1

Interlinked seasonal variation in biogenic nutrient fluxes and pore-water nutrient 1

concentrations in intertidal sediments 2

3

4

P. Magni1,2,*, S. Como1, S. Montani3, H. Tsutsumi4 5

6

1Consiglio Nazionale delle Ricerche – Istituto per l’Ambiente Marino Costiero (CNR-IAMC), Località Sa 7

Mardini, Torregrande, 09170 Oristano, Italy 8

2Consiglio Nazionale delle Ricerche – Istituto di Scienze Marine (CNR-ISMAR), Arsenale-Tesa 104, 9

Castello 2737/F, 30122 Venezia, Italy 10

3Graduate School of Environmental Science, Hokkaido University, N10W5 Sapporo, Hokkaido 060-0810, 11

Japan 12

4Faculty of Environmental and Symbiotic Science, Prefectural University of Kumamoto, Tsukide 3-1-100, 13

Kumamoto 862-8502, Japan 14

15

*Corresponding author: Phone: +39-0783-229135; Fax: +39-0783-229139; E-mail: paolo.magni@cnr.it 16

17

2

Abstract. The seasonal variation in biogenic fluxes of NH4+, PO4

3- and SiO2 calculated from the nutrient 18

excretion rates of dominant bivalves (Ruditapes philippinarum and Arcuatula [=Musculista] senhousia), and 19

pore-water nutrient (NH4+, PO4

3-, SiO2 and NO3-+NO2

-), sedimentary acid-volatile sulfide (AVS) and benthic 20

chlorophyll-a (Chl-a) concentrations was assessed on an intertidal sandflat in the Seto Inland Sea (Japan) 21

from summer 1994 to autumn 1995. In spite of the large variability between sampling dates and stations, 22

significant correlations between biogenic nutrient fluxes and pore-water nutrient concentrations were found, 23

suggesting a seasonal linkage between bivalve-mediated biological processes and chemical features of 24

sediments. This linkage was stronger in surface (0-0.5 cm) than subsurface (0.5-2 cm) sediments, consistent 25

with the autoecological characteristics of R. philippinarum and A. senhousia inhabiting the uppermost 26

sediment layer. Significant temporal variation in pore-water NO3-+NO2

-, AVS and Chl-a concentrations was 27

also found, which was related to both occasional extreme events (e.g., dystrophy) and alternating periods of 28

production and decomposition. This study may serve two-fold in (1) contributing to unravel the ecological 29

structure and functioning of natural tidal flats, and the scale of seasonal variability in biotic and sedimentary 30

parameters; and (2) providing useful information for assessing the effectiveness of the physico-chemical and 31

biological structure of artificial tidal flats which are growing in number and extension worldwide. 32

33

Key words: pore-water nutrients, biogenic flux, benthic macroinvertebrates, bivalves, tidal flat, Ruditapes 34

philippinarum, Arcuatula [=Musculista] senhousia, Seto Inland Sea. 35

36

3

Introduction 37

Tidal flats rich in benthic microalgae and macroinvertebrates (Guarini et al. 1998; Komorita et al. 2010; 38

Donadi et al. 2013) are known to provide important ecosystem services, such as nutrient cycling, primary 39

production and natural products (Prather et al. 2013; Hutchison et al. in press). Pore-water nutrients are a 40

major component in nutrient cycling, fueling benthic primary production (Kuwae et al. 1998; Trimmer et al. 41

1998) and sustaining indirectly primary consumers that feed on benthic and resuspended microalgae (Kasai 42

et al. 2004; Komorita et al. 2014). Benthic animals, particularly abundant suspension feeders, are an 43

important food source for higher trophic levels (Kuwae et al. 2012) and are known to contribute strongly to 44

the regeneration of ammonium and reactive phosphorous, through excretion, bioturbation and bio-deposition 45

(Bartoli et al. 2001; Welsh 2003). Fewer studies have shown that some bivalves (e.g., Mytilus edulis, 46

Ruditapes philippinarum, Arcuatula [=Musculista] senhousia) and polychaetes (e.g., Hediste diversicolor) 47

can also stimulate strongly reactive silica regeneration (Asmus et al. 1990; Bartoli et al. 2009). 48

In tidal flats, pore-water nutrients (Lerat et al. 1990; Kuwae et al. 2003; Wang et al. 2011), benthic 49

microalgae (Davoult et al. 2009; Spilmont et al. 2011) and macrofaunal communities (Castel et al. 1989; 50

Giménez et al. 2006; Magni et al. 2006) display marked variation at different spatial and temporal scales. 51

Consistently, several experimental studies have shown, to varying extent, a link between nutrient fluxes, 52

microphytobenthos activity and benthic animals (e.g., Mortimer et al. 1999; Asmus et al. 2000; Sandwell et 53

al. 2009). However, the extent of temporal interactions between biological processes and geochemical 54

variables in tidal flats remains little explored (Prins and Smaal 1994; Rysgaard et al. 1995). To the best of 55

our knowledge, no studies have quantified the linkage and the scale of seasonal variability in macrofauna-56

mediated biogenic fluxes of nutrients and pore-water nutrient concentrations in intertidal sediments. Such 57

knowledge may help furthering the understanding of the ecological structure and functioning of tidal flats. 58

In addition, in spite of their importance, natural tidal flats have drastically declined over the past 59

century in many coastal areas worldwide owing to coastal development (e.g., Nam et al. 2010; Yang et al. 60

