<figure><img src="image_68.png" /><figcaption>Figure 2: CO₂ fluxes measured at the sediment-air interface in the dark emersion chambers (normalised to the temperature of 20°C, see text) in the various areas and sub-areas studied, in June (a) and October (b).</figcaption></figure>

In October, a similar pattern was observed, with the highest normalized values recorded in control/unploughed sediments, ranging from 37.41 ± 14.75 µmol.m⁻².s⁻¹ for sediments with *Salicornia sp.* to 89.70 ± 45.71 µmol.m⁻².s⁻¹ for sediments with *Suaeda maritima*. The lowest values were consistently recorded on sediment ploughed either in 2022-23 (bare sediments, sediments with *Salicornia sp.* and *Suaeda maritima*) or in 2023-24 (sediments with *Spartina anglica*). Overall fluxes were very low in the area ploughed in 2022-23, with average values falling within the range 2.65-7.12 µmol.m⁻².s⁻¹ regardless of sediment type. In contrast, sediments ploughed in 2023-24 exhibited greater variability, with values ranging from 3.39 to 24.51 µmol.m⁻².s⁻¹, depending on the sediment type.

A three-ways nested ANOVA, with ‘seasons’ as the first level, ‘areas’ as the second level and ‘sub-areas’ as the third level, was performed on all the normalized flux data (N=72). The analysis revealed significant differences between seasons (p<0.05*), between areas (p<0.001***), and between sub-areas (p<0.05*). The most pronounced effect was observed at the area level, highlighting the greater influence of ploughing activity.

## 3.3 Discussion

Carbon dioxide fluxes at the sediment-atmosphere interface were measured using a semi-automatic opaque benthic chamber across different plant covers. As such, this approach does not account for primary production in carbon budgets, but it nonetheless provides an excellent estimate of the overall intensity of metabolism in surface sediments. It is important to note that the carbon dioxide exchange intensities recorded are higher than those observed in the sediments of intertidal mudflats, and are of the similar magnitude to those measured in other intertidal salt marshes along the eastern English Channel (Voltz, 2020). However, the measurements taken in the Canche estuary rarely exceed an intensity of 10 µmolCO2.m⁻².s⁻¹ (Voltz, 2020), which suggests a very active metabolism in the salt marshes studied in the Somme estuary. This is likely linked to the fact that all these measurements were conducted in late spring
(June) and mid-autumn (October), when metabolic activity is close to its seasonal maximum, due to higher temperatures, sunshine intensity (day length and light intensity) and nutrient availability.

The results clearly show a reduction in the metabolic intensity associated with ploughing, regardless of the type of vegetation present. Metabolism is lower, as it is that for plants in the area ploughed in 2023-2024 (with the exception of S. maritima). Moreover, it is evident that more recent ploughing results in lower metabolism, which highlights two key points:

i. the intensity of the disturbance associated with ploughing: the rate of carbon mineralisation is, on average, halved, except in the case of bare sediment, where post-ploughing metabolism is reduced by a factor of more than 10.

ii. the resilience of this system: despite a significant disturbance, sediment metabolism gradually returns to its equilibrium state. It should be noted, however, that following a disturbance, the return to equilibrium is very slow, and the difference remains substantial even more than a year and a half after ploughing.

The fluxes measured in October on sediments ploughed in 2022-23 were generally much lower than those recorded on sediments ploughed in 2023-24, which is inconsistent with the results obtained in June, when a clear decrease was observed from the control area to the area ploughed in 2022-23, and then further to the area ploughed in 2023-24. This may be due to the exceptionally low air temperatures recorded in October 2024, particularly on 3-4 and 11-12 October, with minimum temperatures ranged from 3-5°C. These temperatures may have sharply and profoundly altered the 'summer' functioning of the biota in the salt marsh environment.

During the winter 2023-24 ploughing was delayed due to adverse weather conditions (regular and heavy rains causing flooding), and authorities were granted special authorization to carry out tillage until 8 March. In terrestrial systems, tillage is known to result in bursts of CO2 emissions from the sediments within few hours after tillage, due to a rapid equilibration of soil CO2 with atmospheric CO2. Then, CO2 emissions generally decrease until an equilibrium state observed after a few weeks (Zhang et al., 2008). However, the measurements acquired during this study, even in June (3 months after winter 2023-24 ploughing), very probably took place after the relaxation of the sediments, i.e. the period during which, after a disturbance, the sediments return to a stable biogeochemical functioning. In other words, this corresponds to the time required for the communities of macrofauna, meiofauna and above all bacteria, which are responsible for most of the mineralisation mechanisms, to be reconstituted and rearranged, and for the different mineralisation sequences (aerobic, anaerobic) to return to equilibrium. This was noted on site by the colouration of the sediments and the bedding patterns. However, furrows remained visible on the ploughed sites, with residual topography and the development of plant formations in parallel lines. Rather than returning to the original CO2 flux, as is generally described in terrestrial areas (Zhang et al., 2008), we observed a clear decrease in CO2 emission after tillage. Numerous studies in terrestrial environments highlight rainfall as key control factor for carbon dioxide emission from soils (Delsarte et al., 2021), but do not identify organic matter leaching as a significant factor influencing subsequent CO2 exchanges. However, in intertidal areas, the current and waves during high tide may also lead to significant leaching of surficial sediment after tillage, which could substantially affect the amount and availability of organic matter, thereby reducing mineralisation rates over a period of 5 to 10 years, based on annual estimates of resilience rates.

In addition to the work conducted in this study, further research should include the characteristics (physiology, biomass...) of halophitic plants, as well as an analysis of organic matter (quantity and quality) in surficial sediments after tillage. Furthermore, the phenomenon of photosynthesis, measured using a transparent benthic chamber, should complement these analyses in order to quantify the carbon balance. The next step will be to quantify methane (CH4) gas exchange at the sediment/air interface. Although methane fluxes are generally lower than those of carbon dioxide (Wei et al., 2020) methane is a greenhouse gas that is 28 times more potent than CO2 over a century. This comprehensive approach will enable a definitive quantification of the impact of tillage on the dynamics of carbon storage in surface sediments.

## 4 CONCLUSION

Tillage significantly reduces the intensity of organic matter mineralisation in surface sediments, as measured by CO2 fluxes at the sediment-atmosphere interface in the salt marshes of the Somme estuary. This effect is observed across all the main plant species encountered in this area, as well as on bare sediments, suggesting a broad impact of tillage on the availability of organic matter and/or on organisms (primarily microorganisms) in the surficial sediments, rather than a specific effect on the physiology or growth of halophitic plants.

While some resilience is observed in the system - with metabolism measured in the ploughed area in 2022-23 being intermediate between the unploughed state and the ploughed zone in 2023-24 - it is important to note that the return to the original state is very slow. Almost a year and a half after ploughing, the impact of
this disturbance remains highly significant. Although resilience appears consistent, we estimate that full
recovery to the initial state may take between 5 to 10 years.

5 ACKNOWLEDGMENT

This work received financial support from OFB -PNM-EPMO as well as CPER IDEAL program. This study is part of project CABESTAN of the exploratory research program FairCarboN and received government funding managed by the Agence Nationale de la Recherche under the France 2030 program, reference ANR-22-PEXF-0007. This paper was reviewed by Valérie Mesnage.

6 REFERENCES

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Becuwe, E., Delame, L., McConnon, F., Stien, F., 2024. Suivi des travaux de labours entre 2023 et 2024 et évolution des travaux depuis 2017 au sein des concessions de culture de végétaux marins. GEMEL Report n°24-08. 27 p. + appendix.

Boscolo-Galazzo, F., Crichton, K.A., Barker, S., Pearson, P.N., 2018. Temperature dependency of metabolic rates in the upper ocean: A positive feedback to global climate change?, Global and Planetary Change, 170, 201-212.

Delsarte, I., Cohen, G.J.V., Mommbrun, M., Höhener, P., Atteia, O., 2021. Soil carbon dioxide fluxes to atmosphere: The role of rainfall to control CO2 transport. Applied Geochemistry, 127, 104854.

Endo, T., Otani, S., 2019. Carbon Storage in Tidal Flats. In: Kuwae T., Hori M. (eds) Blue Carbon in shallow coastal ecosystems. Springer, Singapore, pp 129–151.

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Meisinger, J.J., Palmer, R.E., Timlin, D.J., 2015. Effects of tillage practices on drainage and nitrate leaching from winter wheat in the northern Atlantic coastal-plain USA. Soil & Tillage Research, 151, 18–27.

Roberts, A., Pullin, A.S., 2008. The effectiveness of management interventions for the control of *Spartina* species: a systematic review and meta-analysis. Aquatic Conservation: Marine and Freshwater Ecosystems, 18, 592-618.

Voltz, B., 2020. Dynamique de la matière organique dans les estuaires picards : caractérisation des faciès biosédimentaires et quantification des processus de minéralisation benthique. PhD memoir, University of Lille, 267 p.

Wei, S., Han, G., Chu, X., Song, W., He, W., Xia, J., Wu, H., 2020. Effect of tidal flooding on ecosystem CO2 and CH4 fluxes in a salt marsh in the Yellow River delta. Estuarine, Coastal and Shelf Science, 232, 106512.

Zhang, J., Changchun, S., Shenmin, W., 2008. Short-term dynamics of carbon and nitrogen after tillage in a freshwater marsh of northeast China. Soil & Tillage Research, 99, 149-157.
# DEEP-TIME ASTRONOMICAL FORCING ON ORGANIC CARBON ISOTOPE VALUES: A TOOL FOR GLOBAL CORRELATION AT 10⁵-YEAR SCALE

Minka Moreau-Ledegen<fnref n="1" />,<fnref n="2" />, Mathieu Martinez<fnref n="2" />, Beatriz Aguirre-Urreta<fnref n="3" />, Eleys Ben Saïd<fnref n="2" />, Brieuc Rabu<fnref n="2" />, Pierre Pellenard<fnref n="1" /> and Anne-Catherine Pierson-Wickmann<fnref n="2" />

Corresponding author: mathieu.martinez@univ-rennes.fr

**Abstract:** Carbon-isotope ratios have been measured on organic matter from two sections astronomically-calibrated. We show that eccentricity cycles can be observed in the δ¹³Corg signal and correlated at global scale. The record of the orbital cycles in δ¹³Corg appears to be a powerful tool for deep-time fine correlations of widely distributed sections.

