Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

Bon Joo Koo; Jaehwan Seo

doi:10.1371/journal.pone.0228873

. 2020 Feb 10;15(2):e0228873. doi: 10.1371/journal.pone.0228873

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

Bon Joo Koo ^1,^2,^*, Jaehwan Seo ^1,²

Editor: Ismael Aaron Kimirei³

PMCID: PMC7010307 PMID: 32040527

Abstract

The manila clam Ruditapes philippinarum is widely distributed in the sandy mud sediments of tidal flats and plays a role in seawater purification by filtering suspended organic matter. This study was designed to evaluate differences in seawater purification based on the filtration rate of the manila clam in terms of particulate organic matter (POM) between two tidal flats with different hydrographic regimes. In situ experiments were carried out at Geunso and Sihwa tidal flats, along the west coast of Korea. The amount of POM removed from the water column by the feeding activity of the clam was measured in the field using a closed circulation chamber. The filtration rate of clams for POM at Sihwa tidal flat (2.86 for POC, 2.29 for PON and 5.46 L h^-1 gDW^-1 for Chl a) was higher than that at Geunso tidal flat (0.61 for POC, 0.89 for PON and 2.54 L h^-1 gDW^-1 for Chl a) which resulted from differences in the hydrographic regime, including tide characteristics, current speed and submergence time, and food quantity and quality. The current speed was much greater at Geunso tidal flat than at Sihwa tidal flat, but the submergence time by tide was longer at the latter site than the former, resulting in different feeding times for clams. The food quantity in terms of chlorophyll a was higher at Sihwa tidal flat than at Geunso tidal flat, and the food quality based on the C/N ratio of POM was better at the former site than the latter, with values of 12.8 and 15.6, respectively. These findings suggest that hydrographic regime could be important in understanding in situ filtration rates of R. philippinarum.

Introduction

The manila clam, Ruditapes philippinarum, is widely distributed in the sandy mud sediments of tidal flats and shallows, with a geographic range including the west coasts of Korea and Japan, China, the northwestern USA, and European Atlantic coasts [1,2]. This clam is one of the most important shellfish in the Korean fisheries industry due to its high nutritional value and function of water purification in tidal flats [3]. In 2009, a total of 40,393 metric tons (MT) of clams were produced in Korea through aquaculture and fisheries, constituting the second greatest shellfish production, after oysters [4].

R. philippinarum is a filter-feeding bivalve responsible for the transport of materials at the sediment-water interface. It plays a role in seawater purification by filtering suspended organic materials from seawater, storing the filtered materials as assimilation products, and depositing excess organic matter onto the sediment as pseudofeces after ingestion [5]. The pseudofeces deposited onto sediments act as an important food source for deposit feeders. Bivalves such as R. philippinarum are characterized by relatively long lifespans and high biomass [6,7], which enables them to function as an organic matter reservoir in the material cycle of the tidal flat. In addition, the presence of an individual bivalve enhances biodiversity, as a space is formed between the particles of the matrix, providing habitat for various marine organisms.

Organic matter introduced into the marine ecosystem supports primary productivity in the water column, and enters the food chain through consumers; thus, it serves as a major link in the ecosystem’s food chain, and is therefore a significant factor in maintaining ecosystem productivity. However, the high organic matter content of a contaminated water column is not transferred up through the food chain, but instead flows immediately into the sediment, causing a hypoxic layer in the water column and producing toxic substances such as sulfides in the sediments. Such sediments are the main culprit in deterioration of the environment near the seafloor. Filter feeders such as R. philippinarum block the capacity of organic matter to act as a contamination source by consuming organic matter from the water column as a food source [8].

Sihwa lake is an artificial lake formed by the construction of the Sihwa dike as part of the Great Reclamation Comprehensive Development Project, and is a representative area that underwent major environmental changes. Sihwa dike caused dissipation of intertidal zone with severe water quality deterioration. Thus, the government withdrew desalination and started the seawater exchange through sluice gate operation, as a result, tidal flat was partially created with repeated submergence and emergence. On the Sihwa tidal flat, introduction of organic matter directly into the sediment can cause pollution of the bottom-water environment, but the presence of filter feeders can prevent pollution by organic matter, as they decrease the amount of organic matter flowing into the sediment and are themselves consumed by higher-level consumers. In other words, the presence of filter feeders in Sihwa tidal flat can reduce the pollution load in the water column due to their use of organic matter that flows into the sediment as a food source, transferring it into the food chain. Therefore, the seawater purification capacity of filter feeders is essential as a method that can mitigate nutrient pollution.

Previous studies have reported effects of environmental factors such as temperature, salinity, particle size, and prey type on the filtration rate of this clam. The filtration of the clam increased with temperature up to 25°C, with maximum rates in the range of 15–25°C, and also increased with salinity up to 35 psu [3,9]. The filtration rate of this species for phytoplankton was much higher than that for bacteria, mainly due to differences in the size and biomass of these prey items [10]. In addition, the ingestion rate of the clam was much higher for small particles than for large particles due to the increased quantity of organic matter per unit volume [11]. However, scant research has been conducted on how the filtration rate of this species differs among hydrographic regimes.

Thus, the purpose of the present study is to evaluate differences in the seawater purification effect, based on the filtration rate of particulate organic matter (POM) by R. philippinarum, between two tidal flats with different hydrographic regimes.

Study area

In situ experiments were carried out in two tidal flats along the west coast of Korea, Geunso (36°44′7.43″N, 126°10′40.12″E) and Sihwa (37°17′10.01″N, 126°40′57.54″E), with different hydrographic regimes (Fig 1). A permission to conduct research in the two tidal flats was issued by the Ministry of Land, Transport and Maritime Affairs.

Fig 1 — Locations and layout of the study sites: (A) Sihwa tidal flat and (B) Geunso tidal flat.

The manila clam, Ruditapes philippinarum, is widely distributed on the lower zone of these flats. Geunso tidal flat has a semidiurnal macro-tidal regime with a mean tidal range of 600 cm [12]. In the main tidal channel during spring tides, flood-current velocity ranges between 80 and 90 cm s^-1, and ebb-current velocity between 60 and 70 cm s^-1 [13]. While Geunso tidal flat is characterized by a macro-tidal regime, Sihwa tidal flat has an artificially controlled tide with a maximum range of 112 cm (Table 1).