2011; Zainal et al. 2012). As a mitigation measure, in recent years, artificial tidal flats and wetlands are being 61

constructed at many sites (Yamochi 2008; Ohmura et al. 2010; Smith and Jacinthe 2014). There is a growing 62

debate nowadays regarding the functioning and effectiveness of the physico-chemical and biological 63

4

structure of artificial tidal flats as compared to the natural ones (Lee et al. 1998; Aldous et al. 2005; Ishi et al. 64

2008). It is therefore important to assess the biological structure and functioning of remaining natural tidal 65

flats as a baseline to evaluate whether artificial ones are able to provide similar goods and services (Aoki et 66

al. 2011). 67

To fill these gaps, we selected historical and unpublished data sets collected from a small-size 68

temperate estuarine sandflat of Japan, where several investigations have been conducted over the past 20 69

years (e.g., Magni and Montani 1997; Montani et al. 1998; Ichimi et al. 2012). In our associated pieces of 70

work, we conducted laboratory experiments on the excretion rates of NH4+, PO4

3- and SiO2 by dominant 71

bivalves, R. philippinarum and A. senhousia (Magni et al. 2000; Magni and Montani 2005). The results, 72

extrapolated to a field relevant situation, demonstrated that R. philippinarum and A. senhousia contribute up 73

to 90% of NH4+, PO4

3- and SiO2 total upward fluxes of nutrients, the bivalve standing stock playing a major 74

role in the magnitude of these fluxes (Magni et al. 2000; Magni and Montani 2006). 75

In this study, we describe the seasonal changes in biogenic fluxes of nutrients generated by dominant 76

bivalves, R. philippinarum and A. senhousia, and pore-water nutrient concentrations to investigate the 77

processes of nutrient accumulation and transformation in the sandflat. The seasonal variation in sedimentary 78

acid-volatile sulfide (AVS) and benthic chlorophyll-a (Chl-a) concentrations are also studied as a proxy of 79

decomposition processes and microphytobenthic development, respectively. In particular, the main 80

objectives of this study are: 1) to investigate the magnitude and scale of seasonal variation in bivalve-81

mediated biogenic fluxes of nutrients and their relation with pore-water/sediment variables; 2) to discuss the 82

implications of our study in terms of ecosystem functioning and ecological structure and restoration of tidal 83

flats. 84

85

Materials and Methods 86

Study area 87

This study was conducted on a tidal flat located in the Shinkawa-Kasugakawa estuary in the Seto Inland Sea, 88

Japan (Fig. 1). Major variations in the hydrological features occur along this estuary on a time scale of 1-2 89

5

hours, as related to the ebb-flood and neap-spring components of the tidal cycle (Montani et al. 1998; Magni 90

et al. 2002). On the tidal flat, the sediments are sandy with a mud fraction <3% (Magni and Montani 1997). 91

Total organic carbon content of surface sediments ranges between about 6-12 mg g-1, with the tendency to be 92

relatively higher in summer and autumn than in spring and winter (Magni et al. 2006). Microphytobenthos 93

biomass and primary production show large temporal variations, ranging between about 2-20 µg Chl-a g-1 94

and 0.32-3.0 g C m-2 d-1, respectively, and accounting for up to 50% of the overall primary production 95

(Montani et al. 2003). Macrobenthic assemblages are dominated by the filter-feeding bivalves R. 96

philippinarum and A. senhousia (Magni et al. 2000; 2006). 97

Field sampling 98

Field investigations on macrobenthic assemblages, pore-water nutrients (NH4+, PO4

3-, SiO2 and NO3-+NO2

-), 99

sedimentary acid-volatile sulfide (AVS) and benthic chlorophyll-a (Chl-a) were conducted on the tidal flat at 100

low-tide soon after sediment emersion. Sampling was conducted in 6 consecutive seasons between 1994 and 101

1995 (summer [Su94] and autumn [A94] 1994, winter 1994-1995 [W], and spring [Sp], summer [Su95] and 102

autumn [A95] 1995). In each season, two sampling dates were randomly chosen, avoiding the 2 weeks at the 103

beginning and at the end of the season in order to ensure disjunctive boundaries between seasons and to 104

prevent confounding effects (Morrisey et al. 1992). At each date, samples were collected at four stations 105

randomly chosen within a surface area of ca. 50 × 50 m. 106

On each sampling date and station, benthic macroinvertebrates were collected using a 100 cm2 107

stainless steel core (10 cm in depth) from two plots of sediment randomly chosen m’s apart and sieved on a 108

mesh size of 1 mm. The residues were then fixed in a 10% buffered formaldehyde solution. At each station, 109

one sediment sample for chemical analysis was collected using an acrylic core tube (3 cm in diameter) gently 110

pushed by hand into the sediment. The sediment was carefully extruded and the surface (0 to 0.5 cm) and 111

subsurface (0.5 to 2 cm) layers sliced off. Sediment samples were brought to the laboratory within 2 h for 112

further treatment. 113

6

Monthly data of temperature, salinity and dissolved oxygen and nutrient (NH4+, PO4

3-, SiO2 and NO3-114

+NO2-) concentrations in creek water adjacent to the tidal flat were obtained from Magni and Montani (2000) 115

as an additional environmental characterisation of the study area. 116

Laboratory procedure and analysis 117

Benthic macroinvertebrates were separated from the residue and transferred into a 75% ethanol and 2.5% 118

ethylene-glycol solution. The animals were sorted and counted under a stereo-microscope (Olympus, Wild 119

M3Z). They were weighed as a total wet weight (WW), including the shell for bivalves. The dry weight 120

