# Influence of cockle bioturbation on microphytobenthic primary producers: habitat and density-dependent effects

Anais Richard, Francis Orvain, Annabelle Dairain, Jérôme Morelle, Alicia Roméro-Ramirez, Tiffany Rodolfo Damiano, Xavier de Montaudouin, Olivier Maire

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Anais Richard, Francis Orvain, Annabelle Dairain, Jérôme Morelle, Alicia Roméro-Ramirez, et al. Influence of cockle bioturbation on microphytobenthic primary producers: habitat and density-dependent effects. Marine Biology, 2025, 172 (5), pp.70. 10.1007/s00227-025-04629-0. hal-05026490

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# Influence of cockle bioturbation on microphytobenthic primary producers: habitat and density-dependent effects

Anaïs Richard<fnref n="1" />,<fnref n="2" />✉ · Francis Orvain<fnref n="3" /> · Annabelle Dairain<fnref n="3" />,<fnref n="4" /> · Jérôme Morelle<fnref n="3" />,<fnref n="5" /> · Alicia Romero-Ramirez<fnref n="1" /> ·
Tiffany Rodolfo-Damiano<fnref n="1" /> · Xavier de Montaudouin<fnref n="1" /> · Olivier Maire<fnref n="1" />

Received: 31 October 2024 / Accepted: 12 March 2025

© The Author(s) 2025

## Abstract

While the global impact of the edible cockle *Cerastoderma edule* on microphytobenthos (MPB) dynamics has been extensively studied, some underlying processes remain unknown, such as their non-trophic interactions. For this purpose, a laboratory mesocosm experimental approach was used to quantify the bioturbation generated by *C. edule* adults and their influence on oxygen and nutrient fluxes, MPB biomass and photosynthetic performance at the sediment-water interface. The effects of sediment properties and cockle densities (0, 288, 720 and 1,297 ind. m⁻<fnref n="2" />) were also investigated. Our results showed that cockles exert a net negative density-dependent effect on MPB biomass, with intensity varying according to sediment type. This is mainly due to sediment reworking by cockles and their emergence at the sediment surface, mechanically disrupting MPB biofilms. Nonetheless, the physiological status and photosynthetic performances of the remaining MPB cells were not impacted. This could be explained by cockle bioirrigation which stimulates nutrient fluxes at the sediment surface and cell turnover. In the finest sediment, the stimulation of MPB growth by nutrient fluxes did not compensate algal biomass loss due to burial, leading to a net decrease in MPB biomass. In coarser sediment, no significant impact of cockles on the net total MPB biomass was observed. This indicates a balance between suboptimal sediment condition for MPB, biofilm destruction by cockles, and enhanced bioirrigation rates that increase nutrient availability for MPB biofilms at the sediment-water interface.

**Keywords** Common cockle *Cerastoderma edule* · Bioirrigation · Sediment reworking · Biogeochemical fluxes · Microphytobenthos · Non-trophic interactions

Communicated by M. Huettel.

## Introduction

Intertidal mudflats are among the most productive ecosystems in temperate regions (Heip et al. 1995; Barbier et al. 2011), characterised by a high biomass of benthic macrofauna (Herman et al. 1999; Rodil and Lastra 2022). Along the East Atlantic coastlines, these communities are often dominated by the common cockle *Cerastoderma edule*, a bivalve species inhabiting the upper 5 cm of various sediment types, with adult densities reaching up to 2,000 ind. m⁻<fnref n="2" /> (Tyler-Walters 2007). This ecosystem engineer species (Jones et al. 1994) plays a key role in ecosystem functioning supporting a wide array of ecosystem services. Through seawater filtration, cockles exert top-down control on phytoplankton biomass, regulate water turbidity, and sustain biodiversity by supporting complex food webs (Carss et al. 2020). Cockles also enhance nutrient fluxes at the sediment-water interface by rejecting faeces, pseudofaeces, and

<footnote>✉ Anaïs Richard
anais.richard@biology.sdu.dk</footnote>

<footnote>✉ Olivier Maire
olivier.maire@u-bordeaux.fr</footnote>

<footnote n="1">¹ Univ. Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, Pessac F-33600, France</footnote>

<footnote n="2">² Department of Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark</footnote>

<footnote n="3">³ Laboratoire MERSEA UR 7482, Université de Caen
Normandie, Centre de Recherches en Environnement Côtier,
Luc-sur-Mer 14530, France</footnote>

<footnote n="4">⁴ Sorbonne Université, CNRS, Adaptation et Diversité en
Milieu Marin, Roscoff AD2M, F-29680, France</footnote>

<footnote n="5">⁵ CESAM-Centre for Environmental and Marine Studies,
Department of Biology, University of Aveiro, Campus de
Santiago, 3810-193 Aveiro, Aveiro, Portugal</footnote>
metabolites (Swanberg 1991), and indirectly through their bioturbation activity (Jones et al. 2011; Donadi et al. 2013). This activity involves vertical and horizontal movements (Richardson et al. 1993) that induce sediment reworking and bioirrigation, contributing to particle and solute transport (Kristensen et al. 2012). While the potential of individual bioturbation is moderate (Mermillod-Blondin et al. 2004; Richard et al. 2021), dense cockle populations drive intense sediment reworking (Sandwell et al. 2009; Donadi et al. 2013), enhancing nutrient fluxes (Eriksson et al. 2017) and increasing the roughness and erodibility of the superficial sediment layer (Dairain et al. 2020). These processes facilitate sediment resuspension, disrupting and exporting benthic primary producers, commonly referred to as microphytobenthos (MPB).