**Keywords:** Carbon isotopes, organic matter, Milankovitch cycles, chemostratigraphy, astrochronology.

## 1 INTRODUCTION

Carbon-isotope ratios are widely used to constrain deep-time evolution of the carbon cycle and long-distance correlations in order to align paleoclimatic signals (Weissert, 1989). Traditionally, only major events and trends can be used for correlations while low-amplitude, short-lived fluctuations are regarded as noise or linked to basin-scale processes (e.g., Joachimski, 2023). Global fluctuations in deep-time are often regarded as a balance between organic and carbonate carbon buried in the sedimentary column. Basin-wide, the source of carbonate (pelagic producers, neritic aragonitic or calcitic producers, inorganic carbon supply from the continent, remineralization of organic carbon, diagenesis) can overprint the global trend of marine δ¹³C values (e.g. Swart and Eberli, 2003). A recent reappraisal of the time scale from the Valanginian to the Barremian (Early Cretaceous) nonetheless demonstrated the impact of the long orbital cycles (1.2 and 2.4 Ma), with amplitudes as much important as the emplacement of a large igneous province on the evolution of the marine carbonate δ¹³C (Martinez et al., 2023). That raises the opportunity of finding the record of shorter orbital cycles in the δ¹³C, so that it can be used to finely correlate sedimentary record widely distributed in the world. Organic-carbon δ¹³C values have usually higher amplitudes of change than carbonate δ¹³C. They have the ability to record the same trends as observed in marine carbonate δ¹³C, while basin-scale processes that can alter a global trend in organic δ¹³C (δ¹³Corg) series are due to the change of source between terrestrial and aquatic organic matter, the terrestrial organic matter having higher δ¹³C values in the Mesozoic (Suan et al., 2015). Here, we acquire two series of δ¹³Corg from the Vergol-Morénas (Vocontian Basin, France, Fig. 1) and Cerro La Parva (Neuquén Basin, Argentina, Fig. 2). These two sections are geographically remote from each other and encompass the lower-upper Valanginian boundary. The Vergol-Morénas section was deposited in basin environments, while the Cerro La Parva section was deposited in an outer ramp environment. They are both composed of marl and limestone beds, albeit the detrital content is higher in Cerro La Parva. In both cases, the balance between siliciclastic and carbonate content is related to astronomical forcing. We show that in both cases the δ¹³Corg correlates with the 405-ka eccentricity cycle and that both sections can be correlated thanks to these cycles with a precision in time of ca. 100 ka.

## 2 MATERIALS AND METHODS

X-Ray Diffractometry (XRD) was performed on 27 bulk samples of powdered marl and limestone from Vergol-Morénas using a Bruker D8 Endeavor diffractometer to identify and quantify mineral phases. The spectra were corrected for calcite contents, obtained from decarbonation data during the preparation of samples for δ¹³C measurements on organic carbon.

The δ¹³Corg values were measured on 27 samples at Vergol-Morénas and 71 samples at Cerro La Parva. The samples previously crushed were decarbonated using 2N hydrochloric acid. XRD verification was performed to ensure the absence of residual carbonates that could affect the results. After cleaning with pure water, decarbonated powders were dried in an oven (50 °C for 48 h), then crushed again to obtain a fine powder. Each sample, of a specific mass (about 50 mg), was weighed in a tin capsule with a Sartorius MCA6-6S-2S00-M ultrabalance. Samples were analyzed with an elemental analyzer (vario MICRO cube,

<footnote n="1">¹Biogéosciences, UMR 6282 CNRS, Université Bourgogne Europe, 21000 Dijon, France</footnote>

<footnote n="2">²Université de Rennes, CNRS, Géosciences Rennes, UMR 6118, 35000 Rennes, France</footnote>

<footnote n="3">³Instituto de Estudios Andinos Don Pablo Groeber, CONICET & Universidad de Buenos Aires, Ciudad Universitaria, pabellón 2, 1428 Buenos Aires, Argentina</footnote>
Elementar, GISMO analytical platform, Biogéosciences laboratory Dijon) coupled to an isotope ratio mass spectrometer (Isoprime, Elementar). Isotope ratios obtained were compared to international standards USGS40 L-glutamic acid (δ¹³C = -26.39 ±0.04‰ VPDB) and IAEA-600 caffeine (δ13C = -27.77 ±0.04‰ VPDB). For each sample, replicates showed reproducibility better than ±0.15 ‰. The total organic carbon (TOC) content, expressed in wt %, was determined by the elemental analyzer at the same time.

Mass-normalized magnetic susceptibility (MS) was measured on 816 regularly spaced every 0.20 m at Cerro La Parva with a Kappabridge KLY-3. Prior to the measurements, the weight of each of the samples was measured with a weighting scale having an accuracy of 0.01 mg. The volume measurements were corrected from the blank. The blank MS here corresponds to the volume MS of the container and the analysis room. The blank-corrected MS values were then normalized to the weight of the sample and a volume of 10 cc. Values are reported in m³.kg⁻¹.

## 3 RESULTS AND DISCUSSION

The δ¹³Corg values from Vergol-Morénas range from -29.3 ‰ to -27.1 ‰ PDB (Fig. 1d). The values increase from the *Karakaschiceras inostranzewi* to the *Karakaschiceras pronecostatum* subzones, which reflect the Weissert Carbon Isotope Excursion (CIE). Within this trend, four local maxima are observed at levels 89 m, 101 m, 121 m, and 146 m, near the boundaries of the 405-ka eccentricity cycles identified in Martinez et al. (2013; Fig. 1c). These boundaries were located on maxima of detrital supply identified by spectral analyses through the basin. XRD analyses shows a lower part of the studied interval, until level 113 m (Figs. 1e-g), dominated by carbonate. Then, the detrital fraction rapidly increases in the *Saynoceras verrucosum* Subzone and remains maximal until level 145 m. The carbonate fraction then progressively increases toward the top of the series. This trend in detrital content can be easily observed in the field, as a thick marl series with thin intercalated limestone beds replaces regular marl-limestone alternations in the *S. verrucosum* Zone. Within this trend, the four local maxima in δ¹³Corg associated with 405-ka cycle boundaries correspond to local maxima in the detrital fraction. Despite their low amplitude, the whole δ¹³Corg shows a cyclic trend in line with the mineralogy and the 405-ka eccentricity cycles sequenced in Martinez et al. (2013). Thus, the record of the 405-ka eccentricity cycle is observed both in δ¹³Corg and detrital fraction. In the Vocontian Basin, increased detrital fraction was due to increased chemical weathering and runoff. Clay mineral analyses shown that this feature could be observed at different timescales from precession to long eccentricity cycles (Deconinck and Debrabant, 1985; Martinez et al., 2020).

<figure><img src="image_69.png" /><figcaption>**Figure 1.** Location and results of the data acquired at Vergol-Morénas. A. Location of the Vergol-Morénas section within the Vocontian Basin. B. Synthesis of δ¹³Ccarb data published on the studied interval of Vergol-Morénas (from Martinez et al., 2020). C. Gamma-Ray Total Count (GRTC; Martinez et al., 2013). D. δ¹³Corg. E. CaCO₃ content from X-Ray Diffractometry. F. Quartz content from X-Ray Diffractometry. G. Clay content from X-Ray Diffractometry.</figcaption></figure>

In Cerro la Parva, the MS signal has values ranging from 23 to 170 x 10⁻⁹ m³.kg⁻¹ and displays a trend toward decreasing values from the bottom to the top of the series (Fig. 2d). The trend displayed in Fig. 2d was calculated using the LOWESS method (Cleveland, 1979) with a coefficient of 0.5. The δ¹³Corg values range from -24.7 ‰ to -21.2 ‰ PDB. The series starts with the second highest values of the series at the base of *Karakaschiceras attenuatus* Subzone. The values then decrease toward the minimum values of the series until level 50 m, and then increase until level 85 m. The trend in the average values (calculated using the LOWESS method with a coefficient of 0.5) then stabilizes until level 150 m, where it initiates a trend toward decreasing values at the top of the series. The values of the base of the series are higher than the values
acquired at the top of the Vaca Muerta Formation (Weger et al., 2022), while the lowermost values acquired here match the maximum values of Weger et al. (2022). Regarding the stratigraphic context and the trends of the δ¹³Corg series, the lowermost part of the series corresponds to the peak in δ¹³Carb of the Weissert CIE, within the *S. verrucosum* Tethyan ammonite Subzone. The rest of the series lies within the Plateau Phase of the Weissert CIE, which probably ends at Cerro La Parva at level 150 m.

<figure><img src="image_70.png" /><figcaption>**Figure 2.** Location and results of the data acquired at Cerro La Parva. A. Location of the Cerro La Parva section within the Neuquén Basin. B. Residual magnetic susceptibility (data to which the trend in average was subtracted) calibrated to time after correlations of the δ¹³Corg record from Cerro La Parva to the synthetic δ¹³Carb from the Vocontian Basin. C. Residual δ¹³Corg calibrated to time after correlation to the compiled δ¹³Carb data from the Vocontian Basin. D. Mass-normalized magnetic susceptibility. The red curve represents the long-term trend in average. E. δ¹³Corg. The black curve represents the long-term trend in average. F. Time scale of the Valanginian-Hauterivian time (from Martinez et al., 2023). G. Compilation of δ¹³Carb from the Vocontian Basin (from Martinez et al., 2023). The red curve forms the trend of the δ¹³Carb series calculated using the LOWESS method with a coefficient of 3.5 %.</figcaption></figure>

Within the Plateau Phase, the trend of the δ¹³Corg series shows two minima of the trend at levels 50 m and 125 m correspond to two minima, respectively dated at 133.32 and 132.65 Ma (Martinez et al., 2023), in the trend of the compilation of δ¹³Carb of the Vocontian Basin (Fig. 2). Two maxima at Cerro La Parva 85 and 145 m correspond to two maxima of the compilation of the Vocontian Basin, dated 133.04 and 132.37 Ma. Spectral analyses were made on the MS and δ¹³Corg series of Cerro La Parva, which have been first calibrated into time with the age model just described above, linearly interpolated to their average sampling time (2 ka for the MS, 25 ka for the δ¹³Corg), and which trend has been subtracted to the series. The spectra were calculated with the multi-taper method applying three 2π-tapers (Thomson, 1982).

<figure><img src="image_71.png" /><figcaption>**Figure 3.** Spectra of the δ¹³Corg (A) and MS (B) series of the Cerro La Parva section calibrated into time from the correlation with the δ¹³C from the Cerro La Parva section to the Vocontian Basin. Periods are labelled in thousands of years. The red curves are the filters then displayed in Figures 2A and 2B</figcaption></figure>

The spectrum of the MS shows a spectral peak at 300 ka (Fig. 3b). The spectrum of the δ¹³Corg series shows spectral peaks at 370 ka and 120 ka (Fig. 3a), likely respectively corresponding to the 405-ka and the ~100-ka eccentricity cycles. The application of the filters on the MS and δ¹³Corg series here guided the identification of the boundaries of the 405-ka eccentricity cycles, especially the lower boundary of V11, around which the data are more scattered than in Vergol. From these boundaries, a refined age model of the section can be proposed (Fig. 3e; Tab. 1), allowing in turn the age of the base of ammonite zones recorded in the section to be calculated (Tab. 2). Interestingly, one of the implications of this age model is the synchronicity between
Moreau-Ledegen et al., Astronomic forcing on δ¹³Corg records for global correlation at 10⁵yrs time scale.

the base of the *P. angulatiformis* Andean ammonite zone (131.96 Ma) and the *Criosarasinella furcillata* Tethyan ammonite zone (131.92 Ma; Martinez et al., 2023), as previously inferred from astrochronology at El Portón and La Charce sections (Aguirre-Urreta et al., 2019).

