columns. The output solution was sampled every 5 days, giving for each column 5 samples (fractions F1 to F5) of approximately 250 mL each. For each column, a 105 composite sample was formed by proportional mixing of the fractions. Input solution and percolates were analyzed according to the following techniques. The DOC was determined by TOCmeter (TOC-V, SHIMADZU) coupled to an ASI-V automatic sampler. The dissolved organic matter (DOM) was 115 characterized by 3D fluorescence (Excitation Emission Matrix Fluorimetry, EEMF) (HITACHI F-4500 spectrometer), this method allowing a rapid characterization of fluorophores associated with humic matter and proteins (Chen et al., 2003;Nebbioso and Piccolo, 2013). Major anions and cations were determined by ion chromatography (Dionex DX 120), using 9 mmol L -1 NaHCO 3 for cation elution and 10 mmol L -1 methane sulfonic acid for anion elution. Si, Al, Fe were quantified by ICP-AES. SOA (formic, oxalic, malic ...) were determined and quantified by high performance ion chromatography (Dionex 120 ICS-3000) coupled to a mass spectrometer (MSQ Plus, Thermo Scientific) driven by Chromeleon® (6.80 version) and equipped with an AG11-HC guard column (Dionex), an IonPac AS11-HC column (4x250 mm, Dionex) and using a 25µL loop injection valve. Analysis were performed in a gradient mode (from 1 to 5 mM NaOH in helium sparged demineralized water in 40 min.) at 30 °C, with a flow rate set at 0.8 mL.min-1. To improve the signal-to-noise ratio of the measurement, an external flow electrochemical suppressor system (ACRS 500 4 mm) was added to the analytic system.

Transfers of carbon and major elements
The pH of the percolating solution was 4.0, it was not modified by passing through the columns. For each column, the variations in DOC concentration between fractions throughout the experiment remained much lower than the differences between columns and did not exhibit clear trends (Fig. 2); the same evolution was observed for Al and Fe concentrations. 130 There were therefore no significant changes within a given column experiment in the behavior of the columns which allows us to discuss the results using the composite sample compositions given in Table 3. We can consider that the output of the Q-Bh type columns corresponded to the input into the kaolinitic material of the Q-Bh-K type columns, as sketched in Fig. 3. If there was a relatively large variation between columns of the same type, the differences between columns remained consistent of different types remained consistent. 140 Table 3. C, Al, Fe and Si transferred in dissolved phase in the column experiments. Output values correspond to the composite samples. Analytical percent error: C, 1.4; Al, 6.0; Fe, 4.8; Si, 1.9. na: not analyzed.  Regarding DOC, the lowest column outlet concentration (1.9 mg L -1 ) was observed for the Q-K column (Table 3), where 150 94% of the carbon introduced at the top of the column was retained; consistently, the upper part of the kaolinitic layer acquired a light brown color. The highest DOC outlet concentrations (17.1 and 11.5 mg L -1 ) were observed for the two Q-Bh type columns, which was expected but where, however, 46 and 63% of the carbon introduced were retained. The DOC concentrations at the outlet of the three Q-Bh-K type columns were intermediate (8.4; 3.9 and 3.9 mg L -1 ), corresponding to the retention of 81, 88 and 89% of the introduced carbon, respectively. In summary, the Bh retained a part of the introduced DOC and the kaolinitic material retained most of the DOC of the solution which percolated through. The presence of a Bh, however, increased the proportion of carbon which passed through the kaolinitic material. The nature of the DOM released by the Bhs was therefore probably different from that of the DOM of the input, i.e. of the black water from the perched water table; this was likely due to a specific microbial activity within the Bh in the columns.
Regarding Si, the column outlet concentrations were always higher (393 to 1182 µg L -1 ) than the inlet concentration ( material only (Q-K) resulted in a partial retention of Al (Al concentration at outlet 17 µg L -1 ). As for DOC, however, the previous percolation through the Bh (Q-Bh-K columns) increased the proportion of Al which passed through the kaolinitic material. The discrepancy between Si and Al behavior shows that these elements were not controlled by a congruent dissolution of kaolinite. Iron concentration pattern was quite similar to Al, but with lower concentrations at column outlets.
The behavior of Fe and Al could be explained by the release either of OM-complexes (organo-metallic complexes) 170 (Lucas, 2001;Patel-Sorrentino et al., 2006), or mineral colloids (kaolinite, gibbsite, goethite) (Cheng and Saiers, 2015) during percolation through the Bh, these complexes or colloids being subsequently partially retained during percolation through the kaolinitic material. In the Bh output, Si and Al were released in a stoichiometry equivalent to that of kaolinite, which would be compatible with a release of colloidal kaolinite. Recent studies, however, suggested that Si and Al can be transferred as ternary OM-Al-Si complexes (Merdy et al., 2020). Anyway, the behavior of these two elements during the 175 subsequent percolation through the kaolinitic material diverged completely: the kaolinitic material retained Al while it released Si, suggesting that Al was released by the Bh as OM-complexes. This hypothesis is strongly supported by the high correlation between DOC and Al (Fig. 4). Fe behaved similarly, but with a weaker correlation with DOC, which suggests that Al was only transferred as DOM-Al complexes when Fe could also be transferred as mineral colloids.