MPB consists in a complex assemblage of photosynthetic microalgae, cyanobacteria, and flagellates forming biofilms at the sediment-water interface, colonizing extensive zones of intertidal mudflats (MacIntyre et al. 1996). MPB plays a major role in shallow soft-bottom environments (Hope et al. 2020), contributing over 50% of total primary production (Underwood and Kromkamp 1999) and supporting energy transfer to higher trophic levels, including benthic macrofauna (Daggers et al. 2020). MPB biofilms also stabilize sediment surfaces by secreting a mucilage matrices (Stal 2010). The temporal and spatial MPB dynamics are influenced by abiotic factors such as surface temperature (Blanchard et al. 1996), light availability (MacIntyre et al. 2002), sediment grain size (Ubertini et al. 2015), and nutrient levels (Pascal et al. 2022). Biotic factors, including trophic (e.g., grazing, predation, and filtration) and non-trophic interactions (e.g., spatial competition, facilitation, and bioturbation which can influence these interactions) with macrofaunal communities, also significantly impact MPB (Savelli et al. 2018; Giorgini et al. 2019). Grazing activity by deposit- and suspension feeders is a key factor limiting MPB biomass (Miller et al. 1996; Savelli et al. 2018; Richard et al. 2023). For instance, the total grazing pressure exerted by macrofauna communities dominated by the amphipod *Bathyporeia* sp., the bivalve *Macoma balthica*, or the gastropod *Peringia ulvae*, is 1.3 to 10 times higher than the production of MPB, thus drastically limiting their biomass (Daggers et al. 2020). Regarding non-trophic interactions, including biogenic sediment mixing, their effects on MPB dynamics remain poorly understood. The few studies with cockles (*C. edule* or *Austrovenus stutchburyi*) have suggested both positive and negative effects on MPB biomass and photosynthetic performance due to their direct and indirect modifications of the physical and chemical properties of sediments (Richard et al. 2023; Morelle et al. 2024). Biodeposits and excretory products from cockles can stimulate MPB growth by increasing sediment surface stability

(Andersen et al. 2010; Donadi et al. 2013) and nutrients availability (Swanberg 1991; Sandwell et al. 2009; Thomas et al. 2021). Conversely, sediment reworking by dense cockle populations reduces MPB biomass, with erosion flux depending on cockle density (Rakotomalala et al. 2015). Despite these findings, evidence of the net effects of cockle activity on MPB dynamics remains limited, warranting simultaneous quantification of bioturbation rates, nutrient fluxes at the sediment-water interface, and MPB biomass and photosynthetic performance.

This study aims to characterize the intricate interactions between cockles and MPB by simultaneously measuring (1) bioturbation processes (both sediment reworking and bioirrigation rates of cockles), (2) oxygen and nutrient fluxes at the sediment-water interface, and (3) MPB biomass and photosynthetic performance. Given the variability in cockle density and sediment type, these factors were also considered as potential modulators of their impact on particle and solute transport and MPB dynamics. In general, the global net effect of cockles on MPB remains challenging to predict due to complex interactions between particle transport, which disrupt the sediment-water interface and MPB biofilms, and bioirrigation activity, which affects both oxygen penetration and nutrient availability.

# Materials and methods

## Field sampling

Samples collection took place on two intertidal flats, at Banc d'Arguin (Arcachon Bay, 44°35'N, 1°14'W), and Baie des Veys (49°21'N, 1°08'W), in France. Two types of sediments naturally colonised by microphytobenthos biofilms (MPB) and cockles *Cerastoderma edule* (pers. obs.) were collected. The first type of sediment was characterised by medium sand ("MS", $d_{50}=389$ µm, 2% of silt and clay) while the second was characterised by fine sand ("FS", $d_{50}=129$ µm, 24% of silt and clay). After sampling, sediments were separately sieved through a 2 mm mesh to remove any large macrofauna, debris and shells, homogenized and kept under darkness during one day in covered boxes to avoid photosynthetic growth. Adult cockles were collected by hand in sandy mudflats from Arcachon Bay and stored in tanks with running seawater for 6 days. A total of 287 individuals with a shell length ranging between 24 and 26 mm were selected. The biomass was estimated from the allometric relation found by Gam et al. 2009; from the same sampling location (Arcachon Bay) and time period (February): $log_{10}(W)=2.6091 \times log_{10}(L) -4.6987$; with flesh dry weight W in g and shell size L in mm. Estimated biomass of selected cockles ranged from 0.08 to 0.1 g. MPB biofilms
were collected by sampling the top 5-mm of the sediment surface using a spatula. In the laboratory, MPB samples were spread on plastic trays with a 100-µm mesh net on the surface. Over 3 days, an artificial light with a photosynthetic photon flux density (PPFD) of 114 µmol photon m⁻² s⁻¹ was applied for 6 h daily to induce formation of the biofilm onto the net (Orvain et al. 2003). Then, the net was mixed with natural seawater from Arcachon Bay, and a MPB inoculum was obtained after decantation. Inoculum was added to each of the two sieved sediments and kept under the same artificial light until experiment started (up to 48 h).

## Experimental procedure

Fifty experimental units consisting of PVC tubes (height=20 cm, internal diameter=9.3 cm), were filled up to 19 cm height with homogenized non-inoculated sieved sediment. From these units, 25 were filled with the MS sediment and 25 with the FS sediment. Units were then positioned in a tidal mesocosm containing unfiltered seawater

from Arcachon Bay (total volume=500 L, closed system). An artificial tidal cycle (6 h: 6 h) was generated by pumping out (or in) 200 L of seawater toward (or from) a second tank. The water velocity transition between high and low tides was adjusted to prevent sediment resuspension and corresponded to a variation of approximately 20 cm in the water level height. The temperature (12.2±1 °C), and the salinity (30.9±0.4) remained stable during the experiment.

Cockles were added in experimental units 24 h after the sediment. For each sediment type, three treatments with different densities of cockles were studied (Fig. 1): “low density” with 288 ind·m⁻² (2 cockles per unit), “medium density” with 720 ind·m⁻² (5 cockles per unit), and “high density” with 1297 ind·m⁻² (9 cockles per unit). Densities were chosen according to the range of natural abundances commonly reported for this species (Dabouineau and Ponsero 2009; Beukema and Dekker 2015). For each sediment type, each density was repeated in 6 replicates (Fig. 1), and 6 experimental units without cockles were used as control.

<figure><img src="image_2.png" /><figcaption>Fig. 1 Design of the experiment and parameters measured at the end of the 6 days of incubation</figcaption></figure>
An additional unit without cockles for medium and fine sand was used for sediment porosity measurement.

MPB was added in each experimental unit 24 h after the cockle addition by depositing a layer of 1 cm of inoculated sediment at the surface of each unit.