<table><caption>Table 1. Final age model of the studied interval of the Cerro La Parva section</caption><thead><tr><td>Label</td><td>Level (m)</td><td>Age (Ma)</td></tr></thead><tbody><tr><td>Base V8</td><td>6.0</td><td>133.87</td></tr><tr><td>Base V9</td><td>41.4</td><td>133.46</td></tr><tr><td>Base V10</td><td>83.0</td><td>133.06</td></tr><tr><td>Base V11</td><td>114.6</td><td>132.65</td></tr><tr><td>Base V12</td><td>142.0</td><td>132.25</td></tr></tbody></table>

<table><caption>Table 2. Calculated ages of ammonite zones in the studied interval of Cerro La Parva</caption><thead><tr><td>Ammonite zone</td><td>Level base (m)</td><td>Age base (Ma)</td></tr></thead><tbody><tr><td><i>K. attenuatus</i></td><td>0</td><td>133.94</td></tr><tr><td><i>V. permolestum</i></td><td>145.5</td><td>132.2</td></tr><tr><td><i>P. angulatiformis</i></td><td>161.7</td><td>131.96</td></tr></tbody></table>

It remains to be understood whether the orbital forcing on the carbon-isotope ratios in these basins result from regional or global processes. Regional processes could arise from the change in source of terrestrial or marine organic carbon. Global processes could arise from global changes in primary productivity and sequestration of organic carbon. In both basins, maxima in δ¹³Corg at 405-ka scale are related to increase in siliciclastic input, which is, at least in the Vocontian Basin, related to increased runoff conditions (Deconinck and Debrabant, 1985), which favored primary productivity. In Cerro La Parva, the filters of MS and δ¹³Corg show a maximum difference of 0.06 Ma and are broadly in phase (Figs. 2b, c), so increase in δ¹³Corg was also related to increased siliciclastic input, likely for the same reasons as in the Vocontian Basin. The filter of the 405-ka cycle in the δ¹³Corg signal correlates with the 405-ka cycles defined in the Vocontian Basin, which, in turn, allows the ages of the biozones at Cerro La Parva to be refined. From this age model, the base of the *K. attenuatus* Subzone is dated at 133.94 Ma, the base of the *V. permolestum* Subzone at 132.20 Ma, and the base of the *P. angulatiformis* Zone at 131.96 Ma. At this stage, we thus suggest that the orbital control observed on the δ¹³Corg in both basins results from a global control on the carbon cycle. In this case, carbon isotope stages can be defined at eccentricity and then constitute a powerful correlation method to align with high precision paleoclimatic signals at global scale.

## 4 CONCLUSION

The carbon-isotope signals measured on two sections, Cerro La Parva (Argentina) and Vergol-Morénas (France), and calibrated by astrochronology, demonstrate the pacing of the δ¹³Corg signal by the 405-ka eccentricity cycle, and possibly by the 100-ka eccentricity cycle. In both cases, the cycles in δ¹³Corg are broadly in line with the detrital cycles, implying that increase in δ¹³Corg occurred during increased runoff conditions, possibly under more humid conditions that also favored increased primary productivity. Orbitally-controlled carbon isotope stages can then be defined for long-distance and fine correlations, which then constitutes a powerful method for deep-time calibration of paleoclimatic series.

## 5 ACKNOWLEDGMENT

MM thanks CNRS INSU SYSTER grant WOAW (Widespread Oceanic Anoxia during the Weissert event?) and University of Rennes Défis Scientifiques project: Quantification de la surface des zones anoxiques marines à travers les temps géologiques for supporting this project. Adoum Ali Kodo is thanked for helping in
the data acquisition. The authors thank the GISMO platform and its staff (Biogéosciences, Université Bourgogne Europe, UMR CNRS 6282, France), as well as the GeOHeLiS platform and his staff (Géosciences Rennes, UMR 6118, France). They also thank Michael Joachimski and Nicolas Tribovillard for having reviewed the earlier version of the manuscript.

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# MEASURING ALKALINITY FLUXES AT THE WATER-SEDIMENT INTERFACE IN AQUATIC ECOSYSTEMS OF THE TUNISIAN LITTORAL: IMPACT ON PH AND INORGANIC CARBON STORAGE

Walid OUESLATI<fnref n="1" />, Haifa BEN MNA<fnref n="1" />, Mohamed Amine HELALI<fnref n="1" />, Takwa MGHIRBI<fnref n="1" />, Amina BEN MEFTEH<fnref n="1" />, Sarah BEN JEDDI<fnref n="1" /> and Valérie MESNAGE<fnref n="2" />

Corresponding author: walid.oueslati@fst.utm.tn

**Abstract:** In the context of climate change and rising atmospheric CO₂, coastal marine sediments, rich in algae and seagrass beds, are potential blue carbon sinks and thus contribute to climate mitigation. However, ocean acidification due to CO₂ fluxes has been the subject of scientific debate for over 20 years. Indeed, sediments, through the anaerobic reactions of early diagenesis, promote the production of alkalinity in pore water, which can mitigate the effects of open-water acidification. The aim of this research is to model pore water alkalinity concentrations in four different Tunisian coastal ecosystems, in order to quantify the release of benthic alkalinity and the precipitation of authigenic carbonates. The results show that these sediments are an important source of alkalinity, with fluxes ranging from 1.1 to 7.27 mmol m⁻<fnref n="2" /> d⁻<fnref n="1" />. Additionally, the elevated alkalinity can promote the precipitation of carbonates, enabling the sediments to act as a sink for inorganic carbon.

**Key words:** sediment-water interface, alkalinity, blue carbon, inorganic carbon sequestration, Tunisia

## 1 INTRODUCTION

The ocean plays an important role as a natural carbon sink, absorbing nearly, a third of atmospheric CO₂ through ecosystems such as seagrasses, macroalgae, phytoplankton, mangroves and salt marshes (Wang et al., 2023), helping to mitigate the effects of climate change. However, the release of CO₂ into seawater leads to ocean acidification, which affects calcifying organisms such as corals and molluscs. Coastal marine sediments play a buffering role against this acidification, helping to sequester inorganic carbon. Coastal wetlands, which cover about 6% of the earth's surface, are particularly vulnerable to human impacts from agriculture and tourism. Recent studies have analysed the impact of acidification on the dissolution of calcium carbonate in the sediments (Eyre et al., 2014; Nesbit & Mucci, 2020). Traditionally sediments have been considered as zones of carbonate dissolution. However, recent research suggests that coastal sediments can also act as inorganic carbon sinks (Sun & Turchyn, 2014; Oueslati et al., 2019) models show that authigenic carbonate precipitation in marine sediments can play an important role in the global carbon cycle (Schrag et al., 2013). Furthermore Oueslati et al. (2019) have shown that the sediments of the Ghar El Melh lagoon in Tunisia generate alkalinity and therefore contribute to the acidification of the coastal waters in this area. In this study, we wish to examine the role of other coastal wetlands in Tunisia by diversifying the types of ecosystems studied, in particular lagoons (Korba and Lake North of Tunis), a coastal Sabkha (Sabkha of Ariana in Greater Tunis) and an open sea (Monastir Bay in central Tunisia, characterized by an abundance of *Posidonia Oceanica*). The Ghar El Melh lagoon was also reassessed in this study. The methodology adopted is based on modelling alkalinity concentrations in porewaters, using a computer code (PROFILE, Berg et al. 1998) to calculate flows at the sediment-water interface.

## 2 MATERIALS AND METHODS

### 2.1 Study sites

This research work complements the results of Oueslati et al. (2019) on the Ghar El Melh lagoon, by studying how the sedimentary compartment contributes to the generation of alkalinity, likely to diffuse into the water column to mitigate the acidification of overlying waters, while promoting the precipitation of authigenic calcite and the sequestration of inorganic carbon. The sites studied (Fig. 1) are (1) lagoons (Korba lagoon and Tunis northern lake), (2) a coastal sabkha (Ariana sabkha) and (3) open sea (Monastir Bay, rich in *Posidonia Oceanica*, which promotes blue carbon sequestration). We also re-examined the Ghar El Melh lagoon.

<footnote n="1">¹ LR01ES06 Ressources Minérales et Environnement, Université de Tunis El Manar (Tunisie)</footnote>

<footnote n="2">² UMR 6143 M2C, Université Rouen Normandie, Université Caen Normandie, CNRS</footnote>
<figure><img src="image_72.png" /><figcaption>Figure 1: Study sites</figcaption></figure>

## 2.2 Sampling, pore water alkalinity determination and mathematical modelling

At each study site, a sediment core was taken using a 9 cm PVC corer; except for the Monastir Bay site, where two sediment cores were taken. The sediment cores were then cut into sub-samples under a nitrogen flow. The resulting samples were then centrifuged at 2,500 rpm at low temperature to extract the pore water. Total alkalinity (TA) was measured on this pore water using the method of Rodier et al. (1997). Sediment porosity was calculated on the basis of the water content obtained by drying at 110°C. To assess the diffusion flux of total alkalinity at the sediment-water interface, as well as its rate of production or consumption, we modelled total alkalinity (TA) concentrations. Assuming steady-state, compaction and the absence of bioirrigation (Boudreau, 1997), the equilibrium equation for the alkalinity in pore water is expressed as follows:

$$ \left[ \frac{\partial \phi[TA]}{\partial t} \right]_x = \frac{\partial}{\partial x} \left[ \phi(D_s + D_B) \frac{\partial[TA]}{\partial x} \right] + R_{net}^{TA} $$

where $x$ represents the depth (in cm), $\phi$ the porosity and $t$ the time (in s). $R_{net}^{TA}$ is the rate of net production (positive) or consumption (negative) associated with reactions (in nmol cm$^{-3}$.s$^{-1}$) in the entire sediment, including particles and pore water, for the alkalinity TA present in the pore water. This equation is solved numerically for $R_{net}^{TA}$ using PROFILE code (Berg et al., 1998). For input values to the program, we used porosity data as well as a data set of the average pore water alkalinity. DS and DB values required in PROFILE were estimated according to Li and Gregory (1974).

# 3 RESULTS AND DISCUSSION

Figure 2 illustrates alkalinity profiles, with an increase of concentrations from the sediment-water interface to the first 10-15 centimeters, indicating TA production in this layer of sediment. Beyond this depth, with the exception of the northern lake of Tunis, concentrations show a marked decrease. Alkalinity production rates in pore water (Table 1) are significant. They amount to 3.5 mmol m$^{-2}$ d$^{-1}$ in the Ghar El Melh lagoon, 3.71 mmol m$^{-2}$ d$^{-1}$ (on average) in Monastir Bay, 1.5 mmol m$^{-2}$ d$^{-1}$ in Ariana de Sabkha, and 8.66 mmol m$^{-2}$ d$^{-1}$ and 0.47 mmol m$^{-2}$ d$^{-1}$ in Lac Nord de Tunis. Detailed modeling of diagenesis in the Ghar El Melh lagoon (Oueslati et al., 2019) shows that the chemical precipitation of carbonates is facilitated by the production of alkalinity during the processes of anaerobic mineralization and framboidal pyrite formation.