(DW) biomass of the soft tissues of R. philippinarum and A. senhousia was calculated as 3.6% and 4.7% of 121

the total (live) weight, respectively (Magni et al. 2000). The DW biomass of polychaetes and that of other 122

minor and/or uncommon taxa was calculated as 20% of their WW biomass (Ricciardi and Bourget 1998). 123

The fluxes of nutrients (NH4+, PO4

3- and SiO2) generated by R. philippinarum and A. senhousia were 124

calculated from the nutrient excretion rates of the two bivalves obtained from our associated pieces of work 125

conducted on the same tidal flat (Magni et al. 2000; Magni and Montani 2005), where species-specific 126

excretion rate is applied to the DW biomass of each bivalve species in each replicate sample. Following the 127

approach used in Magni et al. (2000), the excretion rates of R. philippinarum and A. senhousia obtained in 128

our laboratory experiments were applied to the biomass of each bivalve species found in spring and summer 129

(Table S1, Supplementary Material). These rates were considered to be representative of bivalve-mediated 130

biogenic fluxes on the tidal flat during spring and summer. To cope with seasonal changes in temperature, 131

environmental conditions and physiological energetics of bivalves, reduced excretions rates were calculated 132

and applied to the bivalve biomass found in autumn and winter (Table S1, Supplementary Material). The 133

original rates were multiplied by 0.9 (autumn) and 0.6 (winter) for R. philippinarum and by 0.7 (autumn) and 134

0.5 (winter) for A. senhousia. This calculation was based both on our extended knowledge of the 135

environmental features and bivalve condition in our study area (Magni et al. 2000, 2006; Magni and Montani 136

2000), and on relevant literature (Bayne and Scullard 1977, Mann and Glomb 1978, Goulletquer et al. 1989, 137

Schlüter and Josefsen 1994, Nizzoli et al. 2011), as detailed in Magni et al. (2000). While we acknowledge 138

that this is an approximation, we infer that an additional manipulation of bivalve excretion rates would 139

7

introduce inaccuracy. In addition, we found that the use of relatively different excretion rates did not 140

markedly vary the seasonal pattern of biogenic fluxes of nutrients due to the strong temporal change in 141

bivalve biomass (Magni et al. 2000). 142

For the determination of pore-water nutrient concentrations, part of fresh sediment sample was 143

centrifuged at 1000 x g and the extracted pore-water was filtered immediately, to minimize exposure to the 144

atmosphere and sample oxidation, on disposable filters (0.45 µm) fitted to a 10 ml sterile syringe and 145

transferred into polystyrene test tubes. The filtrate was stored at 20°C for nutrient analysis, carried out within 146

2–3 weeks with a Technicon autoanalyzer II, according to Strickland and Parsons (1972). From the same 147

pool of fresh sediment, the AVS concentrations, expressed as mg S g-1 of the DW, were determined in 148

duplicate (ca. 1 g) by acidifying the sediment with 18 N H2SO4 and subsequently trapping the released H2S 149

with an H2S absorbent column (Gastec, Hedrotek 201, Kagawa Science, Japan). Chl-a was extracted from 150

duplicate subsamples of wet sediment (ca. 1 g) using 90% acetone. After 24 h of darkness at 4 °C, the 151

samples were sonicated for 5 min, centrifuged at 1000 x g for 10 min, and extracts were 152

spectrophotometrically analyzed (Jasco, Uvidec-320). Chl-a concentrations, expressed as µg g-1 of the DW, 153

were obtained according to Lorenzen’s (1967) method, as described by Parsons et al. (1984). 154

Data analysis 155

Differences in the abundance and biomass of R. philippinarum and A. senhousia and associated biogenic 156

fluxes of nutrients (NH4+, PO4

3- and SiO2) were assessed using a three-way nested ANOVA model with 157

‘Season’ (fixed), ‘Dates’ (random, nested in season), “Stations” (random, nested in dates) as factors and 158

stations as replicates. Differences in pore-water nutrient (NH4+, PO4

3-, SiO2 and NO3-+NO2

-), sedimentary 159

AVS and benthic Chl-a concentrations were assessed in surface and subsurface sediment layers, separately, 160

using a two-way nested ANOVA model with ‘Season’ (fixed), ‘Dates’ (random, nested in season) as factors 161

and stations as replicates. When significant differences among seasons were found (P < 0.05), a posteriori 162

comparisons were done using SNK test (Underwood 1997), as indicated in the Results section. Homogeneity 163

of variances was checked using Cochran’s C-test (Winer et al. 1991), and when necessary, data were 164

transformed to remove the heterogeneous variances. In some cases, variances were heterogeneous even after 165

8

several transformations of the data. The analyses were done anyway because ANOVA is robust to deviations 166

from this assumption when there are several independent estimates of the residual variance (see Underwood 167

1997 for details). Differences in pore-water nutrient (NH4+, PO4

3-, SiO2 and NO3-+NO2

-), sedimentary AVS 168

and benthic Chl-a concentrations between surface and subsurface sediment layers were tested using the 169

Wilcoxon paired-sample test. A paired-sample test was used since data were not independent (Zar 2010). In 170

order to evaluate the relationships between biogenic fluxes generated by the excretion of R. philippinarum 171

and A. senhousia and pore-water nutrient concentrations in surface and subsurface sediments a simple linear 172

regression analysis was fitted to data. To account for multiple simultaneous analyses, the level of 173

significance was adjusted using the sequential Bonferroni technique (Rice 1989). All analyses were done 174

using Statistica (StatSoft 6.1). 175

176

Results 177

Seasonal variation in biogenic fluxes of nutrients and pore-water nutrient concentrations 178