To quantify sediment reworking activity of cockles, fluorescent particle tracers were homogeneously spread (10 g DW of luminophores per unit, with a $d_{50}$ of 160–200 µm when used in MS sediment and a $d_{50}$ of 100–160 µm for luminophores used in FS sediment, with 2.6 g cm$^{-3}$ density for both luminophore types) at the sediment-water interface of 24 experimental units from batch 1, corresponding to three replicates for each sediment and density (Fig. 1). No luminophores were added to the experimental units from batch 2 to not interfere with measurement of MPB chlorophyll fluorescence. To measure bioirrigation induced by cockles, 490 g of sodium bromide (NaBr) were dissolved, at the same time as luminophores, in the 500 L seawater of the tidal mesocosm, with a measured final concentration of 10.5 mM.

Over 6 days, the mesocosm was exposed to a daily 6-h photoperiod (average PPFD of 114 µmol photons m$^{-2}$ s$^{-1}$) using neon tubes (Philips, WT060C LED36S/840) during the diurnal low tide to mimic natural conditions and stimulate MPB growth. Cockles were fed every day with the planktonic microalgae *Isochrysis galbana* (approx. 4.10⁶ cells mL$^{-1}$ corresponding to a Chl *a* concentration of 0.47±0.02 µg L$^{-1}$). At the same time, 87 g of wet muddy sediment ($d_{50}$=22 µm, 71% mud and 29% sand) was added to the water of the tidal mesocosm, to mimic natural conditions within an estuary. It corresponded to a concentration of suspended particulate matter of 0.13±0.06 mg L$^{-1}$. Both Chl *a* water concentration and turbidity value were in the range of those commonly reported in Banc d'Arguin (Liénart et al. 2017) and Baie des Veys (Jouenne et al. 2007).

After 6 days, MPB surface biomass and photosynthetic parameters were measured during the diurnal low tide in experimental units of batch 2 (without luminophores, Fig. 1). Immediately afterwards, experimental units were sealed, and benthic fluxes were measured. After incubations, the first centimetre of sediment was sampled to assess sediment characteristics, chlorophyll *a*, and phaeopigment contents. The 6-day incubation was chosen because it corresponds to the exponential growth phase of MPB (Orvain et al. 2003), while providing enough time to observe the non-trophic effects of cockle bioturbation, yet remaining within the limits to measure the dynamics of sediment reworking and bioirrigation.

## Sediment characteristics

After core slicing, subsample of the first centimetre of all units of batch 2 was taken to measure water content, dry bulk density and total organic carbon and nitrogen. The water content (ω; %) of the top 1st centimetre of the sediment column was estimated based on the weight difference before (wet–weight) and after (dry–weight) freeze-drying, divided by the wet weight of the measured sediment column. The dry bulk density (Csed, kg m$^{-3}$) was then computed according to the following equation:

$$C_{sed} = \frac{(\gamma_s \times 1000)}{\frac{\omega}{100} \times \gamma_s + 1000} \quad (1)$$

where $γ_s$ is the constant sediment grain density of each sediment type ($γ_s$=2 540 and 2 600 kg m$^{-3}$ for the MS and FS sediments, respectively).

Sediment total organic carbon (TOC) and total nitrogen (TN) content were assessed using a CN auto-analyzer (Thermoscientific FlashSmart, U.S.) on freeze-dried homogenized sieved samples of each sediment type. Prior to measurement, TOC samples were decarbonated with 1.2 M HCl (Kennedy et al. 2005).

A vertical profile of porosity ($φ$) was measured after slicing the sediment into two additional experimental units in 0.5 cm thick layers for the top 5 cm, and 1 cm thick layers from 5 to 15 cm depth. The porosity was then calculated in each slice from the loss of water after 2 days at 60 °C, and corrected for sea-salt content and sediment grain density (2.54 g cm$^{-3}$ for MS sediment and 2.60 g cm$^{-3}$ for FS sediment) (Burdige 2006).

## Quantification of bioturbation rates

### Bioirrigation

After six days of incubation, rhizon samplers (length 9 cm, mean pore size 0.15 µm) were used to extract porewater from the experimental units of batch 1. Extraction was performed every centimetre from 0.5 to 9.5 cm depth and every 2 cm from 10.5 to 15.5 cm. Extracted porewater samples were stored at 4 °C. Bromide concentration was assessed in each porewater sample and in the overlying water (before and after NaBr addition) by spectrophotometry (Shimadzu UV–1280) (Lepore and Barak 2009). An enhanced diffusion factor ($ε$) was estimated by fitting depth profiles of bromide concentrations with an enhanced diffusion model described by Eq. (2) (Andersson et al. 2006):

$$\varphi \frac{\partial C}{\partial t} = \frac{\partial}{\partial x} \left( \varepsilon D_s \varphi \frac{\partial C}{\partial x} \right) \quad (2)$$
where C is the bromide concentration in the sediment (mM), t the incubation time (day), x the depth in the sediment (cm), Ds the molecular diffusion coefficient, φ the porosity, and ε a factor of enhanced diffusion (i.e., ε is equal to 1 when solute transport is exclusively driven by molecular diffusion). ε was estimated by convergent iterations and weighted least-squared regressions of model prediction on observed vertical bromide profiles.

## Sediment reworking

After porewater extraction, each unit was vertically sliced into 0.5 cm sections for the top 5 cm, and into 1 cm sections from 5 to 10 cm depth. Each slice was freeze-dried and homogenized. A subsample of ca. 3 g was then spread in a Petri dish and photographed under UV light using a digital camera (Nikon D100, Focal F/7.1, shutter speed 1/25s). After a binarization step (based on the RGB level), lumophore pixels were counted for each picture using the Avi-Explore software (Romero-Ramirez et al. 2016). Sediment reworking rates ($D_b$, cm² year⁻¹) were computed by fitting vertical profiles of luminophores with a one-dimensional biodiffusive model (Maire et al. 2008) according to Eq. (3):

where C is the proportion of luminophore (%), t the time (year), and z the vertical depth in the sediment column (cm). $D_b$ was estimated as a function of convergent iterations and weighted least-squared regressions of model prediction on observed vertical luminophore profiles.