Pyrite oxidation and reoxidation of free Fe$^{2+}$ in pore water are very limited. Assuming that chemical carbonate precipitation is the secondary diagenetic reaction responsible for alkalinity consumption in deep sediment layers (Figure 2), authigenic calcite precipitation rates range from 0 to 1.39 mmol m$^{-2}$ d$^{-1}$ (Tab. 1). These rates are relatively low compared with those measured by Zhu et al. (2002) in Amazon delta sediments (4 mmol m$^{-2}$ d$^{-1}$) and higher than those reported by Reimers et al. (1996) in undisturbed muddy sediments from the Santa Barbara Basin (0.2 mmol m$^{-2}$ d$^{-1}$). Although alkalinity is consumed by the precipitation of carbonates and the reoxidation of reduced chemical elements (Fe$^{2+}$ and FeS$_2$), even at low levels, a significant amount remains present in the sediments and may diffuse into the overlying waters. These diffusion fluxes are in the same range as those measured in North Sea sandy sediments (1.7 - 6.6
mmol m⁻² d⁻¹, Brenner et al., 2016), and in Australian lagoon sediments populated by seagrass beds (0 - 2 mmol m⁻² d⁻¹, Eyre and Ferguson, 2002).

<figure><img src="image_73.png" /><figcaption>Figure 2: Vertical profiles of measured (circle) and calculated (solid red line) alkalinity in Sabkha of Ariana (a), Ghar El Melh lagoon (b), Korba lagoon (c), Monastir bay S1 (d), Monastir bay S2 (e) and Lake north of Tunis (f). Alkalinity production rate is presented in solid green line.</figcaption></figure>

<table><caption>Table 1: Diffusion flux and production/consumption rate of alkalinity (mmol m⁻² d⁻¹)</caption><thead><tr><td></td><td>PRODUCTION RATE</td><td>DIFFUSION FLUX</td><td>CONSUMPTION RATE</td></tr></thead><tbody><tr><td>Monastir bay S1</td><td>303</td><td>-2.82</td><td>-0.2</td></tr><tr><td>Monastir bay S2</td><td>4.39</td><td>-3.69</td><td>-0.7</td></tr><tr><td>Lake North of Tunis</td><td>0.47</td><td>-0.47</td><td>0.0</td></tr><tr><td>Sabkha Ariana</td><td>1.5</td><td>-1.1</td><td>-0.4</td></tr><tr><td>Ghar El Melh lagoon</td><td>3.5</td><td>-2.8</td><td>-0.7</td></tr><tr><td>Korba lagoon</td><td>8.66</td><td>-7.27</td><td>-1.39</td></tr></tbody></table>

## 4 CONCLUSION

In this study, by modeling the alkalinity concentrations in the pore water of different coastal marine environments, we demonstrate that early diagenesis of organic matter, generates significant amounts of alkalinity. The latter plays an important role in the global carbon cycle, notably through the precipitation and subsequent burial of authigenic carbonates. On the other hand, it is a source of alkalinity for overlying water and the open ocean, contributing to pH regulation and CO₂ absorption. These positive results, linked to climate change and the dissolution of marine carbonates, could encourage decision-makers to protect these coastal ecosystems, which undoubtedly provide valuable services.
5 REFERENCES

Berg, P., Rysgaard-Petersen, N., Rysgaard, S., 1998. Interpretation of measured concentration profiles in sediment pore water, Limnology and Oceanography, 43, 1500-1510.

Boudreau, B.P., 1997. Diagenetic Models and Their Implementation. Springer. 414 p.

Brenner, H., Braeckman, U., Le Guitton, M., Meysman, F.J.R., 2016. The impact of sedimentary alkalinity release on the water column CO2 system in the North Sea. Biogeosciences Discussions, 12, 12395-12453.

Eyre, B., Andersson, A., Cyronak, T., 2014. Benthic coral reef calcium carbonate dissolution in an acidifying ocean. Nature Climate Change, 4, 969-976.

Eyre, B.D., Ferguson, A.J.P., 2002. Comparison of carbon production and decomposition, benthic nutrient fluxes and denitrification in seagrass, phytoplankton, benthic microalgae and macroalgae dominated warm-temperate Australian lagoons. Marine Ecology Progress Series, 229, 43-59.

Li, Y.H., Gregory, S., 1974. Diffusion of ions in sea water and in deep-sea sediments. Geochimica and Cosmochimica Acta, 38, 703-714.

Nesbitt, W.A., Mucci, A., 2020. Direct evidence of sediment carbonate dissolution in response to bottom-water acidification in the Gulf of St. Lawrence, Canada. Canadian Journal of Earth Sciences, 58.

Oueslati, W., Van de Velde, S., Helali, M.A., Added, A., Aleya, L., Meysman, F., 2019. Carbon, iron and sulphur cycling in the sediments of a Mediterranean lagoon (Ghar El Melh, Tunisia). Estuarine, Coastal and Shelf Science, 221, 156-169.

Reimers, C.E., Ruttenberg, K.C., Canfield, D.E., Christiansen, M.B., Martin, J.B., 1996. Porewater pH and authigenic phases formed in the uppermost sediments of the Santa Barbara Basin. Geochimica and Cosmochimica Acta, 60, 4037-405.

Rodier, J., Bazin, J.P., Chambon, P., Chambaour, H., Rodi, H., 1997. Analyse de l'eau (8e édition), Dunod, 1824 p.

Schrag, D.P., Higgins, J.A., Macdonald, F.A., Johnston, D.T., 2013. Authigenic Carbonate and the History of the Global Carbon Cycle. Science, 339, 540-559.

Sun, X., Turchyn, A.V., 2014. Significant contribution of authigenic carbonate to marine carbon burial. Nature Geoscience, 7, 20-204.

Wang, W., Fu, W., Le Moigne, F.A.C. Letscher, R.T., Liu, Y., Tang, J.M., Primeau, F.W., 2023. Biological carbon pump estimate based on multi-decadal hydrographic data. Nature, 624, 579-585.

Zhu, Z.B., Aller, R.C., Mak, J., 2002. Stable carbon isotope cycling in mobile coastal muds of Amapa, Brazil. Continental Shelf Research, 22, 2065-2079.
# CARBON BUDGETS WITHIN TEMPERATE COASTAL ECOSYSTEMS AND COMPARISON WITH PICARD ESTUARIES (NORTH OF FRANCE)

Kentin Plègue, Nicolas Spilmont and Lionel Denis

Univ. Lille, CNRS, Univ. Littoral Côte d'Opale, IRD, UMR 8187 LOG, Station Marine de Wimereux, F-59000 Lille,
France.

Corresponding author: kentin.plegue@univ-lille.fr

**Abstract:** In estuarine sediments, the balance between organic matter mineralization and primary production determines whether the system is autotrophic (dominated by production processes, i.e. CO₂ sink) or heterotrophic (dominated by respiration/mineralization processes, i.e. CO₂ source). Some autotrophic systems, such as mangroves, seagrass meadows or salt-marshes, are capable of sequestering carbon over long periods, and are referred to as “blue carbon” ecosystems. Salt-marshes are located in the upper part of the estuarine intertidal zone and are only submerged during high tides for a short period. Even if these vegetated areas are generally considered as carbon sinks, the status of the entire intertidal estuarine zone as a “blue carbon” ecosystem is still debated. To accurately assess their carbon sequestration capacity, it is therefore crucial to understand the dynamics of carbon fluxes and stocks in the various typical zones of estuaries (such as channels, mudflats and salt-marshes) across different temporal scales. The aim of this work was to review the knowledge on carbon fluxes and stocks in temperate intertidal ecosystems, particularly within the Picard estuaries (North of France), identify gaps in the current understanding of carbon fluxes and outline objectives and methodologies to quantify the role of these estuaries in carbon sequestration.

**Keywords:** carbon fluxes, salt-marshes, mudflats, intertidal, blue carbon

## 1 INTRODUCTION

Coastal zones, situated at the intersection of land and ocean, include tidal wetlands, estuaries, and continental shelf waters. All of them play a vital role in the global carbon cycle (Duarte et al., 2005). Organic matter mineralization, driven by bacterial activity and respiration, releases carbon as dissolved inorganic carbon (DIC) into the water or as CO₂ into the atmosphere. Conversely, primary production by autotrophs (e.g., microphytobenthos, seagrass, salt-marsh plants) fixes CO₂, with a portion potentially sequestered in sediments. The carbon budget depends on the balance between these processes: a heterotrophic system (carbon source) is characterized by respiration processes exceeding photosynthesis, whereas an autotrophic system (carbon sink) is dominated by photosynthesis. Mcleod et al. (2011) defined carbon stored in marine sediments by primary producers as "Blue Carbon"; despite covering only 0.2% of the ocean surface, "Blue Carbon" systems such as mangroves and seagrass meadows can store up to 50% of oceanic carbon (Duarte et al., 2013, Oueslati et al., 2025). However, only a fraction of fixed CO₂ is ultimately buried, as a portion is released back to the environment by mineralization (Liang et al., 2017). The carbon budget based on autotrophy/heterotrophy reflects short-term exchanges, while long-term sequestration depends on complex dynamics. The high spatio-temporal variability of these processes in intertidal systems makes their contribution to carbon sequestration difficult to quantify. Like mangroves and seagrass, salt-marshes have high primary production and contribute significantly to carbon sequestration (Chmura et al., 2003; Duarte et al., 2005). However, less than 10% of carbon fixed by photosynthesis is buried in salt-marshes (Howes et al., 1985), and their role as carbon sinks remains uncertain, due to limited data and the influence of spatio-temporal variations across estuarine habitats (channels, mudflats, low and high shore) over different time scales (tidal cycles, seasonal changes). This review has multiples aims: (i) synthesize the current knowledge on carbon fluxes in temperate intertidal ecosystems, (ii) compare them with data from the Picard estuaries, defined by a mixed energy regime (influenced by both tides and waves and limited freshwater inputs) and a massive sand-platform at the south of their mouths (Voltz et al., 2021), (iii) identify key research gaps.

## 2 MATERIAL AND METHODS

We mainly focused this review (i) on intertidal temperate ecosystems along the Atlantic coast of Western Europe (Figure 1) and (ii) on studies using one of the main measurement methods described below.
<figure><img src="image_74.png" /><figcaption>Figure 1: Location of the different sites in the considered studies.</figcaption></figure>

**2.1 Measurements at the Sediment/Atmosphere interface**

The studies presented two approaches to quantify CO₂ exchanges between sediment and the atmosphere: enclosed systems and atmospheric micro-meteorological measurements. The first approach involves measuring CO₂ concentration in a flow of air passing over a section of the community enclosed within a benthic chamber. Details on the design and use of this type of chamber are provided by Migné *et al.* (2002). The different measurements were carried out under ambient light and in darkness to estimate Net Community primary Production (NCP) and Community Respiration (CR), respectively. The second approach is based on the eddy-covariance method, which uses wind analysis to quantify net CO₂ exchanges at the ecosystem-atmosphere interface. The details and design of this method are presented in Polsenaere *et al.* (2012) and Mayen *et al.* (2024).