Table 1. Comparison of hydrographic regimes between Geunso and Sihwa tidal flats.

	Geunso tidal flat	Sihwa tidal flat
Hydrographic characteristics	Macro-intertidal	Artificially controlled tide
Tidal range	600 cm (mean) ^a	112 cm (max) ^b
Current speed	~ 90 cm s^-1 ^c	0.1 ~ 33.1 cm s^-1 ^d

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^a[12]

^b[14]

^c[13]

^d[15]

Sihwa tidal flat is located along Lake Sihwa, which is brackish (27 psu). Due to the limited water exchange between the inner lake and outer sea, which occurs only through a narrow sluice gate in accordance with the tidal cycle (see Fig 1), the tidal range in Lake Sihwa is smaller, and its speed slower (33.1 cm s^-1 maximum), compared to Geunso. The experimental sites on these flats also have differing environmental characteristics. The surface sediment type in Sihwa tidal flat is sandy, with a mean grain size of 3.1 to 3.3 Φ, while Geunso tidal flat has sandy silt of 4.2 to 4.8 Φ.

Materials and methods

1) In situ experiments

(1) Experimental equipment

The amount of POM removed from the water column through the feeding activity of R. philippinarum was measured in the field using a closed circulation chamber (Fig 2).

Fig 2 — C1 and C2 represent control chambers without clams and R1 and R2 represent experimental chambers containing clams.

The chamber was constructed from transparent acrylic, with an inner diameter of 0.49 m, height of 0.6 m, cross-sectional area of 0.19 m², and volume of 113 L. The bottom of the chamber was closed and the top was equipped with a lid that could be opened and closed. An agitator was installed inside the chamber, connected to a plastic tube through a hole in the lid, which was intended to prevent natural settling of suspended solids (SS) and stagnation of seawater in the chamber during the experiment, and to maintain homogeneity of the medium. The connection to the lid was waterproofed so that seawater could not flow into the chamber, even when it was flooded. A vacuum pump was connected to the opposite end of a plastic pipe in turn connected to the inside of the chamber, so that seawater in the chamber could be collected from the outside. Due to pressure generated inside the chamber during water sampling, an air pipe connecting the chamber with the atmosphere was installed to equalize the pressure. Dissolved oxygen was monitored to prevent a decrease in metabolic activity due to depletion of dissolved oxygen in the chamber, and when the concentration decreased below 80%, oxygen was supplied using an aerator installed inside the chamber. Because the field experiment was conducted during a submergence period, all devices connected to the chamber were installed on a tower, where an observer was located (Fig 2).

(2) Installation of chambers and sampling

Experiments were carried out in the habitat of R. philippinarum in Geunso and Sihwa tidal flats in October 2009 in the same manner (Fig 3).

Four chambers were installed in the sediments, buried to a depth of 10 cm, and connected to the support structure of the tower with an iron frame to prevent movement due to tidal currents. Inside the chamber, previously dried and prepared defaunated sediments were installed at a depth of 10 cm. Defaunated sediments were prepared by sieving field sediments to remove macroinvertebrates, including Ruditapes, and then dried at a constant temperature of 27°C for 10 days. After the dried sediment was placed in the chamber, the upper part of the chamber was sealed using a plastic sieve with 0.3-mm mesh and acclimated in seawater in the field for 3 days. During the acclimation period, 2- and 3-year-old clams were collected from the field and immediately transplanted into the chamber, and then acclimated for 1 day with the sediments. Fifty individuals were transplanted into each of two chambers (Ruditapes chambers) and none were transplanted into the other two chambers (control chambers). Shortly before starting the experiment, the lid of the chamber was closed and the peristaltic pump, air duct, Fibox-3 oxygen meter, water temperature gauge, agitator, and aerator were installed. Seawater from the experimental site was allowed to flow into the chamber during the flood tide. At this time, to prevent the sediment in the chamber from being disturbed by the influent seawater, silicone plugs installed at various heights in the chamber wall were opened, beginning with the plug in the lowest position. After seawater had filled the chamber, all plugs were closed and the agitator was employed to maintain a constant seawater flow rate in the chamber of 5 cm s^-1. We filled a chamber in the laboratory with the same sediment and seawater as the chamber installed at the experimental site prior to the experiment, and determined the seawater flow rate (5 cm s^-1) from the agitator at which organic matter in the sediment was not suspended and natural sedimentation of POM was prevented. Based on the flow rate obtained under laboratory conditions, the flow rate of seawater in the chamber was maintained during the experimental period.

After 10 min of agitator operation, seawater samples were collected from the four chambers using a vacuum pump. Water sample collection was performed four times (Sihwa tidal flat) or five times (Geunso tidal flat) at regular intervals until the experimental area was exposed during the ebb tide to determine the mean concentration of POM in the experimental shore region, a seawater sample was simultaneously collected at the same depth as the experimental chamber. At the time of collection of each seawater sample, 1 L of seawater was sampled from each chamber and from the surrounding water column. From these samples, 750 mL of seawater was placed into a polyethylene container to measure the concentrations of SS, particulate organic carbon (POC), and particulate organic nitrogen (PON), and the remaining 250 mL was filtered using GF/F filter paper, then frozen and stored for chlorophyll a analysis. Chambers were equipped with aerators (“bubblers” in Fig 2) to prevent dissolved oxygen from dropping below 80% and inhibiting the metabolic activity of clams, but these were not necessary to activate during the experiment at either site. After the experiment, clams were captured for measurement of shell length, flesh wet weight, and flesh dry weight. During the experiment, the dissolved oxygen, and water temperature of the seawater were simultaneously measured at the same depth as the chamber (CTD SBE-19; Sea-Bird Electronics, USA).

2) Sample analysis

(1) Total suspended solids

First, 0.7-μm Whatman GF/F filter paper was washed three times with 20 mL of distilled water in a vacuum filter apparatus, dried at 103–105°C for 1 hour, and weighed using an electronic scale. At the experimental site, seawater collected in plastic bottles (polypropylene [PP]; Nalgene, USA) was mixed thoroughly and 100 ml was filtered onto GF/F filter paper. The filter paper was washed three times with 10 ml distilled water for desalination. The remaining material on the filter paper was dried at 105–110°C for 2 hours and then weighed. The SS content was determined from the difference between the wet and dry weights of the filter paper.