## Microphytobenthos photosynthetic parameters

The chlorophyll a fluorescence was measured on the sediment surface of the experimental units without luminophores (batch 2) during the last low tide using the MAXI version of an Imaging Pulse Amplitude Modulated (PAM) fluorometer (Heinz Walz GmbH, Effeltrich, Germany). Following a dark adaptation time of 5 min, corresponding to a compromise between oxidation of Quinone pool and downward migration of MPB (Perkins et al. 2010), pictures of the different fluorescence values were recorded. First, a low intensity light pulse (1 µmol photons m⁻² s⁻¹, 450 nm) was applied to measure the minimal fluorescence values ($F_0$). Then a saturating light pulse (0.8 s, 6 000 µmol photons m⁻² s⁻¹, 450 nm) was applied to record the maximal fluorescence values ($F_m$). The maximum quantum yield of fluorescence was calculated as $F_\gamma/F_m = (F_m' - F_0)/F_m$. Subsequently, 13 incremental intensities of actinic lights (from 0 to 701 µmol photons m⁻² s⁻¹) (E) were applied with 20-seconds steps.

After each actinic light step, the steady-state fluorescence ($F_s$) and the maximum fluorescence ($F_m'$) were measured. The efficient quantum yield of fluorescence was calculated for each actinic light as $\Delta F/F_m' = (F_m' - F_s)/F_m'$. (Genty et al. 1989).

The numerical values of fluorescence for each pixel of the fluorescence pictures were extracted using the Matlab® routine developed by Morelle et al. (2021). The photosynthetically active Chl a biomass ($F_0$ Chl a; mg Chl a m⁻²) and the maximum relative electron transport rate of PSII (rETRmax, relative unit) were estimated from the fluorescence values. $F_0$ was considered valid above 0.04 corresponding to the detection limit (see device manual). $F_0$ Chl a was estimated from $F_0$ values ($F_0$ Chl a = 10.29 × $F_0$, R²=0.94). The $F_0$ Chl a value in cockle treatments were expressed as percentage based on the $F_0$ Chla value estimated in the control treatment. The rETRmax values were extracted from rETR (ΔF/Fm' x E) vs E curves fitted using the model of Eilers and Peeters (1988).

## Benthic flux measurements

Oxygen ($O_2$), ammonium ($NH_4^+$), oxidized nitrogen forms ($NO_2^-$ + $NO_3^-$, noted $NO_x$ hereafter), dissolved silicate (dSi), and orthophosphate ($H_2PO_4^-$, $HPO_4^{2-}$, and $PO_4^{3-}$, written $PO_4$ hereafter) fluxes were quantified at the sediment-water interface. Experimental units of batch 2 were removed from the tank, hermetically sealed with a PVC lid and then filled with 500 mL of seawater from the tidal mesocosm. Measurements were performed in darkness to prevent oxygen production and nutrient assimilation by MPB. A mini-optode (OXROB10, Pyroscience®) connected to a Firesting Oxygen Meter (Pyroscience®) was introduced in each experimental unit and placed at ca. 5 cm above the benthic interface to continuously record $O_2$ concentration. Linear calibration of the optode was previously achieved between the $O_2$ concentration of the overlaying water (100% saturation) measured by Winkler titration and a sodium ascorbate solution (0% saturation). During the incubation, the overlying water was homogenized by a Teflon-coated magnetic stirring bar (~100 rpm) without sediment resuspension. Incubations were stopped when the $O_2$ concentration reached 75% of its initial value (after 0.6 to 2.6 h of incubation, depending on the treatment). At the beginning and at the end of incubation, a sample of 20 mL of overlying water was collected and filtered over a 0.2 µm cellulose filter to quantify nutrient concentrations. Silicates and phosphates were measured by gas-segmented continuous flow analysers (SFA) for the determination of dissolved nutrients (AA3-HR, SEAL ANALYTICAL). Nitrates, nitrites and ammonium were analysed by QuAAtro (QuAAtro, SEAL ANALYTICAL).
Total oxygen uptake (TOU) and nutrient fluxes (mmol m⁻² day⁻¹) were computed from the concentration decreases in each experimental unit according to the following Eq. (5):

$$Fluxes = \frac{a \times V}{S} \qquad (4)$$

where a is the slope (mmol m⁻³ day⁻¹) of O₂ or nutrient concentrations as a function of time (day), V the volume of overlying water (m³), and S the surface area of the sediment-water interface (m²).

## Chlorophyll a and phaeopigment

Chlorophyll a (Chl a) and phaeopigment were quantified from the top 1st centimetre of the units from batch 2, following the method of Lorenzen (1967). After being freeze-dried, 1 g of sediment was weighted and 10 mL of 90% acetone were added for photopigments extraction. Extraction was performed at 4 °C under continuous mixing for 18 h in the dark. After centrifugation (3000 rpm, 5 min, 4 °C), the fluorescence of the supernatant was measured, before and after acidification (10 µL of HCl, 0.3 M for 1 mL of acetone), using a Turner Designs 10-AU fluorometer. The content of Chl a and phaeopigments (µg gDW⁻¹) was converted in mg m⁻² considering the dry bulk density (Csed, kg m⁻³), to eliminate the effect of particle size, and consider sediment compaction process due to dewatering (Jesus et al. 2006). Phaeopigments were then expressed as a percentage of total photopigments to better indicate the loss of fresh Chl a (Cartaxana et al. 2003) (6):

$$Phaeopigments \ percentage = \left[ \frac{Pheopigment}{(Chl a + Pheopigment)} \right] * 100 \quad (5)$$

## Data analysis

Results were reported as the mean ± standard error (SE) of n replicate measurements, except for the sediment characteristics (mean ± SD). The effects of sediment type and cockle density on (1) bioirrigation and sediment reworking, (2) oxygen and nutrient fluxes, and (3) surficial and total biomass and photosynthetic parameters of MPB, were assessed using a two-way PERmutational Multivariate ANalyses Of VAriances (PERMANOVAs) without data transformation (Anderson 2001). The PERMANOVA design consisted of two fixed factors: the density of cockles (null, low, medium, and high) and the sediment type (medium or fine sand). Euclidean distance was used for the calculation of the distance matrix. A PERMDISP procedure was performed for each factor to test for the homogeneity of dispersion (i.e., variability among replicates) (Anderson 2006). For each

PERMANOVA (permutations=999), the correction of Monte Carlo from the asymptotic permutation distribution was used because of the few possible numbers of permutation. If the test was significant (p<0.05), pairwise tests were carried out using Monte Carlo correction. All statistics were performed using the PRIMER v6 software with the PERMANOVA+add-on package (Anderson et al. 2008).