Results of the analysis of variance (ANOVA) and a posteriori SNK comparisons of biogenic fluxes of 179

nutrients revealed significant differences between seasons for both R. philippinarum and A. senhousia (Fig. 180

2; Table 1a, b). In particular, the fluxes of NH4+, PO4

3- and SiO2 due to R. philippinarum were highest in 181

Su94, intermediate in Su95 and A95, and lowest in A94 and W and Sp. For A. senhousia, the fluxes of NH4+, 182

PO43- and SiO2 was higher in Su94, Su95 and A95 than in A94, W and Sp. The magnitude and seasonal pattern of 183

biogenic nutrient fluxes was influenced to a large extent by the strong seasonal variation in bivalve standing 184

stock, dominant in our study area (Fig. S1; Table S2a, b, Supplementary Material). 185

The pore-water nutrient concentrations showed a temporal pattern similar to that of biogenic fluxes of 186

nutrients (Fig. 3a,b). Analysis of pore-water nutrient concentrations in surface (0-0.5 cm) sediments 187

indicated significant differences between seasons for PO43- which was lowest in W and A94 (Table 2a). 188

Similarly to PO43-, pore-water SiO2 concentrations tended to be lower in winter than in the other seasons 189

(Table 2a). However, the analysis detected a significant effect of Dates but not Seasons in pore-water SiO2 190

concentrations (Table 2a). Although there were no significant differences (judged at P < 0.01, due to 191

9

heterogeneous variances; Table 2a), pore-water NH4+ concentrations showed a tendency to be lower in W 192

and A94 than in the other seasons (Fig. 3a). Pore-water NH4+, PO4

3- and SiO2 concentrations in subsurface 193

(0.5-2 cm) sediments were higher than those at the surface (Wilcoxon test: Z = 5.18, 2.56 and 5.04, 194

respectively, P < 0.05), yet following a similar seasonal pattern with the tendency to be lower in winter than 195

in the other seasons (Fig. 3b). However, the analysis of variance detected a significant effect of Dates but not 196

Seasons for NH4+ and SiO2, whereas no significant differences (judged at P < 0.01 due to heterogeneous 197

variances) were found for PO43- (Table 2b). 198

The linear regression analysis showed significant relationships between the biogenic fluxes of NH4+ 199

and PO43- generated by both R. philippinarum and A. senhousia and the pore-water nutrient concentrations of 200

the same nutrient species in surface sediments (Table 3a). To a lesser extent, significant relationships were 201

also found between biogenic fluxes of nutrients and pore-water nutrient concentrations in subsurface 202

sediments (Table 3b). These results suggest a stronger linkage of bivalve populations and related biological 203

processes with surface sediments. 204

Seasonal variation in complementary pore-water/sediment variables 205

In surface sediments, significant seasonal variation was also found for pore-water NO3-+NO2

-, highest in 206

winter, and sedimentary AVS and benthic Chl-a concentrations, highest and lowest, respectively, in A94 (Fig. 207

4a; Table 4a). In subsurface sediments, NO3-+NO2

- and Chl-a concentrations were lower than those in 208

surface sediments (Wilcoxon test: Z = 5.61 and 6.03, respectively, P < 0.00), while AVS concentrations 209

increased with depth (Z = 5.70, P < 0.00), with a major peak in the first sampling date of A94 as related to the 210

occurrence of a major dystrophic event (Magni et al. 2006). In subsurface sediments, the analysis of 211

variance detected a significant effect of Dates but not Seasons for pore-water NO2-+NO3

- and sedimentary 212

AVS, whereas no significant differences were found in benthic Chl-a (Fig. 4b; Table 4b). 213

In tidal creek water, temperature and salinity showed large temporal variation, ranging 5.8–32.2 °C 214

and 5.9–32.1 psu, respectively (Fig. 5). NH4+, PO4

3- and SiO2 concentrations did not show clear temporal 215

trends, but were slightly higher in 1994 than in 1995 (Fig. 5). In contrast, NO3-+NO2

- concentrations peaked 216

between May and July 1995, in coincidence with a major decrease of salinity. Accordingly, NO3-+NO2

-, but 217

10

not NH4+, PO4

3- and SiO2, was correlated negatively with salinity (R = 0.90; P = 0.000), indicating the creek 218

water as a major source of NO3-+NO2

- at low tide. 219

220

Discussion 221

Seasonal variation in biogenic nutrient fluxes and pore-water/sediment variables 222

Using an indirect approach where excretion rates of dominant bivalves have been applied to a field-relevant 223

situation on a tidal flat, our study provides a proxy of the magnitude and seasonal variation in the biogenic 224

fluxes of three major nutrient species, i.e., NH4+, PO4

3- and SiO2. Much work on biogenic fluxes of nutrients 225

by intertidal bivalves has been carried out through short-term laboratory or field incubation experiments. 226

Relatively few studies conducted in tidal flats have considered the extent of seasonal variability (Prins and 227

Smaal 1994; Rysgaard et al. 1995; Mortimer et al. 1999). This is also due to a considerable lack of 228

information on the temporal distribution of natural populations of macrozoobenthos in tidal flats. This study 229

thus highlights the importance and the need of extended field surveys on community composition, standing 230

stock and temporal variability when assessing the contribution of benthic populations to the processes of 231

nutrient regeneration. Our study also provides novel evidence of the importance of the often overlooked SiO2 232

in nutrient cycling in tidal flats. Until now most work on macrofauna-induced stimulation of benthic nutrient 233

cycling in intertidal sediments has focused on nitrogen species (e.g., NH4+ and NO3