Figure 4. Relationships between DOC and dissolved Al, Fe and Si in column percolates. The cross represents the concentration in the input solution. Values are from composite samples for Si and from individual fractions for Al and Fe.
To assess the consistency of these interpretations, we built a Si-Al diagram considering the data obtained from the 185 studied solutions, taking into account metal complexation by the DOM in one hand, or not taking it into account in the other hand, (Fig. 5). The parameters of the dissolved organic matter necessary for the quantification of the complexation were a site density equal to 27 µmol mg -1 (Lucas et al., 2012) and a DOM-Al conditional stability constant equal to 10 5 (Lee, 1985;Hagvall et al., 2015). The "Kaolinite 1" line corresponds to the stability of kaolinite calculated with the WATEQ4F database, which uses a solubility product (Ksp = 10 3.705 ) identical to that proposed by Tardy and Nahon, (1985)

Fluorescence properties of percolation solutions
Fluorescence spectroscopy is an appropriate tool to characterize natural organic matter whose fluorophores give specific signals. Excitation-emission fluorescence matrix (EEFM) of input and output solutions are given in Fig. 6. The peak A corresponds to fulvic-like humic compounds, the peak C to humic-like humic compounds, the peak P to protein-like 205 compounds that indicate an active bacterial activity (Coble, 1996), these peaks being characteristics of natural terrestrial DOM. The peaks S1 and S2 have been related to non-humic like, labile matter related to microbial activity (Singh et al., 2010) or to fulvic-like compounds (Stedmon and Markager, 2005).

Figure 6. Excitation-emission fluorescence matrix of the solutions, arbitrary units. The letters identify the usual position of the peaks P (protein-like), C (humic-like), A (fulvic-like), S1 and S2 (non-humic or fulvic like).
The EEFM of the input solution was typical of humified DOM with a dominant C peak, a marked A peak and a small P peak. After the input solution has passed through kaolinitic material (Q-K output), there is hardly any humified DOM and a 215 very reduced signal of the protein-like DOM: nearly all fluorescent DOM was retained in the kaolinitic material. After the input solution has passed through the Bh (Q-Bh1 output), there was a reversal of the C/A intensity ratio, which indicates a partial retention of the most condensed DOM in the Bh, an a higher P peak, which indicates a bacterial activity within the Bh. After the input solution has passed through both Bh and kaolinitic material (Q-Bh1-K(a) output), the DOM exhibited some humified character, more fulvic than humic, and protein-like feature. 220 These observations confirmed that the DOM released by the Bh was different from the DOM of the input solution. The Bh retained the most humified DOM compounds and released compounds capable of being transferred through clay materials, more fulvic-like or protein-like as issued from active bacterial activity.