(2) Particulate organic carbon and particulate organic nitrogen

To determine the concentrations of POC and PON, 0.7-μm Whatman GF/F filter paper, through which 500 ml of seawater had been filtered (burned at 550°C for 5 hours before use), was lyophilized; carbon and nitrogen were then removed by placing the filter in a desiccator containing hydrochloric acid solution for 24 hours. Aliquots were placed into tin cups after measuring their weight, and concentration analysis was performed using an elemental analyzer (EA1110; CE Instruments, UK).

(3) Chlorophyll a

Using 0.7-μm Whatman GF/F filter paper, 250 mL of the seawater sample was filtered, and the filter was transferred to the laboratory frozen. The filter paper was placed in a centrifuge tube with 10 mL of 90% acetone and then shaken thoroughly, and the filter paper was allowed to react in a dark, refrigerated room for 24 hours. The extracted chlorophyll a was measured using a spectrophotometer (Cary 50; Varian, USA).

3) Calculation of POM filtration rate in the water column

Assuming that the feeding activity of the clams is constant, POM in the chamber should be removed at a constant rate such that its concentration should decrease exponentially over time. In this experiment, chlorophyll a, POC and PON were used as indicators of POM. If the initial concentration of the indicator substance is C0 and the coefficient of the concentration decrease is Z, the concentration Ct after t time is defined by the following equation.

C t = C 0 \cdot e^{‐ z t}

The coefficient of the concentration decrease, Z, is defined as follows:

Z = ‐ l n (C t / C 0) \cdot t

The concentration decrease rate, d, per unit time of the indicators in water is expressed as follows:

d = 1 ‐ e^{‐ z}

The filtration rate (FR) per unit time is given by the following equation, where V is the volume of the chamber and h is the unit of time.

F R (L / h) = V \cdot d = V \cdot (1 ‐ e^{‐ z})

Because the amount of particulate matter in the chamber is limited, as the observation time becomes longer, the rate of concentration decrease becomes smaller, and thus the filtration rate can be underestimated. In this study, the filtration rate was calculated for the period in which the maximum filtration was observed from among the time periods. The filtration rate of POM was adjusted by natural sedimentation rate from control chamber.

A two-sample t test was used to determine differences between samples. The results were considered statistically significant when p<0.05.

Results

1) Water temperature and dissolved oxygen

In Sihwa tidal flat, the water temperatures in the Ruditapes chamber, the control chamber, and the surrounding seawater did not change significantly during the experiment, and ranged from 16.3 to 17.3°C (Fig 4). The water temperature in the Ruditapes chamber was highest during experiment with mean values of 17.7±0.2°C, followed by control chamber (17.1±0.2°C) and seawater (16.8±0.2°C). Although, there was statistical significance (p<0.05), but difference of temperature was not differ greatly between the Ruditapes chamber and the control chamber; due to the closed environment, the water temperature inside the chamber was slightly higher than that of the seawater around the chamber (p<0.05).

Fig 4 — Variations in temperature and dissolved oxygen concentration in two experimental chambers and seawater over the entire study period at Geunso and Sihwa tidal flats: (A) temperature at Geunso tidal flat, (B) dissolved oxygen at Geunso tidal flat, (C) temperature at Sihwa tidal flat, (D) dissolved oxygen at Sihwa tidal flat. Green triangles represent sampling times.

In Geunso tidal flat, the seawater temperature showed greater changes than in Sihwa tidal flat. During the flood tide, as the current flowed at shallow depths, the water temperature reached 19.1°C, but as the water depth increased, the water temperature decreased to 17.2°C. Thereafter, the temperature rose rapidly due to rising atmospheric temperature and tended to increase gradually from around the time of slack tide. During the ebb tide, as surface water that had been heated during the daytime passed through the experimental area, the water temperature increased to 21.2°C (Fig 4). Unlike Sihwa tidal flat, the mean water temperature was not significantly different among the Ruditapes chamber, the control chamber, and the seawater with values of 18.9±0.3, 18.9±0.3 and 19.0±0.7°C in Geunso tidal flat, respectively (p<0.05). The mean water temperatures of Ruditapes chamber, the control chamber, and the seawater were significantly higher in Geunso tidal flat than in Sihwa tidal flat in entire experiment (p<0.05) and chambers were less variable in water temperature compared to the seawater.

During the experiment, dissolved oxygen exceeded 100% in all chambers at both study sites. In Sihwa tidal flat, dissolved oxygen in the Ruditapes chamber and the control chamber increased continuously compared to their initial values, and the control chamber showed a larger increase. However, the dissolved oxygen concentration in the chamber showed a different tendency in the Geunso tidal flat. The control chamber maintained a constant concentration, while the Ruditapes chamber showed a decreasing trend. Changes in the dissolved oxygen concentration of seawater were greater in the Geunso tidal flat than Sihwa tidal flat. The dissolved oxygen in the seawater was generally lower than in the chambers during entire experiment in both tidal flats.

2) Suspended solids

The concentration of SS in the seawater of Sihwa tidal flat did not change much during the experiment. At the beginning of the experiment, the SS concentration was 0.10 g L^-1. After 1 hour, the SS concentration increased slightly to 0.12 g L^-1 and then decreased to 0.091 g L^-1 at the end of the experiment (Fig 5A).

Fig 5 — Variation in suspended solids (SS) and particulate organic matter (POM) in two experimental chambers and seawater over the entire study period at Sihwa tidal flat: (A) SS (B) particulate organic carbon (POC) (C) particulate organic nitrogen (PON) and (D) chlorophyll a. The error bars represent 95% confidence intervals.

The SS concentrations of the Ruditapes and control chambers were both about 0.10g L^-1, similar to that of the seawater at the study site. The control chamber did not show significant changes in SS concentration over time, while the Ruditapes chamber showed a pronounced decrease in SS concentration. In particular, there was a significant decrease in SS concentration (0.076 g L^-1) over a period of 1 hour, beginning after 1 hour had elapsed in the experiment.