-) (see review by Stief 234

2013), and to a lesser extent on PO43- (e.g., Table 5 in Magni et al. 2000). Among the few studies conducted 235

in tidal flat on SiO2 cycling, Dame et al. (1991) suggested that SiO2 release from mussel beds results from 236

phytoplankton cells breaking down as they are metabolized by the mussels. They argued that the longer 237

turnover time for SiO2, compared to NH4+ and PO4

3-, implies a lesser role for the mussel beds in recycling 238

this nutrient species in the two estuaries under investigation. Instead, other studies including mesocosm 239

experiments using large tanks (Doering et al. 1987) or field measurements (Asmus et al. 1990; Bartoli et al. 240

2001) found evidence of increased levels of SiO2 flux in the presence of bivalves. In our study, the 241

magnitude of biogenic regeneration of SiO2 (together with that of NH4+ and PO4

3-) was comparable to the 242

highest upward fluxes of nutrients reported for mussel beds or oyster reefs (e.g., Dame et al. 1984; Prins and 243

11

Smaal 1994). This indicates that abundant populations of bivalves are able to recycle rapidly and efficiently 244

the dissolved forms of these three major bioelements, playing a primary role in the development of benthic 245

primary producers within the tidal flat. These results are consistent with the development of abundant 246

microphytobenthos on the studied tidal flat, among which dominant diatoms characterized by extremely high 247

growth rates are found (Montani et al. 2003; Ichimi et al. 2012). 248

In our study area, the seasonal variation in pore-water nutrient concentrations was also marked. 249

Several studies have shown that the seasonal variation in pore-water nutrients in coastal sediments is strongly 250

dependent on temperature, as related to microbial processes (e.g., Klump and Martens 1989; Marinelli et al. 251

1998). In intertidal sediments, Lerat et al. (1990) found a temporal and spatial mismatch in SiO2 and NH4+ 252

distribution within an oyster bed sediment, SiO2 peaking in summer with concentrations progressively 253

increasing down to 10 cm depth, while NH4+ increasing in late autumn at intermediate (3–6 cm) layers. The 254

authors indicated that pore-water SiO2 was regenerated by dissolution of biogenic compounds and was 255

highly correlated with temperature. Whereas, pore-water NH4+ accumulation within the sediment column 256

was suggested to be the result of mineralisation of sedimentary organic nitrogen. In that case, nutrient 257

regeneration would occur at different periods of the year. In our study area, pore-water NH4+, PO4

3- and SiO2 258

concentrations in both surface and subsurface sediments showed similar temporal patterns, with a marked 259

tendency to be lower in winter than in the other seasons. Some differences in the extent of seasonal variation 260

between different pore-water nutrient species were also found. In particular, variability at the scale of dates 261

may have masked differences between seasons in surface and subsurface SiO2 concentrations and subsurface 262

NH4+ concentrations. Instead, variability among replicate stations within the study area may have reduced the 263

ability to detect differences in Seasons and Dates in surface NH4+ and subsurface PO4

3- concentrations. The 264

main difference between the three pore-water nutrient species was found in autumn (A94) when SiO2 265

concentrations in both surface and subsurface sediments tended to remain as high as in summer, while NH4+ 266

and PO43- concentrations had substantially decreased. In the same season (A94), a major peak of SiO2 267

concentrations was concurrently observed in low-tide creek water. The fact that low-tide creek water was a 268

major source of NO3-+NO2

-, but not of SiO2 nor NH4+ and PO4

3-, further indicates the importance of internal 269

12

nutrient regeneration within the tidal flat. Overall, our results indicated that major events of nutrient 270

production and regeneration within the sediments are governed by concurrent processes. 271

In spite of the large environmental variability of tidal flats in macrotidal systems (see also Montani et 272

al. 1998; Magni et al. 2002), our study indicates a seasonal linkage between bivalve-mediated upward fluxes 273

of nutrients and pore-water nutrient concentrations. We acknowledge that demonstration of direct cause-274

effect relationships between biological processes and sediment/pore-water variables would require 275

contemporary measurements of all different variables. Yet, such attempts are very scarce in intertidal 276

sediments, especially on a seasonal basis; this is also due to temporal and financial restrictions and concrete 277

difficulties in extrapolating physiological measurements to long-term trends (Montani et al. 2003). Although 278

the variance explained by the regression analysis was modest due to wide scatter in the raw data, the F-279

statistic from the regression analysis was significant in several cases, suggesting a significant overall similar 280

pattern in seasonal variation (regardless of the exact mode of causality). A stronger linkage of bivalve-281

mediated nutrient fluxes with surface (rather than subsurface) pore-water nutrient concentrations was 282

consistent with the autoecological characteristics of both R. philippinarum and A. senhousia. These bivalves 283

live semiburied in the intertidal and shallow subtidal soft sediments of bays and estuaries and are often found 284

in close association, as in our study area (Magni et al. 2006). In particular, R. philippinarum (also named 285

‘short-neck’ clam) is characterized by two short siphons which are in close contact with the uppermost layer 286

of sediment and the near-bottom water, as per its feeding and excretory activities (e.g., Kasai et al. 2004; 287

Komorita et al. 2014). Similarly, the Asian date mussel A. senhousia secretes byssal threads to attach to 288

conspecifics forming large mats on the seabed surface and strongly modifying sediment properties (Crooks 289