## Results

### Sediment characteristics

The water content (ω) and the dry bulk density (Csed) were significantly different between Medium Sand “MS” (ω=15.4±0.4% and Csed=1,825±15 kg m⁻³) and Fine Sand “FS” sediments (ω=30.3±0.7% and Csed=1,455±15 kg m⁻³) (PERMANOVA, p<0.05). Cockle density showed no impact on those parameters with no interactive effect (PERMANOVA, p>0.05 for ω and Csed). Sediment organic carbon (TOC) and total nitrogen (TN) contents were respectively 0.05% and 0.01% for MS, and 0.04% and 0.06% for FS. The porosity of MS slightly increased with depth, averaging 0.290±0.014 within the top 5 cm and 0.310±0.004 from 5 to 15 cm depth. For FS, the porosity slightly decreased from 0.478±0.034 within the top 2 cm to 0.420±0.003 between 2 and 15 cm of depth.

### Bioturbation rates

#### Bioirrigation

An exponential decrease in the excess bromide concentration was observed with depth for all treatments (Fig. 2). The natural background bromide concentration (0.8 mM) was reached deeper in MS (between 7 and 8 cm of depth) (Fig. 2A) than in FS (between 5 and 6 cm depth) (Fig. 2B).

Effects of cockle densities on bromide concentrations in the top 5 cm were contrasting in the two sediments. For MS, the bromide concentration excess was 4.1±0.7 mM in the top 5 cm of the control, while it reached 6.2±0.4 mM, 5.5±0.5 mM, and 6.5±0.5 mM with low, medium and high cockle density, respectively. For FS, the bromide concentration excess was 3.6±0.7 mM in the top 5 cm of the control, and reached 4.1±0.6 mM, 4.5±0.7 mM, and 5.0±0.7 mM with low, medium and high density, respectively.

For MS and FS, the mean value of the bioirrigation rate ε (the enhanced biodiffusion coefficient) was close to 1 in control treatments indicating that solute transport was only controlled by molecular diffusion (Fig. 3A). Significant single and interactive effects of cockle density and sediment grain on ε were observed (Table S1). In MS, ε values measured
<figure><img src="image_3.png" /><figcaption>**Fig. 2** Excess bromide concentration profiles in experimental units (mean±SE) for the Medium Sand “MS” (A) and Fine Sand “FS” sediments (B) after 6 days of incubation, for the four treatments (“control”, “low density”, “medium density”, and “high density”; n=3 per treatment)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3** Estimation of the (A) enhanced biodiffusion (ε) and (B) sediment reworking (Db) rates for the Medium Sand “MS” (light grey) and Fine Sand “FS” sediments (dark grey) after 6 days of incubation, for</figcaption></figure>

at low, medium and high cockle density were significantly higher than in the control (Table S2). In addition, the values reached at high density were significantly higher than at medium density. In FS, ε was significantly enhanced with the highest density of cockles compared to control treatment (Table S2). Finally, ε was significantly higher in MS than in FS for the low and high cockle density treatments.

the four treatments (“control”, “low density”, “medium density”, and “high density”; n=3 per treatment). Data are represented by crosses, and circles correspond to the mean value±SE

## Sediment reworking

In the FS and MS control treatments, almost 100% of the luminophores remained in the surficial sediment layer after the 6-day incubation period (Fig. 4). The presence of cockle induced a biodiffusive-like sediment reworking, as revealed by the exponential shape of luminophore depth profiles in both sediment types (Fig. 4). In MS, the maximum luminophore penetration depth (MPD) was 1.5, 2, and 2.5 cm, at low, medium and high density, respectively (Fig. 4A).
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Luminophore depth profiles (mean ± SE) for the Medium Sand “MS” (A) and Fine Sand “FS” sediments (B) after 6 days of incubation, for the four treatments (“control”, “low density”, “medium density”, and “high density”; n = 3 per treatment)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5** Total oxygen uptake at the sediment-water interface for the Medium Sand “MS” (light grey) and Fine Sand “FS” sediments (dark grey), for the four treatments (“control”, “low density”, “medium density”, and “high density”; n = 3 per treatment), after 6 days of incubation. Data are represented by crosses, and circles correspond to the mean value ± SE. Values on Y axis are reversed</figcaption></figure>

This incorporated 99% of buried tracers. The proportion of luminophores buried below 0.5 cm reached 4±2, 22±6, and 46±1% at low, medium and high density, respectively. In FS, the MPD was 2.5, 2.5, and 1.5 cm at low, medium and high density, respectively. (Fig. 4B). The proportion of luminophores buried below 0.5 cm reached 6±2, 31±8, and 53±8% with low, medium and high density, respectively.

Significant effects of cockle density, sediment grain size, and an interactive effect of both factors were observed on the sediment reworking rates (Db; Table S1). Db at high density was significantly enhanced in comparison with the low and medium cockle densities. The Db value reached at high density was significantly higher in FS than in MS (Table S2). In MS, Db was significantly enhanced at high density (9.90±0.40 cm² y⁻¹) compared with other treatments (Fig. 3B; Table S2). In FS, Db reached at low and high cockle density were significantly higher than control treatment (Table S2).

## Benthic fluxes

The total oxygen uptake (TOU) significantly increased with increasing cockle density, ranging from −15 mmol m⁻² d⁻¹ at low density to −146 mmol m⁻² d⁻¹ at high density (Fig. 5), without significant differences between the two types of sediment (Table S1).

Cockle density had a significant effect on NH₄⁺ effluxes, which differed between sediment type (Fig. 6A; Table S1). In MS, NH₄⁺ effluxes at high density were enhanced compared to other treatments (Table S2). In addition, the NH₄⁺ efflux was significantly higher in the medium density compared to the control (Table S2). For FS, NH₄⁺ effluxes increased gradually with cockle density and, except between medium and high densities, all values were significantly different from each other (Table S2). For NOₓ fluxes, values were mainly negative and only the density effect was significant
<figure><img src="image_7.png" /><figcaption>**Fig. 6** Ammonium (A), nitrate and nitrite (B), phosphate (C) and dissolved silicate (D) fluxes across the sediment-water interface for the Medium Sand “MS” (light grey) and Fine Sand “FS” sediments (dark grey), for the four treatments (“control”, “low density”, “medium den-</figcaption></figure>

(Fig. 6B; Table S1). The consumption of NOₓ in sediment increased with cockle density. The value reached with high density was significantly larger than the value in control and with low density. Low and medium densities were also significantly different (Table S2).