Changes in SS concentrations at Geunso tidal flat differed significantly from those observed in Sihwa tidal flat. The SS concentrations in the two chambers, were initially identical at 0.14 g L^-1, higher than those observed in Sihwa tidal flat (Fig 6A).

Fig 6 — Variation in SS and POM in two experimental chambers and seawater over the entire study period at Geunso tidal flat: (A) SS (B) POC (C) PON and (D) chlorophyll a. The error bars represent 95% confidence intervals.

The concentration in the seawater around the study site was highest at the beginning of the experiment, then decreased rapidly over time, and increased again at the end of the experiment. This pattern can be attributed to the fact that the flood current during the flood tide and the ebb current during the ebb tide caused the sediment to be resuspended. The SS concentrations in the Ruditapes and control chambers decreased rapidly until 1 hour and 30 minutes had elapsed. After this time point, the changes in SS concentrations in the two chambers showed differing tendencies. There was no significant change in SS concentration in the control chamber, while SS content decreased continuously in the Ruditapes chamber. The sharp decrease in SS concentration during the initial stage of the experiment was attributed to the seawater flow rate in the chamber (5 cm s^-1 in this experiment) being insufficient to prevent deposition of SS resuspended by the flood current. However, after a certain amount of SS settled, a constant SS concentration was maintained in the control chamber during the experimental period, while it continued to decrease in the Ruditapes chamber, leading to concentration differences between the two chambers, as observed at Sihwa tidal flat.

3) POC and PON

Concentrations of POC and PON in the seawater ranged from 0.88 to 2.18 mg L^-1 and 0.05 to 0.14 mg L^-1, respectively, during the experiment period at Sihwa tidal flat (Fig 5B). The concentrations of these substances in the seawater tended to gradually decrease and then increase. The POC and PON concentrations showed similar tendencies in both chambers (Fig 5B and 5C). In the control chamber, there was no significant change in the concentrations of the two indicators during the experimental period, whereas there was a large decrease in these concentrations due to the filter-feeding activity of clams in the Ruditapes chamber. During the experimental period, the POC concentration decreased from an initial value of 2.40 to 0.89 mg L^-1, and the PON concentration decreased from an initial value of 0.22 to 0.07 mg L^-1. For both indicators, the greatest decrease in concentration occurred between the second and third sampling periods, when the concentration of POC decreased from 2.15 to 1.27 mg L^-1 and the concentration of PON decreased from 0.16 to 0.08 mg L^-1.

The initial concentrations of POC and PON at Geunso tidal flat were higher than those at Sihwa tidal flat. POC and PON concentrations in the seawater were in the range of 1.00 to 6.56 mg L^-1 and 0.05 to 0.43 mg L^-1, respectively (Fig 6B and 6C). In seawater at the study site, the concentration of each indicator tended to gradually decrease and then increase. In the chamber, there was a sharp decrease in these concentrations at the beginning of the experiment, as observed for suspended matter, and a difference in concentration between the Ruditapes chamber and the control chamber arose during the second sampling period. After the second sampling event, no significant change in the concentrations of the two indicators was observed in the control chamber, but their concentrations decreased sharply due to the filter-feeding activity of clams in the Ruditapes chamber. In the Ruditapes chamber, the POC concentration decreased from 2.49 to 0.89 mg L^-1, and the PON concentration decreased from 0.15 to 0.06 mg L^-1.

4) Chlorophyll a

The chlorophyll a concentration in seawater at the Sihwa tidal flat was 2.37 μg L^-1. This value was relatively high due to resuspension of sediment during the initial phase of the experiment, and a constant concentration of about 2.0 μg L^-1 was maintained thereafter (Fig 5D). At the beginning of the experiment, the concentration was about 3.3 μg L^-1 in both the control and Ruditapes chambers, but the difference in chlorophyll a concentration between the two chambers increased gradually over time. In the control chamber, little change in the chlorophyll a concentration occurred during the first 2 hours (3.24 to 3.27 μg L^-1), but it then increased dramatically to 4.54 μg L^-1 at the end of the experiment. On the other hand, in the Ruditapes chamber, a large decrease in concentration was observed, down to 1.49 μg L^-1 at the end of the experiment.

At Geunso tidal flat, the chlorophyll a concentration was relatively high at the beginning and end of the experiment due to resuspension caused by the flood and ebb currents. The chlorophyll a concentration at the beginning and the end of the experiment was 4.79 and 6.10 μg L^-1, respectively (Fig 6D). The chlorophyll a concentration showed a sharply decreasing tendency during the initial part of the experiment, followed by a constant concentration around the time of the high-water stand, and then increased sharply during the ebb tide. In the Ruditapes and control chambers, a sharp decrease in the chlorophyll a concentration took place during the beginning of the experiment, as described for POC and PON concentrations. At the second sampling time, the chlorophyll a concentration was similar between the Ruditapes and control chambers, at 2.36 and 2.56 μg L^-1, respectively. The difference in chlorophyll a concentration between the two chambers at the third sampling time was 1.27 μg L^-1, with a lower concentration in the Ruditapes chamber. Thereafter, no significant changes in the chlorophyll a concentration in the Ruditapes chamber occurred, while that in the control chamber tended to increase (S1 Table).

5) The filtration rate of R. philippinarum

The morphometric dimensions of R. philippinarum were compared between Geunso and Sihwa tidal flats (Table 2). The shell length, height and width were significantly higher in Geunso tidal flat than in Sihwa tidal flat. The flesh wet weight and flesh dry weight were also higher in the former than in the latter with significance

Table 2. Comparison of morphometric data of Ruditapes philippinarum between Geunso and Sihwa tidal flats.

	Shell length (mm)	Shell height (mm)	Shell width (mm)	Flesh wet weight (g)	Flesh dry weight (g)
Geunso (n = 94)	38.5±3.1	27.3±2.4	19.2±2.1	6.2±1.6	0.4±0.1
Sihwa (n = 93)	33.2±2.3	22.7±1.6	15.3±1.2	2.2±0.6	0.2±0.1
P value	<0.05	<0.05	<0.05	<0.05	<0.05

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Significant differences by t test at 0.05

The filtration rate of R. philippinarum was calculated from the changes in the concentrations of POC, PON, and chlorophyll a in the Ruditapes chamber. As noted above, because the supply of POM introduced with food source materials at the beginning of the experiment was limited in the closed chamber, the filtration rate may be underestimated due to the reduced concentration with increased observation time. Therefore, the filtration rate was calculated for the period in which the maximum filtration was observed from among the time periods. The filtration rate at Sihwa tidal flat was 2.86±1.28 L h^-1 gDW^-1 for POC, 2.29±1.85 L h^-1 gDW^-1 for PON, and 5.46±1.07 L h^-1 gDW^-1 for chlorophyll a (Table 3).