1998; Crooks and Khim 1999). The main difference between bivalve-mediated biogenic processes and pore-290

water nutrients was observed in spring (Sp) when biogenic fluxes of nutrients were low, while pore-water 291

nutrient concentrations were high. This can be explained by the fact that while bivalve recruitment had 292

already occurred, leading to a sharp increase in the bivalve abundance, animal biomass was still low. At the 293

same time, increasing temperature may have had an effect on the nutrient regeneration within the sediments. 294

Enhanced bioturbation by abundant polychaetes in spring (Magni et al. 2006) may also have played a role in 295

pore-water nutrient mobilisation from lower sediment layers (Huettel 1990; Magni and Montani 2006). In 296

13

addition, nutrient utilization by microphytobenthos in the uppermost few millimeters of sediments, as being 297

related to other concurrent variables (e.g., temperature, light availability, sediment emersion time), may 298

influence the temporal variability in pore-water nutrient concentrations (Davoult et al. 2009; see also Magni 299

and Montani 1997, 2006). 300

As related to production and decomposition processes, we also demonstrated a large seasonal variation 301

in NO3-+NO2

-, Chl-a and AVS concentrations. Consistently, NO3-+NO2

- and Chl-a concentrations in surface 302

sediments were found to be correlated positively with one another and negatively with AVS through the 303

study period (see also Magni and Montani 2006). In addition, as a result of year-to-year variation, AVS and 304

Chl-a concentrations were considerably higher and lower, respectively, in A94 than in A95. This was related to 305

a major dystrophic event that occurred in late summer 1994, also indicated by a major peak of AVS in the 306

subsurface sediments and a massive mortality of R. philippinarum (Magni et al. 2006). Thus, both occasional 307

extreme events and alternating periods of production and decomposition occurring on a tidal flat may play a 308

major role in the temporal variability of benthic/sedimentary variables which are dependent upon biological 309

processes. 310

Implications for ecosystem functioning and ecological restoration of tidal flats 311

The analyses of the linkage of seasonal variation in bivalve populations, associated biogenic fluxes of 312

nutrients and sediment/pore-water variables provided by this study may be useful for evaluating the 313

functioning of artificial tidal flats which are being constructed in recent years at many sites in Japan and 314

worldwide (Yamochi 2008; Ohmura et al. 2010; Smith and Jacinthe 2014). There is a growing debate 315

nowadays regarding whether the habitats created by the artificial tidal flats have similar ecological functions 316

to the natural habitats (Lee et al. 1998; Ishi et al. 2008; Yamochi 2013), and whether they are able to provide 317

similar goods and services (Aoki et al. 2011). Unfortunately, ecological studies conducted in artificial or 318

constructed tidal flats are still few, mostly from Japan. As an example, topics include dissolved oxygen 319

consumption (Eguchi et al. 2002), nitrogen and phosphorus budgets (Yamochi et al. 2003; Yamochi 2008; 320

Hatano et al. 2013), outbreak of green algae (Yamochi 2013) and benthic food web structure (Kanaya et al. 321

2013). However, none of them has taken into account the link between biogenic fluxes of nutrients and 322

14

sediment/pore-water variables as a tool to assess the effectiveness of the physico-chemical or biological 323

structure of artificial tidal flats. Few attempts comparing benthic populations in natural and artificial tidal 324

flats are available (Imanaka et al. 2002; Aoki et al. 2011; Nishijima et al. 2014). For instance, Aoki et al. 325

(2001) found no differences between bivalve assemblages of artificial and natural tidal flats in inner Tokyo 326

Bay. They showed that bivalve assemblage was determined not by sediment characteristics of a tidal flat but 327

by the location in the bay. Nishijima et al. (2014) found higher density of macrobenthic assemblage in the 328

man-made intertidal sandflat than in three natural intertidal sandflats within the Ohta River Estuary (Japan), 329

which was explained by a higher elevation of the former one. Differences in benthic assemblage between 330

constructed and natural areas at comparable sites have been demonstrated on the southeastern coast of New 331

South Wales (Australia), being infauna at constructed locations intermediate in abundance and species 332

richness, typical of an early successional stage (French et al. 2004). Although the above studies are valuable 333

attempts to assess the ecological structure of artificial tidal flats, little inference can be extrapolated in terms 334

of their biological processes and ecosystem functions as compared to natural ones, for which reason more 335

studies on both natural and artificial tidal flats are needed. 336

In conclusion, our study contributes new knowledge to the linkage and scale of seasonal variation in 337

bivalve populations, associated nutrient fluxes and sediment/pore-water parameters in a tidal flat, most work 338

on these aspects being conducted in subtidal sediments (e.g., Bartoli et al. 2001; Nizzoli et al. 2011; Zhang et 339

al. 2013). This study may serve two-fold in (1) contributing to unravel the ecological structure and 340

functioning of natural tidal flats, recognizing their importance in providing fundamental ecosystem services 341

within an estuary, and (2) providing useful information for assessing the effectiveness of the physico-342

chemical or biological structure of artificial tidal flats which are growing in number and extension 343

worldwide. 344

345

Acknowledgments This study was partly supported by a research fund (No. 22380102) from the 346

Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid of Scientific 347

Research) and by the Flagship Project RITMARE – The Italian Research for the Sea – coordinated by the 348

Italian National Research Council and funded by the Italian Ministry of Education, University and Research. 349

15

We gratefully acknowledge the constructive criticisms of two anonymous reviewers which helped us 350

improve manuscript’s clarity. 351

352

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537

23

Table 1. Analysis of the effects of Seasons and Dates on biogenic fluxes of ammonium [NH4+], reactive phosphorous 538

[PO43-] and reactive silica [SiO2] generated by the excretion of Ruditapes philippinarum (a) and Arcuatula [Musculista] 539

senhousia (b). 540

(a) R. philippinarum nutrient fluxes (mmol m-2 h-1)