**2.2 Measurements at the Sediment/Water interface**

Benthic chambers are also employed to quantify DIC fluxes, using underwater versions of the systems. There is a type of benthic chamber known as “flow through” chamber, which allows continuous measurement of DIC fluxes measurement (Ouisse *et al*, 2014). Another technique involves core sampling and subsequent ex-situ whole-core incubations (Cores incubations) in the dark (Denis *et al.*, 2001).

**3 RESULTS**

During emersion periods, the ecosystems analyzed exhibited negative CO₂ fluxes under light conditions (i.e., CO₂ uptakes, Table 1) and positive fluxes under dark conditions (i.e., CO₂ efflux, Table 1). There was spatial variability both between the different study sites and among the sediment types within each site (Table 1). For example, the highest CO₂ uptake was measured in the Fier d'Ars salt-marshes, and the highest CO₂ efflux was measured in the low part of the Authie salt-marsh. Moreover, Canche and Authie salt-marshes showed higher CO₂ efflux at dark in summer than the other ecosystems. A seasonal pattern was observed, with more intense CO₂ fluxes during the summer, except in Mont Saint-Michel and Fier d'Ars salt-marshes. During immersion periods, a seasonal pattern was also evident, with higher DIC fluxes in summer compared to spring, when data were available for both seasons (Table 2). A spatial variability was again observed, both between the different study sites and among sediment types within each site. For instance, DIC fluxes were higher in the mudflat and low salt-marsh in Canche estuary during summer compared to other systems. A clear gradient was also observed along the Canche estuary transect, with DIC fluxes decreasing from the mudflat to the high salt-marsh during spring and summer. However, the DIC fluxes measured in Canche remained similar to those in other systems, such as the *Zostera noltei* beds in Roscoff or the sand colonized by *Ruditapes philippinarum* in the Rance estuary during spring.

**4 DISCUSSION**

A part of the variability in carbon fluxes observed between studies can be attributed to methodological differences. Benthic chambers provide localized, low-frequency measurements over small areas (dm²), limiting the integration of large macrophytes, whereas eddy-covariance covers large surface areas (hundreds of m²) but is sensitive to physical variables such as friction velocity.
<table><caption>Table 1: Mean CO₂ fluxes (in mmol C m⁻² h⁻¹) during emersion as reported in the literature on French intertidal ecosystems along the Atlantic coast including the locations, sediment types, methods used (Benthic Chambers: BC; Eddy-Covariance: EC) and corresponding references. Negative values indicate a flux from the atmosphere to the sediment, while positive values indicate a flux from the sediment to the atmosphere.</caption><thead><tr><th rowspan="2">Location</th><th rowspan="2">Sediment type</th><th rowspan="2">Method</th><th colspan="2">Winter</th><th colspan="2">Summer</th></tr><tr><th>Light</th><th>Dark</th><th>Light</th><th>Dark</th></tr></thead><tbody><tr><td>Roscoff</td><td>Sand</td><td>BC (Migné et al., 2011)</td><td>-0.2</td><td>0.31</td><td>-1.18</td><td>0.69</td></tr><tr><td>Seine</td><td>Mud</td><td>BC (Spilmont et al., 2006)</td><td>-2.65</td><td>0.48</td><td>-3.54</td><td>1.93</td></tr><tr><td>Seine</td><td>Sand + Mud</td><td>BC (Spilmont et al., 2006)</td><td>-1.76</td><td>0.54</td><td>-5.44</td><td>2.54</td></tr><tr><td>St Michel</td><td>Sand</td><td>BC (Davoult et al., 2009)</td><td>-0.59</td><td>0.17</td><td>-0.54</td><td>0.19</td></tr><tr><td>St Michel</td><td>Mud</td><td>BC (Davoult et al., 2009)</td><td>-1.36</td><td>1.61</td><td>-1.55</td><td>0.77</td></tr><tr><td>Moraix</td><td>Sand + Zostera noltei</td><td>BC (Ouisse et al., 2010)</td><td>-6.4</td><td>1.1</td><td>-4.79</td><td>3.93</td></tr><tr><td>Arcachon</td><td>Sand</td><td>BC (Migné et al., 2016)</td><td>-0.15</td><td>0.17</td><td>-1.33</td><td>0.73</td></tr><tr><td>Arcachon</td><td>Mud</td><td>BC (Migné et al., 2016)</td><td>-2.17</td><td>0.26</td><td>-6.58</td><td>1.2</td></tr><tr><td>Arcachon</td><td>Sand + Zostera noltei</td><td>BC (Migné et al., 2016)</td><td>-1.97</td><td>1.07</td><td>-3.27</td><td>1.86</td></tr><tr><td>Arcachon</td><td>Sand + Zostera noltei</td><td>EC (Polsenaere et al., 2012)</td><td>NA</td><td>NA</td><td>-1.08</td><td>3.24</td></tr><tr><td>Fier d'Ars</td><td>High Salt-marsh</td><td>EC (Mayen et al., 2024)</td><td>-11.34</td><td>9.72</td><td>-15.9</td><td>7.6</td></tr><tr><td>Canche</td><td>Mud</td><td>BC (Karelus, 2024)</td><td>NA</td><td>NA</td><td>-3.41 ± 2.97<br>n = 5</td><td>3.1 ± 1.54<br>n = 6</td></tr><tr><td>Canche</td><td>Low Salt-marsh</td><td>BC (Karelus, 2024)</td><td>NA</td><td>NA</td><td>-6.73 ± 2.26<br>n = 6</td><td>20.51 ± 7.93<br>n = 6</td></tr><tr><td>Canche</td><td>High Salt-marsh</td><td>BC (Karelus, 2024)</td><td>NA</td><td>NA</td><td>-4.10 ± 2.63<br>n = 4</td><td>12.03 ± 3.32<br>n = 5</td></tr><tr><td>Authie</td><td>Sand</td><td>BC (Voltz et al., 2019)</td><td>NA</td><td>NA</td><td>NA</td><td>9.76</td></tr><tr><td>Authie</td><td>Mud</td><td>BC (Voltz et al., 2019)</td><td>NA</td><td>NA</td><td>NA</td><td>9.47</td></tr><tr><td>Authie</td><td>Low Salt-marsh</td><td>BC (Voltz et al., 2019)</td><td>NA</td><td>NA</td><td>NA</td><td>24.73</td></tr><tr><td>Authie</td><td>High Salt-marsh</td><td>BC (Voltz et al., 2019)</td><td>NA</td><td>NA</td><td>NA</td><td>17.78</td></tr></tbody></table>

<table><caption>Table 2: Mean DIC fluxes (in mmol C m⁻² h⁻¹) measured in various studies during immersion in spring and summer across different temperate intertidal zones in France and Spain, under dark conditions.</caption><thead><tr><th>Location</th><th>Sediment type</th><th>Method</th><th>Spring</th><th>Summer</th></tr></thead><tbody><tr><td>Canche</td><td>Mud</td><td>Cores incubations (Karelus, 2024)</td><td>4.72 ± 2.08<br>n = 8</td><td>27.3 ± 6.35<br>n = 3</td></tr><tr><td>Canche</td><td>Low Salt-marsh</td><td>Cores incubations (Karelus, 2024)</td><td>3.56 ± 1.34<br>n = 9</td><td>20.56 ± 3.83<br>n = 3</td></tr><tr><td>Canche</td><td>High Salt-marsh</td><td>Cores incubations (Karelus, 2024)</td><td>1.33 ± 0.42<br>n = 9</td><td>7.15 ± 1.74<br>n = 3</td></tr><tr><td>Roscoff</td><td>Sand + Zostera marina</td><td>BC (Ouisse et al., 2011)</td><td>NA</td><td>15.50</td></tr><tr><td>Roscoff</td><td>Sand + Zostera noltei</td><td>BC (Ouisse et al., 2011)</td><td>NA</td><td>9.08</td></tr><tr><td>Roscoff</td><td>Sand + Zostera noltei</td><td>Flow through BC (Ouisse et al., 2014)</td><td>5.04</td><td>7.66</td></tr><tr><td>Rance estuary</td><td>Sand + R. philippinarum (High density)</td><td>BC (Migné et al., 2018)</td><td>4.00</td><td>6.00</td></tr><tr><td>Rance estuary</td><td>Sand + R. philippinarum (Low density)</td><td>BC (Migné et al., 2018)</td><td>NA</td><td>2.00</td></tr><tr><td>Bay of Cadiz</td><td>Sand</td><td>BC (Ortega et al., 2002)</td><td>NA</td><td>2.74</td></tr><tr><td>Ason estuary</td><td>Sand + Salt-Marshes</td><td>BC (Ortega et al., 2002)</td><td>NA</td><td>6.80</td></tr><tr><td>Oka estuary</td><td>Sand</td><td>BC (Ortega et al., 2002)</td><td>NA</td><td>15.75</td></tr></tbody></table>

During emersion, spatial variability both between and within sites can be driven by environmental factors such as vegetation cover, species assemblages, and sediment characteristics. Permeable sandy sediments promote advection, while mudflats retain porewater, and these processes can affect carbon fluxes (Migné et al., 2011). For instance, CO₂ fluxes increase faster in sandy flats (maximum values after one hour, Sasaki et
al., 2009) than in muddy flats (maximum values after three hours, Klaassen & Spilmont, 2012). The seasonal pattern observed at emersion can be driven by light and temperature, which influence photosynthesis and respiration. High temperatures enhance bacterial activity, increasing CO₂ fluxes in summer. During immersion, light attenuation due to water depth and turbidity limits photosynthesis, thereby explaining DIC release from sediment to water (Hoch, 1998). These fluxes can be enhanced by the macrofauna activity, such as bioturbation and excretion. For instance, bioturbation by infaunal organisms (e.g., annelids, bivalves) increases solute and oxygen infiltration (Deborde et al., 2008), and stimulates bacterial activity, while excretion recycles nutrients and consequently promotes primary production (Migné et al., 2018). Moreover, some studies showed that infaunal density can drive DIC fluxes in unvegetated environments, particularly in sandy sediments (Dye, 1981; Asmus, 1982) and mesocosm experiments confirmed that macrofauna increases inorganic carbon release (Gribsholt & Kristensen, 2002). Tidal range and immersion duration also contribute to variability, especially in estuarine transects. High salt-marsh zones, like those in the Canche estuary, are inundated only during spring tides. Seasonal variations in environmental factors such as temperature, riverine inputs, and nutrients further influence benthic metabolism, reinforcing heterotrophic activity and can explain a part of the seasonal pattern of DIC fluxes observed. Moreover, organic matter generally demonstrates higher accumulation rate in the lower part of the salt-marsh during immersion, leading to an increase in bacterial respiration and DIC fluxes.