Table 3. Comparison of filtration rate of Ruditapes philippinarum between Geunso and Sihwa tidal flats.

	Filtration rate (L h-1 gDW-1)
	POC	PON	Chl a
Geunso	0.61±0.04	0.89±0.19	3.17±1.72
Sihwa	2.86±1.28	2.29±1.85	5.46±1.07
P value	0.24	0.40	0.17

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POC, particulate organic carbon; PON, particulate organic nitrogen; Chl a, chlorophyll a; Significant differences by t test at 0.05

At Geunso tidal flat, the filtration rates were lower than at Sihwa tidal flat. The filtration rates of POC, PON and chlorophyll a were 0.61±0.04, 0.89±0.19, and 2.54±1.61 L h^-1 gDW^-1, respectively. Although the difference in POM filtration rate was not statistically significant, it was higher at Sihwa tidal flat than that at Geunso tidal flat.

Discussion

In contrast to our expectation that dissolved oxygen would decrease due to the respiration of clams, no significant decrease was observed in any chamber, and it exceeded 100% throughout the experiment. Moreover, oxygen concentration was elevated in the Ruditapes and control chambers placed at Sihwa, presumably because the depth of the chambers was shallow, which allowed photosynthesis by phytoplankton. This result was attributed to the oxygen consumption by the clams being offset by the oxygen production due to photosynthesis in the chamber.

Generally, filtration rate in terms of filtering ability for seawater increase as clam size decrease. This relationship may be due to the decrease with age of the gill surface to body size ratio and to the higher metabolic demands of the younger individuals [16,17]. Segade et al., 2003; Sylvester et al., 2005; Han et al., 2008). Han et al. (2008) reported that the filtration rate of small Ruditapes philippinarum (0.2 gDW) was about 28% higher than that of large ones (0.4 gDW) at 20°C. This indicated that the filtration rate of Ruditapes can be changed by size and biomass, but, the difference in filtration rate is too great to be explained by size and biomass in this study.

One experimental condition that must be considered carefully when calculating the amount of POM removed from seawater by filter feeders using a closed chamber, as indicated in this study, is that the concentration of POM introduced into the chamber at the beginning of the experiment should be maintained. While the concentrations of the indicators in the control chamber at Sihwa tidal flat were constant during the experimental period, the concentrations of those indicators (POC, PON and Chlorophyll a) decreased rapidly in the control chamber at Geunso tidal flat (Fig 6). This result may be due to settling of some of the POM initially introduced into the chamber in the sediments, which is related to the intensity of the flood current in the tidal flats. At Sihwa tidal flat, which has a closed circulation environment, the inflow of resuspended particulate matter from the sediment into the chamber was limited because the intensity of the flood current was low. In contrast, the intensity of the flood current was high at Geunso tidal flat, and resuspended sediments were introduced into the chamber in large quantities. Prior to this experiment, the flow rate in the chamber that prevented resuspension of particulate matter without causing sedimentation of POM from seawater introduced into the chamber was determined under laboratory conditions using sediments and seawater from Sihwa tidal flat (5 cm s^-1). This flow rate was applied to the experiments at both sites, and was slower than in the in situ environment at Geunso tidal flat. However, the concentration of particulate matter in the control chamber remained constant after a certain period of time, whereas that in the Ruditapes chamber tended to decrease; thus, it is possible to obtain a filtration rate based on this difference in concentration. However, it is possible that the filtration rate at Geunso tidal flat measured in this experiment was somewhat underestimated, as we presume that filtration by clams also occurred during the period when the concentration decreased rapidly at the beginning of the experiment.

At Sihwa tidal flat, there is no natural tidal rhythm, and ebb and flood currents are created by artificial manipulation of the sluice gate. These ebb and flood currents are not created twice a day like natural currents, but are instead repeated at an interval of 2 or 3 days. The mean daily submergence time of the study site at Sihwa tidal flat was longer than that at Geunso tidal flat, with values of 21 and 16 h day^-1, respectively. Therefore, clams at the former site can carry out feeding activity more efficiently, and for a longer time, than those at the latter site.

The relatively high filtration rate at Sihwa tidal flat can be attributed to this difference in hydrographic regimes. Another factor affecting the difference in filtration rate is high productivity in the water column at Sihwa tidal flat, which causes a quantitative difference in the food available to Manila clams. The Manila clams at Sihwa tidal flat had a higher growth rate than those at Geunso tidal flat, and the mean chlorophyll a concentration in the water column was significantly higher at the former site than the latter, with values of 3.1 and 52.5 μg L^-1, respectively [18]. This result suggests that the quantity of food available contributed to differences in the filtration rate of this clam between the two tidal flats.

The C/N ratio of POM is an indicator of food quality. High values of the C/N ratio are generally indicative of nutrient starvation, with lower values being recorded under non-limiting nutrient conditions [19]. Optimal feeding by Ruditapes occurred at C/N ratios of 8.4–10.5, with a range of 6.2–12.0, and was correlated with nitrogen rather than carbon content [20]. The mean C/N ratios of the Geunso and Sihwa tidal flats over the entire study period were 15.6 and 12.8, respectively, and the mean PON concentration at Sihwa tidal flat was slightly higher than that at Geunso tidal flat, with values of 0.13 and 0.10 mg L^-1, respectively. These results suggest that the food quality at Sihwa is better than that at Geunso tidal flat, resulting in a difference in filtration rate between these two tidal flats.