NH4+ PO4

3- SiO2 Source of variation df MS F P MS F P MS F P Seasons 5 32.52 30.68 *** 2.30 30.52 *** 27.54 30.11 *** Dates(Seasons)A 6 1.06 0.72 0.08 0.73 0.91 0.73 Stations(Dates(Seasons))B 36 1.47 2.10 * 0.10 2.07 * 1.26 2.09 *

ResidualsC 48 0.70 0.05 0.60 Total 95 Transformation None None None Cochran's test NS NS NS

Seasons: Seasons: Seasons: SNK test

Su94>Su95=A95>A94=W=Sp

Su94>Su95=A95>A94=W=Sp

Su94>Su95=A95>A94=W=Sp

(b) A. senhousia nutrient fluxes (mmol m-2 h-1)

NH4+ PO4

3- SiO2 Source of variation df MS F P MS F P MS F P Seasons 5 324.71 10.04 * 5.34 12.74 *** 124.48 10.01 * Dates(Seasons)A 6 32.33 1.14 0.42 1.11 12.44 1.15 Stations(Dates(Seasons))B 36 28.24 2.34 *** 0.38 2.35 *** 10.84 2.34 ***

ResidualsC 48 12.07 0.16 4.63 Total 95 Transformation None None None Cochran's test NS

§ NS

Seasons: Seasons: Seasons: SNK test Su94=Su95=A95>A94=W=Sp Su94=Su95=A95>A94=W=Sp Su94=Su95=A95>A94=W=Sp A Denominator of Seasons B Denominator of Dates(Seasons) c Denominator of Stations(Dates(Seasons))

df degrees of freedom, MS mean squares, F Fischer’s F, P probability (* P < 0.05; *** P < 0.001; NS = not significant) 541 C-Cochran’s and results from SNK (Student–Newmans–Keuls) tests for differences among Seasons are reported 542 § Transformation failed to remove heterogeneous variances for PO4

3- fluxes by A. senhousia, hence, significance was judged at 543 a more conservative level (α = 0.01); in this case, P < 0.01544

24

Table 2. Analysis of the effects of Seasons and Dates on pore-water ammonium [NH4+], reactive phosphorous [PO4

3-] 545 and reactive silica [SiO2] in surface (0-0-5- cm, a) and subsurface (0.5-2 cm, b) sediments. 546

(a) Surface sediment pore-water nutrients (µM)

NH4+ PO4

3- SiO2 Source of variation df MS F P MS F P MS F P Seasons 5 32829.00 9.03 1.87 22.76 *** 9105.77 3.51 Dates(Seasons)A 6 3633.56 0.96 0.08 1.25 2592.62 15.93 ***

ResidualsB 36 3779.48 0.07 162.71 Total 47 Transformation None Ln(x+1) None Cochran's test

§ NS NS

Seasons: SNK test A94=W<Su94=Sp=Su95=A95 (b) Subsurface sediment pore-water nutrients (µM)

NH4+ PO4

3- SiO2 Source of variation df MS F P MS F P MS F P Seasons 5 32164.70 1.08

245.99 2.92 0.76 1.75

Dates(Seasons)A 6 29853.62 3.72 * 84.22 1.34

0.43 17.02 *** ResidualsB 36 8024.88

62.81

0.03

Total 47 Transformation None None Ln(x+1) Cochran's test NS

§ NS

A Denominator of Seasons B Denominator of Dates(Seasons) c Denominator of Stations(Dates(Seasons))

df degrees of freedom, MS mean squares, F Fischer’s F, P probability (* P < 0.05; *** P < 0.001; NS = not significant) 547 C-Cochran’s and results from SNK (Student–Newmans–Keuls) tests for differences among Seasons are reported 548 § Transformation failed to remove heterogeneous variances for surface pore-water NH4

+ and subsurface pore-water PO43-549

concentrations, hence, significance was judged at a more conservative level (α = 0.01); in this case, P < 0.01. 550 551

25

Table 3. Results of simple linear regression showing the relationship between pore-water nutrients in surface (0-0.5 cm, 552 a) and subsurface (0.5-2 cm, b) sediments and nutrient fluxes generated by the excretion of Ruditapes philippinarum 553 and Arcuatula [Musculista] senhousia (n=48) 554

(a) Surface pore-water nutrients (µM)

NH4+ PO4

3- SiO2

Biogenic fluxes of nutrients (mmol m-2 h-1) R2 F1,46 P R2 F 1,46 P R2 F 1,46 P

R. philippinarum 0.26 17.10 *** 0.21 13.21 *** 0.12 7.63 **

A. senhousia 0.24 15.79 *** 0.24 15.81 *** 0.10 6.25 *

(b) Subsurface pore-water nutrients (µM)

NH4+ PO4

3- SiO2

Biogenic fluxes of nutrients (mmol m-2 h-1) R2 F 1,46 P R2 F 1,46 P R2 F 1,46 P

R. philippinarum 0.11 6.81 * 0.08 4.83 * 0.12 7.16 **

A. senhousia 0.10 6.46 * 0.13 7.76 ** 0.13 7.79 **

F Fischer’s F, P probability (* P < 0.05, ** P < 0.01; *** P < 0.001). Significant correlations after sequential Bonferroni 555 correction (Rice, 1989) are in bold. 556

557

26

Table 4. Analysis of the effects of Seasons and Dates on pore-water nitrite+nitrate [NO2-+NO3