## 5 CONCLUSION

This study revealed a spatio-temporal variability in carbon fluxes which can be explained by various biotic and abiotic factors such as macrofauna density and plant cover, temperature, immersion/emersion periods or the daylength. An inversion in carbon fluxes during the night is observed, with the system becoming a source of CO₂ under dark conditions as photosynthesis ceases. This inversion is also observed during immersion, where the ecosystem shows positive DIC fluxes. Several challenges and factors must be considered for future studies in the Canche estuary to produce the most reliable carbon budgets, given the complexity of the carbon cycle and flux dynamics. The different biotic and abiotic factors that drive carbon flux dynamics need to be more precisely identified. Additionally, effective methods and sampling strategies should be discussed to obtain a comprehensive understanding of this dynamic at various spatial and temporal scales.

## 6 ACKNOWLEDGEMENTS

This work received financial support from OFB-PNM-EPMO as well as CPER IDEAL program. This work is part of project CABESTAN of the exploratory research program FairCarboN and received government funding managed by the Agence Nationale de la Recherche under the France 2030 program, reference ANR-22-PEXF-0007. This paper was reviewed by Valérie Mesnage and Eric Armynot du Châtelet.

## 7 REFERENCES

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Deborde, J., Abril, G., Mouret, A., Jezequel, D., Thouzeau, G., Clavier, J., Bachelet, G., Anschutz, P., 2008. Effects of seasonal dynamics in a Zostera noltii meadow on phosphorus and iron cycles in a tidal mudflat (Arcachon Bay, France). Marine Ecology Progress Series, 355, 59–71.

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Duarte, C.M., Kennedy, H., Marbà, N., Hendriks, I., 2013. Assessing the capacity of seagrass meadows for carbon burial: Current limitations and future strategies. Ocean & Coastal Management, 83, 32–38.

Dye, A.H., 1981. A study of benthic oxygen consumption on exposed sandy beaches. Estuarine, Coastal and Shelf Science, 13, 671e680.
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Howes, B.L., Dacey, J.W.H., Teal, J.M., 1985. Annual Carbon Mineralization and Belowground Production of Spartina Alterniflora in a New England Salt Marsh. Ecology, 66, 595-605.

Karelus, A., 2024. Bilans de carbone aux interfaces sédiment-eau et sédiment-air dans la zone intertidale estuarienne de la Canche. Université de Lille, France.

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Mayen, J., Polsenaere, P., Lamaud, É., Arnaud, M., Kostyrka, P., Bonnefond, J.-M., Geairon, P., Gernigon, J., Chassagne, R., Lacoue-Labarthe, T., Regaudie De Gioux, A., Souchu, P., 2024. Atmospheric CO2 exchanges measured by eddy covariance over a temperate salt marsh and influence of environmental controlling factors. Biogeosciences, 21, 993-1016.

Mcleod, E., Chmura, G. L., Bouillon, S., Salm, R., Björk, M., Duarte, C. M., Lovelock, C. E., Schlesinger, W. H., Silliman, B. R. 2011. A blueprint for blue carbon: toward an improved understanding of the role of vegetated coastal habitats in sequestering CO2. Frontiers in Ecology and the Environment, 9, 552-560.

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Migné, A., Spilmont, N., Boucher, G., Denis, L., Hubas, C., Janquin, M.-A., Rauch, M., Davoult, D., 2009. Annual budget of benthic production in Mont Saint-Michel Bay considering cloudiness, microphytobenthos migration, and variability of respiration rates with tidal conditions. Continental Shelf Research, 29, 2280-2285.

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# MULTI-PROXY SEDIMENT TREND ANALYSIS (MSTA): SEDIMENTARY DYNAMICS FROM SOURCE TO SINK

Emmanuel Poizot<fnref n="1" />,<fnref n="2" />, Yann Méar<fnref n="1" />,<fnref n="2" />, Anne Murat<fnref n="1" />,<fnref n="2" />, Clément Frigola<fnref n="1" />,<fnref n="2" />, Gwendoline Grégoire<fnref n="1" />,<fnref n="2" /> and Benjamin Le Lorec<fnref n="1" />,<fnref n="2" />

Corresponding author: emmanuel-poizot@lecnam.net

**Abstract:** To study the surface sediment dynamics of the Cherbourg roadstead, the grain-size end-members of a set of 199 sediment samples are computed and integrated into a multi-proxy sediment trend analysis (MSTA). Cherbourg harbor is a sheltered area where the finest sediments mix with "fossil" sands, gravels from local erosion and shell debris from outside. Using the finest sediment of the data set, this new approach allows us to highlight marine or continental sources and sinks of this fraction.

**Keywords:** sedimentary dynamic, end-members, GSTA, Cherbourg roadstead

## 1 INTRODUCTION

Sediment is made up of a more or less complex mixture of particles, each of which is the result of a source and erosion, transport and deposition processes (Flemming, 1988). Grain size analysis is widely used to study sediment environments. The spatial variability of granulometric parameters (mean, sorting, skewness, kurtosis) can be used to estimate transport directions (STA®: Sediment Trend Analysis; McLaren, 1981; McLaren and Bowles, 1985) and GSTA (Gao and Collins, 1992). The "End-Members" (EMs) approach was developed by Weltje (1997) for this purpose. This model can determine the number and granulometric spectra of different populations in a granulometric data set of a study area. The analysis of the spatial organization of each EMs and their mixing percentage allows identifying the characteristics of sedimentary environments. The R package 0.9.6 of EMMAGEo (Dietze and Dietze, 2019), facilitates the application of this approach in the decomposition of a multimodal granulometric distribution into its different grain populations. The "End-Members" method is increasingly used and compared jointly with the STA®/GSTA method (Liu et al., 2012; Li and Li, 2018; Yu et al., 2019; Yuan et al., 2021; Chen et al., 2022; Paladino et al., 2022; Dong et al., 2023; Meng et al., 2023). For these authors, modeling of granulometric EMs used in addition to STA®/GSTA analysis allows a better understanding of sediment transport patterns and associated mechanisms, thus facilitating environmental interpretation. The approach proposed here is based on the integration of these two methods (EMs and STA®/GSTA) by replacing historical statistical parameters with EM characteristics. It is applied to the Cherbourg harbor (Fig. 1), a site whose complexity is reflected in the heterogeneity of the surface grain-size patterns, the currents and the anthropogenic morphology (Fig. Erreur! Source du renvoi introuvable.) (Gregoire et al., 2019). The objective is to highlight the dynamics of the establishment of the relative concentrations of EMs recognized in the Cherbourg harbor and, consequently, of the related sedimentary processes.

## 2 MATERIALS AND METHODS

Sediment samples were taken using a Shipek grab and stored still wet at 5°C. The grain-size dataset has been obtained using a Horiba laser particle size analyzer with a measurement range from 0.01 µm to 2000 µm (logarithmically spaced). We used granulometric data (87 classes) in an end-member analysis (Weltje 1997) performed with the EMMAgeo package (Dietze and Dietze, 2019) of the R software. We obtained vector fields showing sediment dynamics based on End-Members through a Multi-proxy Sediment Trend Analysis (MSTA). MSTA is an internal numeric tool developed in Python under QGIS software based on a GSTA like approach as developed by Poizot et al. (2006) and Poizot and Méar (2010). Residual current vector fields were obtained through the MARS 2D (Model for Application at Regional Scale in 2D) numerical model. This model simulates the horizontal movement of oceanic water masses to study and predict the dynamics of coastal and estuarine waters (Lazure and Dumas, 2008).

## 3 RESULTS AND DISCUSSION

The End-Members (EM) analysis of the 199 surface sediment samples collected in 2018 in the Cherbourg roadstead (Fig. Erreur! Source du renvoi introuvable.) recognized 4 typical grain-size distribution patterns, identified as EM1, EM2, EM3 and EM4 (Fig. 2). The 4 EMs explain about 90% of the total variance,

<footnote n="1">¹Conservatoire National des Arts et Métiers, INTECHMER, 50100 Cherbourg, France.</footnote>

<footnote n="2">²Normandie Univ., UNICAEN, Laboratoire des Sciences Appliquées de Cherbourg, EA 4253, 50100 Cherbourg, France.</footnote>
with respectively 39.3% for EM1, 5.6% for EM2, 37.8% EM3 and 17.3% for EM4. The figure 3 is composed
of the spatial distribution maps of the normalized relative concentrations of these 4 EMs.

<figure><img src="image_75.png" /><figcaption>Figure 1: The Cherbourg roadstead.</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>Figure 2. Sediment sample location (2018) and bathymetry of Cherbourg roadstead</figcaption></figure>

The particle size curve of EM3 is characterized by a single mode at 200 µm (Fig. 2). Spatially, EM3 is present in significant concentrations, where the other EMs are not. In other words, EM3 corresponds to the sedimentary bottom in the study area.

EM4 is characteristic of the coarsest sediments, with two almost identical modes at 287 µm and 1 mm (Fig. 2). It is present only in the axis of the West Pass, the area of the strongest hydro-dynamics in Cherbourg roadstead, under the joint action of tidal currents and swells. All grain sizes lower than EM4 are leached in this area.

The main mode of EM2 is at 134 µm (Fig. 2), with two other minor modes at 1 mm and 12 µm. In the Cherbourg roadstead, EM3 is mainly located east of the central dike, gradational between the high
percentages of EM1 to the west and those of EM3 to the east. EM2 represents a transitional sedimentary
cover between two other well-marked ones.

The particle size curve of EM1, whose main mode is at 12 µm (Fig. 2), nevertheless shows a significant asymmetry with 3 other weaker modes in relation to the main modes of the other EMs. This feature indicates that EM1 overlaps the other 3 EMs. This is reinforced with regard to the spatial distribution map, which shows that the concentrations of EM1 (> 30%) cover a large part of the sampled area.

<figure><img src="image_77.png" /><figcaption>Figure 2: The four EMs identified in the sedimentological dataset of the 2018 campaign.</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>Figure 3: Relative percentage of the four EMs in the Cherbourg roadstead.</figcaption></figure>
To highlight the sedimentary dynamics in the Cherbourg roadstead, the MSTA approach is implemented. Taking the example of EM1, MSTA should allow to identify the dynamics of the establishment of the highest concentrations of fine sediments. The evolution case studied corresponds to that highlighting the concentration gradients of EM1 (noted EM1+). Figure 4 shows the vector field of the EM1+ evolution case.

The fine sediments of the Cherbourg harbor come mainly from the outside, passing through the West and East passes. To the west, these sediments are trapped in the Sainte-Anne cove (Fig. 4, zone A). At this location, the resulting currents (Fig. 4, bottom left) form a dextral gyre that participates in the entry of sediment into the cove. The very weak currents inside the cove, favor the sedimentation of the finest particles (EM1). To the east of the roadstead (Fig. 4, zone C), the weak resulting currents allow their sedimentation. The convergence of the EM1+ vectors towards a point close to the new EMR embankments ("Flamands jetty", Fig. 1) here highlights a concentration of fine sediments under the action of a bathymetric control linked to the presence of a dredging trench at the center of the roadstead (Fig. **Érreur! Source du renvoi introuvable.**).