Transfer of small organic acids
Chromatography was used to identify the composition of the DOM present in the column experiments. Lactic and malic 225 acids were the only small organic acids found in the input solution at detectable concentrations (Table 4). After the input solution has passed directly through kaolinitic material (Q-K output), 68% of the carbon from the measured SOA was retained in the kaolinitic material and it only remained a low concentration of lactic acid. Percolation of the input solution through the Bh (Q-Bh output) resulted in an increase in the quantities and variety of SOA, indicating microbial activity during the experiment, which is consistent with fluorescence observations. Comparing Q-K and Q-Bh-K outputs shows that 230 a previous percolation through the Bh increased, as for DOC, the proportion of SOA carbon which passed through the kaolinitic material (Fig. 3). This observation is consistent with fluorescence data and strengthened the hypothesis that the DOM released by the Bhs was different from that of the input solution.

From experiment to field
To what extent can the conclusions of the experiment be extrapolated to field conditions? The column experiments exhibited differences with field usual conditions. 240 lasted only 3 weeks, when at field under usual conditions a quasi-permanent water table is perched over the Bh which has a low hydraulic conductivity. The solution circulating in the E horizon likely percolates very slowly in the Bh throughout the year (Ishida et al., 2014). In the columns the Bh was reworked, which ensured a higher hydraulic conductivity and most likely different soil-solution contact conditions from that at field, and was previously dried out, which may result in a change in microbial activity (Denef et al., 2001). Microbial activity is also sensitive to redox conditions. Our column experiments 245 were conducted without control of the redox potential, when deep in situ Bh can be submitted to reducing conditions (Lucas et al., 2012).
Nevertheless, the column experiment showed negligible variations with time of the percolate characteristics, which suggests a steady state. Results are also consistent with the scarce field data available: • The ratio of input/output average DOC concentration for the Q-Bh-K columns ranged from 0.12 to 0.27, which is in the 250 range of those observed between DOC concentration in E horizons and deep water table (0.13, Lucas et al., 2012), or predicted by podzol genesis modelling (0.14 to 0.35, Doupoux et al., 2017).
• In the column experiments, the DOM that percolates through the kaolinitic material had a higher content of small carboxylic acids and of fulvic-like compounds, i.e. less aromatic than DOM of the input solution. This is consistent with the observations of Lucas et al. (2012), who have found that the DOC of the water table situated under a kaolinitic 255 horizon in a podzolic area had a high proportion of small organic compounds with high complexing capacity.
It is therefore possible to conclude that percolation through the Bh plays a key role in the geochemistry of the system, by producing compounds able to transfer both DOC and metals through kaolinitic materials.

Conclusion
The column experiments led to conclusions shown schematically in Fig. 7. The DOM produced in the acidic upper horizons 260 and circulating in the E horizons would be highly adsorbed, with a complete retention of the humified compounds, if directly percolating through a clayey, kaolinitic material. If this DOM percolates previously through a Bh, it is subjected to transformation in this horizon. The humic-like compounds are retained, and a more fulvic-like, proteinaceous DOM containing small organic acids, which is more likely to percolate through a kaolinitic material, is released. The DOM that percolates in deep horizons is therefore different that the highly humified DOM that circulates in the E horizon. Microbial activity in an in situ Bh may be different from that observed in the columns, but the input/output C ratio of our experiments was in the range of what has been observed in field or predicted by modelling. A DOC concentration 270 around 1.5-2.5 mg L -1 for solutions percolating through deep kaolinitic horizons appeared therefore as a good order of magnitude. The higher proportion of small organic acids in the solution able to percolate through deep kaolinitic horizons confirmed its ability to transfer metals such as Al or Fe as organo-metallic complexes, increasing therefore the leaching in depth of these elements.
These conclusions strength the hypothesis given in Ishida et al. (2014) related to the genesis of tropical podzols. The 275 solution that percolates through the Bh is able to transfer metals through a kaolinitic material, therefore to promote the downward progression of the E/Bh horizons by weathering the upper part of the kaolinitic deep horizons.
Data availability. The data used in this study are available from the corresponding author.