Despite their enhancement in MS and a bell-shaped relationship in FS with cockle density, PO₄³⁻ fluxes were close to zero and no significant effect of the investigated factors was observed (Fig. 6C; Table S1).

For dSi fluxes, the effect of the density and sediment type factors were significant (Fig. 6D), without significant interaction between these two factors (Table S1). Overall, dSi fluxes in FS were significantly higher than in MS. In both sediment type, dSi fluxes were significantly different between densities. Values were negative or almost null in the control and for the low density, and positives with medium and high cockle densities (Table S2).

sity”, and “high density”; n=3 per treatment), after 6 days of incubation. Data are represented by crosses, and circles correspond to the mean value±SE. Dash lines represent the null value (no net consumption or production)

## Fluorescence measurements

The color-coded pictures for surface chlorophyll *a* biomass (F₀ Chl *a*, mg Chl *a* m⁻²), maximum quantum yield of fluorescence (Fv/Fm, relative unit), and maximum relative electron transport rate of PSII (rETRmax, relative unit) in each condition are shown in Fig. 7. The F₀ Chl *a* picture provides a detailed overview of the spatial heterogeneity of MPB biomass on the sediment surface. In the control treatments, the distribution of the biofilm was without disruption in the two types of sediment, reaching high values. The spatial distribution of MPB became more heterogeneous with cockles in both sediments, with an increasing proportion of areas without surfacing MPB. A significant decrease in F₀ Chl *a* value with an increase in cockle density was observed (Table 1). No significant effect of the sediment type, nor any interaction between the two factors, was observed on F₀ Chl *a* (Table S1). Regarding Fv/Fm and rETRmax, there were no
<figure><img src="image_8.png" /><figcaption>**Fig. 7** Representative chlorophyll *a* biomass (F₀ Chl *a*, mg Chl *a* m⁻²), quantum efficiency (yield, relative unit) and maximum relative electron transport rate of PS II (rETRmax, relative unit) at the sediment</figcaption></figure>

surface after 6 days of incubation, for the Medium Sand “MS” and Fine Sand “FS” sediments and the four treatments (“control”, “low density”, “medium density”, and “high density”)
<table><caption>**Table 1** Surface chlorophyll *a* biomass (F₀ Chl *a*) of experimental units expressed as a percentage of the control treatment, and maximum quantum efficiency of PSII (yield) and maximum relative electron transport rate of PSII (rETRmax) of the remaining cells (F₀>0.04), after 6 days of incubation (mean±SE, *n*=3). The corresponding 100% control value for F₀ Chl *a* is 1.1±0.1 and 1.2±0.1 mg Chl *a* m⁻² for the medium sand “MS” and fine sand “FS” sediments, respectively</caption><thead><tr><th rowspan="2">Sediment type</th><th rowspan="2">Cockle density</th><th>F<sub>0</sub> Chl a</th><th>Yield</th><th>rETRmax</th></tr><tr><th>(%)</th><th>rel. unit</th><th>rel. unit</th></tr></thead><tbody><tr><td rowspan="4">MS</td><td>Control</td><td>100</td><td>0.52±0.00</td><td>90.27±1.54</td></tr><tr><td>Low</td><td>39.2±10.6</td><td>0.52±0.01</td><td>94.62±8.48</td></tr><tr><td>Medium</td><td>21.4±7.0</td><td>0.53±0.00</td><td>98.03±5.94</td></tr><tr><td>High</td><td>4.4±2.3</td><td>0.53±0.00</td><td>113.34±15.31</td></tr><tr><td rowspan="4">FS</td><td>Control</td><td>100</td><td>0.55±0.01</td><td>98.84±2.66</td></tr><tr><td>Low</td><td>40.9±13.7</td><td>0.51±0.00</td><td>90.23±1.10</td></tr><tr><td>Medium</td><td>15.3±5.5</td><td>0.51±0.01</td><td>97.29±2.19</td></tr><tr><td>High</td><td>3.9±2.1</td><td>0.47±0.05</td><td>80.67±13.55</td></tr></tbody></table>

significant effects of the sediment type and cockle density (Fig. 7; Table S1).

## Chlorophyll *a* and phaeopigments

The chlorophyll *a* (chl *a*) content in the top first cm varied from 27.0±2.2 to 29.7±2.1 mg m⁻² in MS, and from 38.5±1.7 to 57.3±0.2 mg m⁻² in FS (Fig. 8A). There were a significant effect of the cockle density and sediment grain size on chl *a* content, with a significant interaction between both factors (Table S1). In FS, at medium and high densities of cockles, chl *a* content decreased significantly compared to the control treatment (Table S2). However, in MS, there was no significant differences between the chl *a* content

with the different cockle density (Table S2). Chl *a* content values were significantly higher in FS than in MS, regardless of cockle density (Table S2).

The effects of cockle density and sediment grain size on phaeopigment percentage were significant, with a significant interaction between the two factors (Table S1). The phaeopigment percentages were significantly higher at medium and high densities of cockles than in the control, regardless of the sediment type (Fig. 8B; Table S2). High density of cockles also significantly increased this percentage as compared to low density (Table S1). Phaeopigment percentage was significantly higher in FS than MS (Table S1).

## Discussion

### Variation of cockle bioturbation with sediment and density

In our experiment, the vertical profiles of particulate and soluble tracers confirmed that the common cockle *Cerastoderma edule* functions as a true biodiffusor (François et al. 1999; Richard et al. 2021). The exponential shape of these profiles indicates that the surficial sediment layer is randomly and constantly mixed (Kristensen et al. 2012). The bioirrigation rates ε, representing the enhanced diffusion factor due to cockles, were low to moderate in comparison with species that induce non-local transport, such as the polychaete *Marenzelleria* spp. (Renz and Forster 2014). Bioirrigation rates varied with sediment type and cockle density. In fine sand (FS), rates were lower compared to medium

<figure><img src="image_9.png" /><figcaption>**Fig. 8** Chlorophyll *a* concentration (A) and phaeopigment percentage (B) for the Medium Sand “MS” (light grey) and Fine Sand “FS” sediments (dark grey), for the four treatments (“control”, “low density”, “medium density”, and “high density”; *n*=3 per treatment). Data are represented by crosses, and circles correspond to the mean value±SE</figcaption></figure>

<figure><img src="image_10.png" /></figure>
sand (MS) and unaffected by density. The attenuation of porewater movements generated by shell-valve abduction/ adduction and closure of the exhalant siphon likely explains these results. The lower permeability of muddy sediments compared to sandy sediments accelerates the dissipation of porewater movement energy (Schulz and Zabel 2006; Flach 1996; Woodin et al. 2016). In medium sand (MS), the water transport generated by cockles increased with their density, though not linearly. Spatial competition at higher densities likely results in increased collisions between individuals, modulating their movements and associated porewater transport (Richardson et al. 1993).