Zone B of the figure 4 contains a vector field that converges, in its northern part, towards the center and in its southern part, towards the south (Ténarde buoy, Fig. 1). This organization is the result of two superimposed mechanisms. First, the resulting currents forms two gyres to the east (sinistral) and west (dextral) of this zone B. At the height of the dividing line between these gyres, the currents are weaker and thus allow the fall of fine particles. This has favored the installation of a population of benthic filter organisms *Crepidula fornicata* in high density. As the population has grown (mainly towards the south of zone B), these organisms have promoted, on the one hand, with their feces, the sedimentation and fixation of fine sediments and on the other hand, by their presence, the increase of the seabed apparent roughness and thus further reduced the current speed close to the seabed.

The fine sediments from the main Cherbourg roadstead enter the small one via the pass that makes them communicate (Fig. 4, zone D). The marine influence of this contribution extends to the marina entrance. Thanks to a bathymetric step that goes from -11 m to -3 m at the entrance to the port, the external marine fine sediments are stopped. This configuration leaves the possibility for fine continental sediments, contributions of the Divette (a small coastal river that flows into the port of Cherbourg), to be able to be deposited up to the edge of this step.

<figure><img src="image_79.png" /><figcaption>Figure 4: Vector field corresponding to the concentration gradient of EM1 (EM1+). Bottom left corner: the resultant speed and direction vector field of currents in the Cherbourg roadstead computed by MARS2D.</figcaption></figure>
## 4 CONCLUSION

In this study, we propose a new method called MSTA which integrate various environmental parameters into a GSTA-like approach. As an example, we use results from an End-Members analysis to highlight fine sediment dynamics of the Cherbourg roadstead. We show that fine sediment fraction is mainly of marine origin. Continental sediment deposit is restricted to the inner part of the marina. The main accumulation areas have a dual origin. Anthropogenic action is linked to the construction of dykes and the digging of dredging pits. In both cases, these developments modify the circulation of water masses, facilitating the deposition of fine sediments transported in suspension. The setting up of invasive species of benthic filter-feeders has been facilitated by the existence in the center of the roadstead of an area of weak currents, allowing the sedimentation of fine sediments. Since then, this population has maintained this silted-up area. The results of this study demonstrate that in complex coastal areas, analyzing sediment dynamics based solely on grain-size analysis might provide a partial reconstruction. The MSTA approach allows integrating other environmental proxies that are more specific to the study area.

## 5 ACKNOWLEDGMENT

The sediment samples were obtained during the TPINT-2018-01 cruise. The authors would like to thank the "Flotte Océanographique Française" and the vessel crew of the "Côtes de la Manche". This paper has been reviewed by François Bourrin and Sandric Lesourd.

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Gao, S., Collins, M., 1992. Net sediment transport patterns inferred from grain-size trends, based upon definition of "transport vectors". Sedimentary Geology, 80, 47-60.

Grégoire, G., Méar, Y., Poizot, E., Marion, C., Murat, A., Hebert, B., 2019. The morpho-sedimentology of an artificial roadstead (Cherbourg, France). Journal of Maps, 15, 677-685.

Lazure, P., Dumas, F., 2008. An external–internal mode coupling for a 3D hydrodynamical model for applications at regional scale (MARS). Advances in Water Resources, 31, 233-250.

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Poizot, E., Méar, Y., Thomas, M., Garnaud, S., 2006. The application of geostatistics in defining the characteristic distance for grain size trend analysis. Computers & Geosciences, 32, 360 - 370.

Weltje, G.J., 1997. End-Member Modeling of Compositional Data: Numerical-Statistical Algorithms for Solving the Explicit Mixing Problem. Mathematical Geology, 29, 503-549.

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INFLUENCE OF THE REPRODUCTIVE CYCLE AND SEX ON MICROPLASTIC TOXICITY IN MUSSELS

Marine Uguen<fnref n="1" />, Sylvie Marylène Gaudron<fnref n="1" />,<fnref n="2" />, Lorenzo Cozzolino<fnref n="3" /> and Laurent Seuront<fnref n="1" />,<fnref n="4" />,<fnref n="5" />

Corresponding author: marine.uguen@univ-lille.fr

**Abstract:** Plastic contamination has become a major scientific and societal concern. While research on this issue has been flourishing over the last decade, several aspects remain insufficiently explored, including the chemical impact of plastic leachates (i.e., a complex mix of chemical compounds released by plastic particles). Furthermore, essential biological factors, such as the reproductive cycle and sex, are still largely overlooked, particularly in marine invertebrates, further limiting our ability to fully assess the ecotoxicological risks posed by pollutants. Here, we investigate the influence of these biological factors on organismal sensitivity to microplastic leachates (MPL) using two mussel species, *Mytilus edulis* and *Mytilus galloprovincialis*, which are important ecosystem engineers with significant economic values. The role of reproductive cycle was assessed in *M. edulis* through aggregation assays, while sex-based differences in sensitivity were assessed in *M. galloprovincialis* through byssal thread production assays. *M. edulis* showed increased sensitivity to MPL post-spawning, through a delay in their time to form their first aggregation, while female *M. galloprovincialis* showed higher sensitivity to MPL through increased byssal thread production. The observed effects may result from the high energetic cost of reproduction, coupled with potential endocrine-disrupting effects of plastic additives, highlighting the need for further studies. Our work stresses the importance of incorporating biological factors, such as the reproductive cycle and sex, into pollution risk assessments to enhance their ecological relevance.

**Keywords:** contaminant; vulnerability; bivalve; gametogenesis; gender

# 1 INTRODUCTION

Plastic pollution is a pervasive threat, affecting marine ecosystem globally, from industrialized coastlines to the most remote and pristine regions, spanning from the Arctic to the Antarctic, and from the ocean surface to its deepest trenches (Chiba et al., 2018; Sebille et al., 2020). An estimated 19 to 23 million tons of plastic waste enter aquatic systems annually, making plastics the most common form of marine litter (ca. 80 – 85%; Auta et al., 2017; Borrelle et al., 2020). Among the various forms of plastics found in the marine environment, microplastics—particles smaller than 5 mm—are by far the most abundant (Eriksen et al., 2014) and have been linked to numerous detrimental effects on marine life in experimental studies.

The effects of microplastics on marine organisms have been extensively documented; see Gall and Thompson (2015), Auta et al. (2017) and Delaeter et al. (2022) for reviews. Due to their small size, microplastics are ingested by a wide variety of marine species, from invertebrates at the base of the food web to apex predators (Wright et al., 2013; Setälä et al., 2014). Such ingestion can result in numerous physical damages, such as gastrointestinal obstruction and injuries, alteration of physiological processes, all of which can act on the overall fitness of affected organisms (Franzellitti et al., 2019; Wright et al., 2013). In addition to these well-documented physical impacts, microplastics can also induce chemical effects, that remain comparatively understudied (Delaeter et al., 2022; Seuront et al., 2022). Indeed, plastic particles are associated with a diverse array of potentially toxic chemicals, either incorporated during their manufacture (e.g., functional additives) or accumulated from their surrounding environment (e.g., pesticides, heavy metals; Verla et al., 2019; Wiesinger et al., 2021; Lynch et al., 2022). Once released into the marine environment or directly into organisms following ingestion, these chemicals, referred to as plastic leachates, can induce diverse impacts across biological levels, from subcellular to behavioral alterations (Delaeter et al., 2022).

<footnote n="1">¹ Université de Lille, CNRS, Université du Littoral Côte d'Opale, IRD, UMR 8187, LOG, Laboratoire d'Océanologie et de Géosciences, Station marine de Wimereux, F-59000 Lille, France</footnote>

<footnote n="2">² Sorbonne Université, UFR 927, F-75005 Paris, France</footnote>

<footnote n="3">³ CCMAR-Centro de Ciencias do Mar, CIMAR Laboratório Associado, Universidade do Algarve, Campus de Gambelas, Faro 8005-139, Portugal</footnote>

<footnote n="4">⁴ Department of Zoology and Entomology, Rhodes University, Grahamstown, 6140 South Africa</footnote>

<footnote n="5">⁵ Department of Marine Resources and Energy, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477 Japan</footnote>
While research on microplastic toxicity has become one of the most prolific scientific fields, the role of biological factors, such as sex and the reproductive cycle, in modulating an organism sensitivity to these pollutants remains underexplored. This gap is particularly pronounced in marine invertebrates, despite their ecological importance (Burger, 2007). Importantly, the sex and the reproductive cycle, can influence the bioavailability, fate and effects of contaminants in organisms (Burger, 2007; Langston, 2020; McClellan-Green et al., 2007). Reproduction is an energetically demanding process—especially for females (Hayward and Gillooly, 2011)— and may heighten vulnerability to contaminants depending on individual's sex and reproductive stage. In addition, several plastic additives, such as Bisphenol A and phthalates, are acknowledged to act as endocrine disruptors (Burgos-Aceves et al., 2021; Liu et al., 2021; Oehlmann et al., 2009), interfering with hormonal systems and causing sex- and reproductive stage- specific effects (Burger, 2007; Langston, 2020; McClellan-Green et al., 2007). Such interaction adds complexity to the toxicity of plastic pollution and underscore the necessity of studying how biological factors interact with these contaminants to affect marine species, especially marine invertebrates.

Mussels are keystone species in marine ecosystem that hold significant ecological and economic functions (Avdelas et al., 2021; Borthagaray and Carranza, 2007; Buschbaum et al., 2009). Their high filtration capacity makes them effective sentinel species for monitoring environmental pollution while also making them more susceptible to plastic contamination (Beyer et al., 2017; Uguen et al., 2025). Additionally, as broadcast spawners and gonochorics with a seasonal reproduction (Gosling, 2015), mussels provide an ideal model for investigating how biological factors, such as sex and the reproductive stage, influence their sensitivity to pollutants. In this study, we focused on two mussel species, the blue mussel (*Mytilus edulis*) and the Mediterranean mussel (*Mytilus galloprovincialis*). Specifically, we aimed to examine whether the reproductive cycle and mussel sex affect the sensitivity to microplastic leachates in *M. edulis* and *M. galloprovincialis*, respectively. We hypothesize that sensitivity to these contaminants will increase during the post-spawning period and will be higher in females, reflecting the important energetic cost of reproduction.

## 2 MATERIALS AND METHODS

### 2.1 Impact of the reproductive cycle on mussel sensitivity to microplastic leachates

To study the impact of the reproductive cycle on mussel sensitivity to plastic leachates, adult blue mussels (*Mytilus edulis*; shell length: 2.5 – 3.5 cm; *N* = 180) were collected monthly from March to December 2021 (with the exception of October and November 2021) from a rocky intertidal reef located along the French shores of the eastern English Channel (Pointe aux Oies, Wimereux, France; 50°47′08.3″N, 1°36′03.9″E). Mussels were acclimated for 24 hours in 85 L tanks filled with running aerated natural seawater representative of *in situ* conditions.