-], sedimentary acid-558 volatile sulfide [AVS] and benthic chlorophyll-a [Chl-a] in surface (0-0-5 cm, a) and subsurface (0.5-2 cm, b) 559 sediments. 560

(a)

NO2-+NO3

- (µM)

AVS (mg g-1)

Chl-a (µg g-1) Source of variation df MS F P

MS F P

MS F P

Seasons 5 9.41 8.52 *

0.01 16.90 *

1.60 5.53 *

Dates(Seasons)A 6 1.10 4.76 ***

0.00 3.56

0.29 2.35 ResidualsB 36 0.23

0.00

0.12

Total 47

Transformation

Sqrt(x+1)

None

Sqrt(x+1) Cochran's test

NS

§

NS

Seasons:

Seasons:

Seasons: SNK test

W>Su94=A94=Sp=Su95=A95

A94>Su94=Su95>Sp=W=A95

A94<Su94=W= Sp=Su95=A95

(b)

NO2-+NO3

- (µM)

AVS (mg g-1)

Chl-a (µg g-1) Source of variation df MS F P

MS F P

MS F P

Seasons 5 0.58 2.41

0.18 1.44

10.01 2.32 Dates(Seasons)A 6 0.24 7.68 ***

0.13 4.02 ***

4.31 1.10

ResidualsB 36 0.03

0.03

3.93 Total 47

Transformation

Ln(x+1)

None

None

Cochran's test

NS

§

NS

A Denominator of Seasons B Denominator of Dates(Seasons)

df degrees of freedom, MS mean squares, F Fischer’s F, P probability (* P < 0.05; *** P < 0.001; NS = not significant) 561 C-Cochran’s and results from SNK (Student–Newmans–Keuls) tests for differences among Seasons are reported 562 § Transformation failed to remove heterogeneous variances for surface and subsurface AVS, hence, significance was judged at 563 a more conservative level (α = 0.01); in this case, P < 0.01 564

565

27

Figure captions 566

Fig. 1 Study area and location of the tidal flat in the Shinkawa-Kasugakawa estuary (Seto Inland Sea, Japan) 567

Fig. 2 Seasonal variation (mean of four stations, ±standard error, SE) in biogenic fluxes of ammonium 568

[NH4+], reactive phosphorous [PO4

3-] and reactive silica [SiO2] generated by the excretion of (a) 569

Ruditapes philippinarum and (b) Arcuatula [Musculista] senhousia. Columns: white = summer; light 570

gray = autumn; black = winter; dark gray = spring 571

Fig. 3 Seasonal variation (mean of four stations, ±standard error, SE) in (a) pore-water ammonium [NH4+], 572

reactive phosphorous [PO43-] and reactive silica [SiO2] in surface (0-0-5- cm, a) and subsurface (0.5-2 573

cm, b) sediments. Columns: white = summer; light gray = autumn; black = winter; dark gray = spring 574

Fig. 4 Seasonal variation (mean of four stations, ±standard error, SE) in pore-water nitrite+nitrate [NO2-575

+NO3-], sedimentary acid-volatile sulfide [AVS] and benthic chlorophyll-a [Chl-a] in surface (0-0-5- 576

cm, a) and subsurface (0.5-2 cm, b) sediments 577

Fig. 5 Monthly variation in hydrological variables of low tide creek water (from top panel): salinity and 578

nitrate+nitrite [NO2-+NO3

-], temperature and ammonium [NH4+], and reactive phosphorous [PO4

3-] and 579

reactive silica [SiO2] 580

581

28

Yashima

0 km 5

Takamatsucity

134°00' E

34°25' N

Seto Inland Sea

Yashima

Takamatsucity

34°21' N

Tsumeta River Kasuga River Shin River

Tidal flat

134° 05' E 0 200 400 m

10 m

5 m

2 m

Seto InlandSea

582

Figure 1. 583

584

585

29

0

1.6

0.8

0

1.6

0.8

0

1.0

0.5

0

1.0

0.5

0

0.1

0.2

0

0.1

0.2

R. philippinarum A. senhousia

PO43-

SiO

2N

H4+

PO43-

SiO

2N

H4+

Bio

geni

c flu

x (m

mol

m-2

h-1

)

(a) (b)

Su94 A94 Su95 A95W Sp Su94 A94 Su95 A95W Sp 586

Figure 2. 587

588

30

PO43-

SiO

2

240

120

0

0

17

34

NH

4+

500

250

0

Pore

-wat

er n

utrie

nts (

µM)

Su94 A94 Su95 A95W Sp

Surface sediment

PO43-

SiO

2240

120

0

0

17

34

NH

4+

500

250

0

Subsurface sediment

Su94 A94 Su95 A95W Sp

(a) (b)

589

Figure 3. 590

31

NO

2- +N

O3- (

µM)

13

26

1.2

0.6

AV

S (m

g g-1

)C

hl-a

(µg

g-1)

8

16N

O2- +

NO

3- (µM

)

13

26

1.2

0.6

AV

S (m

g g-1

)C

hl-a

(µg

g-1)

8

16

(a) (b)

Su94 A94 Su95 A95W Sp Su94 A94 Su95 A95W Sp

Surface sediment Subsurface sediment

0

0

0

00

0

591

Figure 4. 592 593

32

18Salinity

(psu)N

H4+

(μM

)

5

Tem

pera

ture

(°C

)N

O2 -+N

O3 (µM

)SiO

2 (µM) PO

43-(µ

M)

36

18

0

150

75

90

45

0

0DN J F M A A S OJ OA S

94M J J N

95

10

45

0

36

90

0

0

594 595

Figure 5. 596