Regarding sediment reworking, rates (Db) generated by *C. edule* were moderate when compared to other biodiffusors, such as *Scrobicularia plana* or *Echinocardium cordatum* (Lohrer et al. 2005; Richard et al. 2023). These rates were consistent with previous studies on *C. edule* (François et al. 1999; Mermillod-Blondin et al. 2004). While sediment type is a critical factor affecting the bioirrigation activity of cockles, it had a lesser effect on their sediment reworking activity. Specifically, the thickness of the reworked sediment layer was similar between the two sediments, approximately 2.5 cm. Indeed, the burying depth of infanual bivalves mainly depends on their siphon length (Zwarts and Wanink 1989) and adult cockles are usually located close to the sediment surface (<5 cm) (Mermillod-Blondin et al. 2004). In both sediments, sediment reworking rates increased exponentially with cockle density, likely due to enhanced collision-induced vertical migration and positional changes within the sediment column (Richardson et al. 1993). However, the sediment habitat can modulate reworking rates depending on cockle density. In FS, sediment reworking was more pronounced compared to MS at higher densities, likely due to enhanced collision-induced vertical migration and positional changes within the sediment column (Wiesebron et al. 2021). This trend is opposite to what was observed for bioirrigation rates. These results highlighted that lower bulk density sediment conditions lead to more macrofaunal movement and could explain the different Db values between the 2 sediment types in the present mesocosm experiment.

### C. edule enhances nutrient fluxes at the sediment-water interface

Bioturbation processes significantly influenced nutrient transport within the sediment and at the sediment-water interface, with effects modulated by both cockle density and sediment properties. These results are in agreement with previous experimental and field studies, underscoring the critical role of macrofauna in shaping sediment biogeochemical dynamics (e.g., Kristensen et al. 2014; Richard

et al. 2023; Morelle et al. 2024). For example, the aerobic heterotrophic activity of suspension-feeding bivalves can account for over 50% of total oxygen uptake (TOU) in sediments (Henriksen et al. 1983; Michaud et al. 2005). Ammonium ($NH_4^+$) effluxes in soft-bottom environments are often attributed to cockle excretion (Swanberg 1991; Sandwell et al. 2009), while bivalve biodeposits (faeces and pseudo-faeces) enrich sediments with organic matter (Newell et al. 2002). Additionally, bivalve bioturbation, particularly bioirrigation, enhances porewater exchange, oxygen penetration into anoxic sediment (Volkenborn et al. 2019), stimulating aerobic and anaerobic organic matter remineralization rates and nutrient fluxes (Henriksen et al. 1983; Camillini et al. 2019). Our study showed that $NH_4^+$ fluxes from the sediment to the water column increased significantly with cockle density in both sediment types. This pattern reflects the combined effects of excretion and bioturbation by cockles. There was also a significant increase in nitrates and nitrites influxes with rising cockle density, likely due to the stimulation of coupled nitrification and denitrification processes, which occur in oxic and anoxic sediment layers, respectively (Laverock et al. 2011). Phosphates fluxes remained near zero across all treatments, unaffected by cockle density or sediment type. Phosphate dynamics are influenced by chemicals reaction, particularly adsorbed to $Fe^{3+}$ in oxic layers, and could explain low phosphate fluxes (Sundby et al. 1992; Sandwell et al. 2009). The excreted phosphorus by cockles would be more use by bacteria or adsorbed, because, living at the top 2 cm sediment surface, they can increase oxygenation of iron in the first cm of the sediment column (Doering et al. 1987; Kauppi et al. 2018). Unlike nitrogen and phosphorus, dissolved silica (dSi) fluxes are not directly related to organic matter remineralization. In control and low cockle density, dSi fluxes were negative, possibly due to assimilation by MPB cells (Leynaert et al. 2009). As *C. edule* density increased, bioirrigation enhanced the release of sediment-stored dSi into the overlying water. Despite higher bioirrigation rates generated by cockles in MS, nitrogen fluxes did not differ between sediments, and dSi fluxes were higher in FS. This pattern may be explained by the higher chlorophyll *a* content observed in the upper centimetre of FS, indicating greater benthic primary producer biomass. The dissolution of benthic diatom frustules (composed of biogenic silica) contributed to dSi accumulation in porewater (Aller 2014). The lower permeability of FS compared to MS also prolongs porewater residence time, allowing for greater dSi accumulation that is then flushed out into the overlying water. In contrast, the more intense bioirrigation in MS could accelerate solute removal, limiting dSi accumulation in the porewater. Overall, the presence of cockles increased nutrient availability at
the sediment-water interface in both sediment types, where they can be assimilated by MPB.

## Impacts of cockles and sediment type on microphytobenthos dynamics

The sediment reworking activity of cockles created spatial heterogeneity and disrupted MPB biofilms at the sediment surface in both MS and FS. This mechanical disruption reduced surficial MPB biomass across treatments, with an intensity that didn't significantly vary between sediment types, despite reworking rates being higher in FS compared to MS at high cockle density. This suggests that once sediment reworking exceeds a certain threshold, further increases in mixing intensity do not have additional effects on surface sediment and MPB biomass, as the entire area has already been disturbed. Additionally, the presence of emerging cockle shells at the surface reduced the available space for MPB biofilms. Similar results were observed in field studies with the New-Zealand cockle *Austrovenus stutchburyi*, where empty cockle shells at the sediment surface reduced chlorophyll a content (Mouritsen and Poulin 2006). Despite MPB biofilms disruption, cockles did not negatively impact the physiology and photosynthetic performance of the remaining microphytobenthic cells. Maximum quantum efficiency of PSII ($F_v/F_m$) and maximum relative electron transport rate of PSII ($rETR_{max}$) values were high, suggesting a good physiological state of photosynthetic cells (McMinn and Hegseth 2004; Morelle et al. 2020). High MPB production was likely maintained due to moderate sediment reworking which generated a high cell turnover and prevented cell aging (Andersen and Kristensen 1988).