The filtration rates obtained in this study, especially at Sihwa tidal flat, were higher than those reported in previous studies. While Lee [21] in similar field experimental settings as the current study reported the filtration rate of the Manila clams ranging from 0.13 to 0.97 L h^-1 gDW^-1, our study reports significantly higher values (2.29 to 5.46 L h^-1 gDW^-1), which could be related to differences in experiment methods. In their experiment, the flow rate in the chamber was maintained at 65 cm s^-1, which was 13 times higher than that used in the present study. At flow rates of 15–20 cm s^-1 or higher, the bottom sediments and feces of bivalves begin to be resuspended [22]. Maintaining a flow rate of 65 cm s^-1 in the chamber caused resuspension of sediments, meaning that POM was continuously supplied from the sediments to the water column during the experiment. Thus, the decrease in POM concentration due to clams was offset by the supply from sediments, and the filtration rate of clams was consequently underestimated.

In another field experiment, Aoyama and Suzuki [23] reported the filtration rate of 3.44 L h^-1 gDW^-1. They conducted their experiment by adding 0.6–1.0 L of diatoms (a mixture of Chaetoceros sp. and Skeletonema sp.) cultured in a laboratory into the culture solution to greatly increase the initial concentrations of indicators in the chamber. These high initial concentrations promoted feeding activity of clams and a relatively high filtration rate was observed. In other words, these findings indirectly suggest that the feeding activity of clams may increase in areas where a large amount of food is available in the seawater, as was the case at Sihwa tidal flat. This likely explains our finding that the filtration rate at Sihwa tidal flat was higher than that at Geunso tidal flat.

In conclusion, this study evaluated differences in the filtration rate of R. philippinarum due to differing hydrographic regimes between two tidal flats that the influence of differing hydrographic conditions on the filtration rates of Manila clams has not been studied previously. Though this study was limited by factors such as salinity, turbidity and particle size that affect variations in filtration rates, the findings provide important information about the effects of hydrographic regime on the filtration rate of this species.

Supporting information

S1 Table. Variations on mean concentration of SS and POM in two experimental chambers and seawater over the entire study period at Geunso and Sihwa tidal flats.

(DOCX)

Click here for additional data file.^{(22.7KB, docx)}

S2 Table. Comparison of filtration rate of Ruditapes philippinarum for POM between Geunso and Sihwa tidal flats over time.

(DOCX)

Click here for additional data file.^{(15.2KB, docx)}

Acknowledgments

We would like to thank Dr. Jennifer Ruesink and anonymous reviewer for their valuable comments.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2018R1D1A1B07049632, KIOST PN67930) and also was supported by a grant from the ‘Development and security of mud shellfish resources in the small-sized tidal flat waterways’, funded by the Ministry of Oceans and Fisheries, Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

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PLoS One. doi: 10.1371/journal.pone.0228873.r001

Decision Letter 0

Ismael Aaron Kimirei

6 Aug 2019

PONE-D-19-15055

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

PLOS ONE

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript provides data from a 4-hour study of water properties in 63-L in situ containers on two tidal flats, comparing how water properties change with and without manila clams. The overall intent involves some clever field equipment to get measurements of what clams are doing in situ. On the other hand, the statistical methods are currently insufficient (and insufficiently-documented) to reveal if a) clams affect water properties, and b) this effect differs between the two sites. (And c) if this effect differs over time during the 4-hour study) Since there are just two replicates of each chamber type, the power to detect a difference is low, but such a statistical approach is essential to scientific assessment of hypotheses. More details on this issue are provided in the line-by-line comments below.

Abstract line 29-30: inconsistent results – stated to be higher chl at Sihwa with 3.1 ug/L, vs. Geunso at 52.5 ug/L – clearly Geunso is higher than Sihwa by the numbers. 52.5 ug/L chl at Geunso is not evident from any results in the paper, for instance, Fig. 6 shows chlorophyll concentrations to be on the order of 5 ug/L. I think it is inappropriate to use 52.5 and 3.1 ug/L in the abstract, as these data were not collected in the present study but are cited from other work.

Line 56: ecosystem homeostasis? Many ecologists – I guess myself included – would take issue with a sense of ecosystems self-regulating to avoid change. The construction of the sentence also implies that organic matter (“it”) contributes to homeostasis. The authors should consider whether they really mean ecosystem productivity supported by organic matter, or ecosystem resilience to nutrient inputs achieved through suspension-feeders?

Line 62: Sihwa tidal flat is first mentioned here in the paper, without any description of where in the world it is, or why its function is worthy of note. I recommend a description here that introduces Sihwa, its size and construction, and its management challenges. This will motivate why clams could serve to mitigate water pollution there.

Line 69: alternative to what? Seems like it’s possible to instead say “as a method that can mitigate nutrient pollution or impaired water quality.”

Line 93 “riparian” refers to the out-of-water habitat adjacent to a water body, usually a river. Thus a tidal flat cannot be a riparian area, since it is covered with water (and also not a river): drop “is a riparian area that”

Line 102: what is the frequency of high and low tides at the Sihwa site? Based on Fig. 3, tidal period is a major feature that distinguishes the two sites!

Line 150: authors need to provide information on the biomass of clams in each chamber. It is stated (line 175) that clams were sacrificed at the end of the experiment for biomass and size measurements; these results are important for readers to know, because filtration rates can change with bivalve size, or high biomasses of clams may undergo exploitation competition that reduces apparent filtration rate.

Line 157 and 164: I think I understand that the seawater flow rate was not a continuous flow through the container, but rather the speed at which water moved around within the container – was this more like a current, or more like turbulence? And from later information I assume that removing 5x 1L from the 63-L chambers did not require adding any new water? So the water sampled in the chamber at the beginning of the submergence period was the same as at the end?

Line 165: more important to state the time between successive samples than how many samples were collected, given that ultimately filtration rate is based on the steepest decline between successive samples, not fitting all data

Line 175: Oxygen did not drop below 80% so the aerator was not used? Or because the aerator acted to elevate oxygen concentrations? I think from the figures it would be accurate to say: “Chambers were equipped with aerators (“bubblers” in Fig. 2) to prevent dissolved oxygen from dropping below 80% and inhibiting the metabolic activity of clams, but these were not necessary to activate during the experiment at either site.”

Line 176-178: very sparse description of the calculations used to calculate removal rate by comparing clam and no-clam containers, and how to distinguish filtration from sedimentation. Do you mean “Removal rate of suspended material from the water by clams was evaluated by the change in water properties in the clam-containing chambers over time, adjusted by changes that were occurring concurrently in the control chambers. Specifically, the change between successive samples in each control chamber was subtracted from the change in the paired clam chamber, because losses from the water column in the control chamber were due to natural sedimentation rate.”