Behavioral experiments were run in 22 cm glass arenas. Mussels (*N* = 15) were placed in the arena, their narrow end facing the center of the arena in two concentric circles, at ca. 1 body length from each other. The arenas were filled with 1.5 L of control seawater (*n* = 6) or microplastic leachate seawater (MPL; *n* = 6). MPL was prepared by incubating commercially available polypropylene pellets (3.80 ± 0.21 mm; Pemmiproducts, Aachen, Germany) in natural aerated seawater (12°C; 24 h) at an experimental concentration of ca 400 pellets per liter (equivalent to 12 g or 20 mL of pellets per liter), a concentration used in previous microplastic leachate studies known to induce effects in molluscs (Seuront, 2018; Uguen et al., 2023). After the incubation, pellets were removed from the solution using a wire mesh. The additive content of polypropylene pellets was analyzed in a previous study (Uguen et al., 2023) that identified a total of 27 additives, including three brominated flame retardants, six phosphorus flame retardants, five antioxidants, and 13 plasticizers.

Mussel positions were recorded every minute for 2 h using GoPro cameras (GoPro HERO7 Black, GoPro Inc., San Mateo, California, USA). At each 1 min interval, mussels were visually classified as either solitary or aggregated, i.e. two or more mussels with their shells in direct physical contact (Seuront et al., 2021; Uguen et al., 2023). The starting time (min) defined as the time needed to form the first aggregation within an arena was then quantified (Uguen et al., 2023; Zardi et al., 2024a).

Wilcoxon-Mann-Whitney tests with continuity correction (hereafter referred to as WMW test) were performed for each month of study to assess the impact of MPL on mussel starting time behavior.

### 2.2 Impact of sex on mussel sensitivity to microplastic leachates

To study the impact of sex on the mussel sensitivity to plastic leachates, adult Mediterranean mussels (*Mytilus galloprovincialis*; shell length: 4.0 – 4.6 cm; *N* = 18) were collected in January 2023 from Praia de Faro (Faro, Southern Portugal; 37°0′33.5″N, 7°59′36.3″W). Mussels were acclimated for 24 hours in 50 L tanks filled with running aerated natural seawater representative of *in situ* conditions.

Byssal thread production experiments were run in sealed glass chambers. Mussels were individually placed in chambers filled with 200 mL of control seawater (*n* = 9) or microplastic leachate seawater (MPL; *n* = 9). MPL was prepared by incubating low density polyethylene pellets collected from the shore of Northern
France (Ambleteuse; 50°80'61.9"N, 1°60'31.34"E; 4.04 ± 0.56 mm) in natural aerated seawater (17°C; 24 h) at an experimental concentration of ca 400 pellets per liter (equivalent to 10 g or 20 mL of pellets per liter), a concentration used in previous microplastic leachate studies known to induce effects in mussels (Uguen & Cozzolino, 2024; Uguen et al., 2024). After the incubation, pellets were removed from the solution using a wire mesh. The additive content of beached French pellets was analyzed in a previous study (Uguen and Cozzolino, 2024) that identified a total of 9 additives, including two antioxidants and seven plasticizers.

After a 12-h exposure, the total byssal thread production was visually quantified for each mussel by counting the total number of byssal plaques in the chamber (Seuront et al., 2021; Zardi et al., 2024b). Mussels were then dissected, and each individual was sexed through gamete observation under a microscope (Motic BA210) following a biopsy of the mantle (Fraser et al., 2016). The control group consisted of three females and six males, while the MPL group consisted of four females and five males.

Wilcoxon-Mann-Whitney tests with continuity correction were performed for each sex to assess the impact of MPL on mussel byssal thread production.

# 3 RESULTS AND DISCUSSION

## 3.1 The reproductive cycle affects mussel sensitivity to microplastic leachates

*Mytilus edulis* starting time was significantly impacted by MPL in September, with a longer time needed by mussels to form the first aggregation (2.2 times longer compared to control; WMW test; p < 0.05; Figure 1). In contrast, no significant effect of MPL was found for any of the other months studied (from March to August and in December; WMW test; p > 0.05; Figure 1). At the study site, the reproductive cycle of *M. edulis* is characterized by a spawning period occurring from May to August, September marking the end of the spawning season with the start of a new reproductive cycle (Uguen, unpublished data). These observations imply that *M. edulis* showed an increased sensitivity to MPL during the post-spawning period.

<figure><img src="image_80.png" /><figcaption>**Figure 1.** Starting time in minutes (min) taken by *Mytilus edulis* to form the first aggregation in control seawater (blue) and in microplastic leachates (red) from commercially available polypropylene pellets for each studied month. Asterisks depict significant difference (p < 0.05; Wilcoxon-Mann-Whitney test with continuity correction) among treatments.</figcaption></figure>

The increased mussel sensitivity during the post-spawning period (i.e. September) likely reflects physiological stress associated with reproduction, a process acknowledged to be energetically costly in mussels (Seed and Suchanek, 1992; Mredul et al., 2024; Osterheld et al., 2024). Spawning can significantly deplete mussel energy reserves, leading to a marked reduction in their condition index during the post-spawning period (Seed and Suchanek, 1992; Mredul et al., 2024), with individuals losing up to 40% of their soft tissue weight (Cossa, 1989). Mussels need time to regenerate their energy reserves following spawning (Lubet, 1959; Myrand et al., 2000; Mredul et al., 2024), while repairing and reorganizing their tissues (Tremblay et al., 1998). Thus, spawning induces a considerable energetic stress, which can make them more vulnerable to other stressors, such as pollutants.
Our results are consistent with previous studies that showed an increase in susceptibility to pollutant and other environmental stressors in post-spawning mussels (e.g. Myrand et al., 2000; Lupo et al., 2021; Laubscher et al., 2023) further corroborating that the reproductive cycle significantly modulates stress responses. Notably, recently spawned mussels exhibit heightened sensitivity to microplastic exposure, as evidenced by a more pronounced reduction in clearance rate compared to their pre-spawn counterparts (Laubscher et al., 2023). The post-spawning stage may increase the mussel mortality risk, especially when subjected to additional stressors (e.g. heat stress, pathogens). This is consistent with field observations of mass mortality in mussels following the spawning period (Charles et al., 2020; Myrand et al., 2000; Ajithkumar et al., 2024). Thus, as the mussel sensitivity to pollutant is not constant throughout the year, mussels may show a "window of sensitivity", i.e. a specific period when the sensitivity of species to contaminants is amplified (Winner et al., 1990; Dissanayake et al., 2011; Finch and Stubblefield, 2016), here at the post-spawning period, highlighting the critical need to take into account the reproductive cycles in pollution assessments and other stressors in general.

## 3.2 Mussel sex affects mussel sensitivity to microplastic leachates

Byssal thread production in female *Mytilus galloprovincialis* was significantly affected by MPL exposure, with females producing ca. 2.0 times more threads when exposed to MPL compared to control (WMW test; p < 0.05; Figure 2). In contrast, no significant effect of MPL was observed in male *M. galloprovincialis* byssal thread production (WMW test; p > 0.05; Figure 2). Thus, *M. galloprovincialis* showed a sex-specific difference in their sensitivity to MPL with an increased sensitivity in females.

<figure><img src="image_81.png" /><figcaption>**Figure 2.** *Mytilus galloprovincialis* byssal thread production in control seawater (blue) and in MPL (red) from beached French pellets for each sex. Asterisks depict significant difference (p < 0.05; Wilcoxon-Mann-Whitney test with continuity correction) among treatments.</figcaption></figure>

The higher sensitivity to MPL of female compared to male mussels can be attributed to distinct energy investment. Female allocate a substantial part of their energy reserves to reproduction compared to male (Hayward and Gillooly, 2011), particularly during gametogenesis, as they produced numerous energy-rich oocytes (Bayne, 1976; Sprung, 1983; Petes et al., 2007). In contrast to males, the high reproductive energy investment in female likely intensifies their vulnerability to environmental stressors, such as MPL exposure. However, the stress response observed here in females is an increase in the byssal thread production, which is energy costly (Roberts et al., 2021). This increase in *M. galloprovincialis* byssal thread production during MPL exposure is consistent with previous observations (Seuront et al., 2021), suggesting that this species use byssal production as a resistance strategy to cope with stressors. By allocating their remaining energy to this behavior, they may prioritize maintaining attachment in stressful environments. Although high byssal thread production could enhance short-term survival under certain stress, e.g. wave disturbance (Carrington, 2002; Carrington et al., 2009) and predation pressure (Côté, 1995; Cheung et al., 2006; Caro et al., 2008), it might also deplete energy reserves, especially in females during gametogenesis, potentially impacting population dynamics and mussel ecological roles.

This sex-dependent effect may also be due to the endocrine-disrupting properties of plastic leachates. Numerous plastic additives have been established to be endocrine-disrupting chemicals (EDCs) due to their ability to interfere with the endocrine system, leading to sex-specific effects (see Oehlmann et al., 2009; Burgos-Aceves et al., 2021 and Liu et al., 2021 for reviews). For example, certain plastic additives can
stimulate gametogenesis and induce early spawning in female *M. galloprovincialis* (Wang et al., 2023). As byssal thread production in mussels follows a seasonal cycle, increasing during gametogenesis and declining during the post-spawning period (Carrington, 2002), MPL exposure could potentially enhance female byssal thread production indirectly by promoting gametogenesis.

## 4 CONCLUSION

This study highlights the significant role of biological factors, here the reproductive cycles and sex, in shaping mussel sensitivity to microplastic leachates. Our findings show that *M. edulis* exhibited an increased sensitivity to MPL during the post-spawning period, while female *M. galloprovincialis* were more sensitive than males. These results stress the need to incorporate biological factors into pollution risk assessments, as they provide a more nuanced understanding of how pollutants, especially microplastics, affect marine organisms. Additionally, this study emphasizes the importance of investigating the bioenergetic and endocrine-disrupting effects of plastic associated chemicals, that could explain the observed responses. As plastic pollution continues to rise, integrating biological factors into pollution studies is essential for developing targeted conservation strategies. This is particularly true for marine invertebrates and engineer species like mussels, where such factors are often overlooked despite their critical roles in supporting ecosystem stability. It is finally stressed that the impact of microplastic leachates on mussel reproductive cycle and sex has been assessed independently using two distinct polymers (i.e. polypropylene and polyethylene, respectively). This warrants the need for further work to decipher the role of distinct polymers, notably the emerging bio-sourced and/or biodegradable alternatives to conventional polymer such as polypropylene and polyethylene considered in the present work.

## 5 ACKNOWLEDGMENT

We thank J. Anquetin, A. Lutrand, A. Rahoui and V. Lefebvre for their technical support. This study was funded by the European Maritime and Fisheries Fund (FEAMP), the France Filière Pêche through the research project SOLACE («Resilience of the mussel *Mytilus edulis* to anthropogenic and climatic stressors»), the Fundação para a Ciência e a Tecnologia (FCT, MEC, Portugal, grant number: UIDB/04326/2022, PhD grant with reference 2020.06671.BD), the Pierre Hubert Curien PESSOA grant 2022-2023 and a PhD fellowship from the University of Lille and the Region-Hauts-de-France. This work was also funded by the Artois-Picardie Water Agency through the research project INFORMER ("Development of indices of ecological status of intertidal rocky ecosystems in response to climatic and anthropogenic forcings"). The manuscript was reviewed by Kévin Tallec.

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19th French Conference of Sedimentology

SHORT PAPERS

ISSN 0990-3925 ISBN

2-907205-83-8

DOI 10.70665/IPJT8001