Regarding MPB biomass in the top centimetre (Chl *a*), the effect of cockles varied with the sediment type. In FS, $F_o$ Chl *a* and Chl *a* were significantly reduced in the presence of cockles compared to the control, and decreased with increasing cockle density. This reduction could be attributed to increased sediment reworking activity of cockles at higher density, which destabilize the sediment surface and favour the resuspension of fine particles in the water column (Rakotomalala et al. 2015; Dairain et al. 2020). However, in our mesocosm, there was no current, which excludes this hypothesis. It is more likely that MPB was transported deeper into the sediment column, beyond the depth of photosynthetic active radiation penetration (generally less than 1 mm depth) (Paterson and Hagerthey 2001; Jesus et al. 2006). Even though not measured all along the sediment core, the high percentages of phaeopigments observed in FS in the first centimetre support this hypothesis, as phaeopigments are the degradation product of chlorophyll pigments. Indeed, despite the ability of epipelic MPB cells to perform

an upward migration toward light to perform photosynthesis (Barnett et al. 2020), cockle bioturbation can transport photosynthetic cells to deep (i.e., aphotic) layers, potentially leading to their death and degradation. Some phaeopigments measured could originate from the degradation of the microalgae (*Isochrysis galbana*) added to the seawater during the 6 days of incubation. However, this degradation did not differ significantly between sediment types.

In MS, chl *a* values were significantly lower than in FS but consistent with those reported for sandy intertidal environments (Seuront and Spilmont 2002; Morelle et al. 2020). This pattern is not surprising since MPB biomass is positively correlated to the fine particle content in sediment (Van de Koppel et al. 2001). The most suitable sediment for MPB development is a combination of sand and mud, as it balances light penetration and nutrient accessibility (Morelle et al. 2020), corresponding more to FS in the present study. In MS, a decrease in $F_o$ Chl *a* was also reported with increasing cockle density, while Chl *a* remained constant. The lack of a net cockle density effect on chl *a* can be explained by the offsetting positive and negative effects generated by cockles. Cockles stimulate porewater transport and fluxes of silica and ammonium, providing MPB with essential nutrients for growth (Swanberg 1991). In contrast, the sediment reworking activities mechanically disrupt the biofilm and bury MPB cells beyond the photic layer. The intensity of this disruption was lower in MS and thus limited the overall negative impact on MPB. The suboptimal conditions for biofilm growth in MS, such as reduced nutrient retention and less favourable sediment texture, may have further limited biofilm establishment and balanced the impact of cockles (Morelle et al. 2020). Field studies in sandy sediments have shown that cockles can stimulate MPB biomass accumulation at the sediment surface through sediment stabilization and increased nutrient availability (Swanberg 1991; Donadi et al. 2013; Eriksson et al. 2017). However, these studies spanned longer durations (extending up to 1 year) and could explain discrepancies with our 6-days mesocosm experiment. Consequently, it would be beneficial to extend our experiment approach with in situ measurements to better understand long-term effect of cockles on MPB, integrating more biotic and abiotic factors.

## Conclusion

This study demonstrates that the bioturbation intensity of *Cerastoderma edule* adults is influenced by both population density and type of sediment. Cockle bioirrigation was more pronounced in medium sand than in fine sand sediment while sediment reworking exhibited an opposing trend. Regardless of the sediment type, bioturbation intensity
increased with cockle density. This bioturbation activity had cascading effects, notably influencing oxygen and nutrient fluxes at the sediment-water interface in both sediment types and ultimately impacting microphytobenthos (MPB) dynamics. The non-trophic interactions between cockles and MPB were both positive and negative, with the balance of these effects varying as a function of sediment type. To our knowledge, this is the first experiment that simultaneously quantified the non-trophic effects of adult cockles on MPB dynamics alongside biogeochemical fluxes at the sediment-water interface. These findings underscore the pivotal role of *C. edule* in shaping benthic ecosystem processes and highlight the importance of integrating sediment characteristics into studies of bioturbation and its ecological consequences.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00227-025-04629-0.

**Acknowledgements** We are grateful to the captain and crewmembers of the R/V Planula IV (CNRS-INSU, Flotte Océanographique Française) for their help during field work. We thank Nicolas Savoye, Nathalie Labourdette and Marie-Ange Cordier for their assistance with laboratory work.

**Author contributions** Conceptualization, AR, FO and OM; Investigation: AR, AD, TRD, XdM and OM; Formal analysis, AR, TRD and ARR; Software, ARR; Writing—original draft, AR; Writing—review and editing, AR, AD, XdM, and OM. All authors read and agreed to the published version of the manuscript.

**Funding** Open access funding provided by University of Southern Denmark

This research was partly funded by the INTERREG-ATLANTIC program through the research project COCKLES (EAPA_458/2016 COCKLES: Cooperation for restoring cockle shellfisheries and their ecosystem services in the Atlantic Area). This work is part of Anaïs Richard's doctoral thesis (University of Bordeaux-2018-SG-D-13) financed by a doctoral grant of the French "Ministère de l'Enseignement Supérieur et de la Recherche". Jérôme Morelle was supported by CESAM (UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020).

**Data availability** The datasets generated during the current study are available. Richard, A., F. Orvain, A. Dairain, J. Morelle, A. Romero-Ramirez, T. Rodolfo-Damiano, X. de Montaudouin, and O. Maire. 2025. Influence of cockle bioturbation on microphytobenthic primary producers: habitat and density-dependent effect ver 1. Environmental Data Initiative. https://doi.org/10.6073/pasta/104a9c5465250d9c236239f5ca400ca3 (Accessed 2025-02-05).

## Declarations

**Conflicts of interest** The authors have no relevant financial or non-financial interests to disclose.

**Ethical approval** No research ethics board approval was required to achieve the objectives of this study, as the experimental work was carried out with an unregulated invertebrate species.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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