Line 179-180: not clear how measuring water column particles before and after experiment in all containers would be a metric of natural sedimentation

Line 197: does the desiccator contain HCl? Or does the desiccator contain beakers of HCl, one for each sample?

Line 171 says 250 ml were used for chl analysis, but line 202 says 300 ml

Lines 208-225 only address calculations for filtration rate, whereas the authors also need to consider how they are going to test statistically whether the patterns in the two treatments and two tidal flats are different. Asserting these differences by examining time series or by comparing the magnitude of two means, as is currently the case in the results, is not sufficient support for the claims and conclusions.

Line 225: maximum slope should be specified to refer to Z (logarithmic change in concentration), rather than the absolute difference in concentration. Also, were these calculations carried out only with the clam-containing containers, or based on the difference from reference containers? And how were the two replicates of each treatment handled? i.e. calculate the average concentration at each time based on two chambers before doing the calculations, or do the calculations for each chamber separately? And if the latter, and if including how the water properties in these chambers change relative to controls, then how were the chambers with and without clams paired up?

Line 231: specify “surrounding seawater”

Line 230-253: Multiple statements exist in these paragraphs about significance, or one group being different from another. Typically such statements in science need to be accompanied by a statistical test. So this section needs some rewriting (or the authors need to set up the statistical tests – I think these are less important here for the physical conditions of the samples, and more important to have statistical tests associated with comparing reference and control chambers and the two sites). Also, some of the major distinctions are not mentioned, for instance: a) thoughout 4 hours, temperature with clams at Geunso is 0.5 degrees warmer than without clams, b) dissolved oxygen outside the chambers is generally below – sometimes a lot below – the concentrations inside the chambers, and c) chambers seem to be less variable in water temperature than the water they are bathed in.

In the discussion section, the authors provide possible explanations for differing filtration rates between the two tidal flats, and why these may differ from other studies. This general content is relevant and important. Several comments here regarding logical connections drawn in the discussion. 1) I find it puzzling that the higher quality of food at Sihwa relative to Geunso can be used as an explanation for higher filtration rate at Sihwa, because in the present study, the food concentrations and quality appear very similar (although no statistical test has been done). The authors need to be careful about using results from other studies (about growth rates and chlorophyll concentrations) to provide a mechanism in the current case, since I think that the filtration rate response for this mechanism is expected to be immediate. 2) The other mechanism that the authors provide for the higher filtration rate at Sihwa is in regards to circatidal rhythms, but it appears that these might also be immediate responses – that is, ebb and flood tides reduce filtration not because of inherent circatidal rhythms but because rapid water motion resuspend sediment that interferes with feeding. 3) Overall in the discussion, the authors attribute differences in filtration to hydrographic regime, but need to keep in mind that the filtration rate measurements were done in closed chambers that had identical water motion conditions, so the clams would have to respond to longer-term factors than what they are experiencing during the experiment; yet the authors look at the changes over time in filtration rate as a response to immediate conditions.

Fig. 5, 6 description – “two experimental chambers” is confusing. Does this refer to replicates or treatments?

Fig. 7 description – insufficient to have the only material about statistical tests here. Statistical tests need to be set up in the methods, with results in tables or supplemental material. The authors need to clarify what is a “replicate” and whether the comparisons indicated by letters a and be are only done within sites or also across sites. I am afraid that the authors are considering “replicates” as filtration rate detected by chl, PON, and POC. Rather, a replicate needs to be a chamber.

Table 2 as currently written is redundant with the text of results.

In all figures, authors need to define what is shown by error bars – I assume SD of two replicates, but have really no idea!

Data availability: authors should provide raw data for measurements of clams and water properties. The data provided in figures are based on a series of calculations.

Reviewer #2: The purpose of this study was to investigate the filtration rates of the Manila clam between two natural sites. The test device is widely suggested to use for the study on the physiology of the benthic shellfish. Because we always test the filtration rate or ammonia excretion rate in the lab. It might be just 1 or 2 factors for the experiment. This paper show us a interesting device to test the filtration rate at the complicate environments, including the temperature, salinity, suspended particles, current speed, tidal flat, and chlorophyll a. The results provide important information for the role of the Manila clam in the ecology and aquaculture. But the grammars of the manuscript should be carefully revised before publication. Some suggestions are as below,

Line 19: manila clam should be Manila clam, the same changes should be made on line 89.

Lines 28 and 30: give more details for the latter and former;

Line 51: In addition, the…. What is purpose to show this sentence at this paragraph?

Line 78-80 this part should be rewritten to show more significant purposes for this study.

Line 174-175 How can you measure the dissolved oxygen, shell length and body weight?

Line 175 body length should be shell length.

Line 229-253 provide more environmental factors in this part, such as salinity, ammonia, nitrogen and phosphate, because these factors are also effected the filtration rate on Manila clam.

Line 452-455 this part should be rewritten to conclude the main results for this manuscript, and explain how to use the findings to do the further study.

The English of the discussion should be polished by the English speaker, and more references should be added to support the findings of this study.

The figures are obscure. The clear figures should be provided for publication.

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Reviewer #1: Yes: Jennifer Ruesink

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PLoS One. 2020 Feb 10;15(2):e0228873. doi: 10.1371/journal.pone.0228873.r002

Author response to Decision Letter 0

25 Oct 2019

5. Review Comments to the Author

→ These values were deleted in abstract (L 35-36).

→ We intended to mean ecosystem productivity, thus, it was corrected to ecosystem productivity (L 62-63).

→ The description of Sihwa tidal flat was added in L 69-74: Sihwa lake is an artificial lake formed by the construction of the Sihwa dike as part of the Great Reclamation Comprehensive Development Project, and is a representative area that underwent major environmental changes. Sihwa dike caused dissipation of intertidal zone with severe water quality deterioration. Thus, the government withdrew desalination and started the seawater circulation through sluice gate operation, as a result, tidal flat were partially created with repeated submergence and emergence.

Line 69: alternative to what? Seems like it’s possible to instead say “as a method that can mitigate nutrient pollution or impaired water quality.”

→ It was corrected according to your suggestion as follows: Therefore, the seawater purification capacity of filter feeders is essential as a method that can mitigate nutrient pollution (L 75-77).

→ It was corrected according to your suggestion.

Line 102: what is the frequency of high and low tides at the Sihwa site? Based on Fig. 3, tidal period is a major feature that distinguishes the two sites!

→ Due to the Sihwa tidal flat has an artificially controlled tide, it is different from tidal cycle of general tidal flat. The mean frequency of high and low tides in Sihwa tidal flat was 7 times and 10 times in the month when we conducted the experiment, respectively.

→ We added the morphometric information of clams in Table 3. Generally, filtration rate in terms of filtering ability for seawater increase as clam size decrease. This relationship may be due to the decrease with age of the gill surface to body size ratio and to the higher metabolic demands of the younger individuals (Segade et al., 2003; Sylvester et al., 2005; Han et al., 2008). Han et al. (2008) reported that the filtration rate of small Ruditapes philippinarum (0.2 g DW) was about 28% higher than that of large one (0.4 g DW) at 20 °C. This indicated that the filtration rate of Ruditapes can be changed by size and biomass, but, the difference on filtration rate is too great to explain by size and biomass in this study. The results and discussion were described in the each section.

→ The seawater flow rate was not a continuous flow but it was more like a current. The volume of each chamber is 113 L, we couldn’t find 63 L chamber volume as you mentioned. But, there was no addition any new water during the experiment.

→ The water samples were collected at regular intervals with an hour for Sihwa tidal flat and an hour and forty mins for Geunso tidal flat, respectively. It is shown in Figs. 5 and 6 at x-axis.

→ Oxygen concentration in the chamber did not drop below 80% during experiment period in both sites. Thus, aerator was not used as you mentioned. The part of oxygen concentration was corrected according to your suggestion as follows: Chambers were equipped with aerators (“bubblers” in Fig. 2) to prevent dissolved oxygen from dropping below 80% and inhibiting the metabolic activity of clams, but these were not necessary to activate during the experiment at either site.

→ We did not measure natural sedimentation, thus, it was deleted.

Line 179-180: not clear how measuring water column particles before and after experiment in all containers would be a metric of natural sedimentation

→ We did not measure natural sedimentation, thus, it was deleted.

Line 197: does the desiccator contain HCl? Or does the desiccator contain beakers of HCl, one for each sample?

→ The desiccator contained beakers of HCL one for each sample.

Line 171 says 250 ml were used for chl analysis, but line 202 says 300 ml

→ It was corrected to 250 mL in L 212.

→ The aim of this study is to evaluate difference on filtration rate between two tidal flats with different hydrographic regimes. Because the amount of particulate matter in the chamber is limited, as the observation time becomes longer, the rate of concentration decrease becomes smaller, and thus the filtration rate can be underestimated as we described. Thus, we think that time series filtration rate can’t reflect actual decreasing tendency of that.

→ The calculations of POM filtration rate in the water column were carried out only with the clam-containing chambers and were calculated based on the average concentration at which maximum slope of the decreasing concentration in two chambers.

Line 231: specify “surrounding seawater”

→ It was corrected to surrounding seawater in L 241.

→ We conducted statistical analysis in water temperature between chambers and seawater as well as both tidal flats and additional results were described in this section as follows : (a) The water temperature in the Ruditapes chamber was highest during experiment with mean values of 17.7±0.2 °C, followed by control chamber (17.1±0.2 °C) and seawater (16.8±0.2 °C). Although, there was statistical significance (p<0.05), but difference of temperature was not differ greatly between between the Ruditapes chamber and the control chamber; due to the closed environment, the water temperature inside the chamber was slightly higher than that of the seawater around the chamber (p<0.05). (b) Unlike Sihwa tidal flat, the mean water temperature was not significantly different among the Ruditapes chamber, the control chamber, and the seawater with values of 18.9±0.3, 18.9±0.3 and 19.0±0.7 °C in Geunso tidal flat, respectively (p<0.05). The mean water temperatures of Ruditapes chamber, the control chamber, and the seawater were significantly higher in Geunso tidal flat than in Sihwa tidal flat in entire experiment (p<0.05) and chambers were less variable in water temperature compared to the seawater. (c) The dissolved oxygen in the seawater was generally lower than in the chambers during entire experiment in both tidal flats.

→ 1) Although, the initial chl a concentration of this study was similar in both tidal flats, however, the annual mean value of that was significantly higher in Sihwa tidal flat than in Geunso tidal flat. This suggests that the differences of food quantity and quality likely caused different feeding activity which resulted in different filtration rate between both tidal flats.

2) We filled a chamber in the laboratory with the same sediment and seawater as the chamber installed at the experimental site prior to the experiment, and determined the seawater flow rate (5 cm s-1) from the agitator at which organic matter in the sediment was not suspended and natural sedimentation of POM was prevented. Thus, we think that the feeding activity of clams was unaffected by that. Kim et al. (1999) have reported that activity rhythms of clams are controlled not only by exogenous factors, but also by an endogenous circatidal periodicity. They showed that manila clams removed from their natural environment and maintained for 9 weeks in continuously immersed conditions exhibited a clear endogenous circatidal rhythm. Therefore, we think that the different filtration rate between two tidal flats was caused by inherent circatidal rhythms not immediate responses.

3) The aim of this study is to evaluate difference on filtration rate between two tidal flats with different hydrographic regimes. Because the amount of particulate matter in the chamber is limited, as the observation time becomes longer, the rate of concentration decrease becomes smaller, and thus the filtration rate can be underestimated as we described. Thus, we think that time series filtration rate can’t reflect actual decreasing tendency of that.

Fig. 5, 6 description – “two experimental chambers” is confusing. Does this refer to replicates or treatments?

→ Two experimental chambers represent replicates.

→ The letters a and b represent comparison of mean filtration rate within sites. The mean filtration rate represents POC, PON and chl a removal rate, thus, the error bars indicate 95% confidence intervals of each filtration rate.

Table 2 as currently written is redundant with the text of results.

In all figures, authors need to define what is shown by error bars – I assume SD of two replicates, but have really no idea!

→ Table 2 (Table 3 in revised manuscript) is a main result in this experiment which can help see it to readers. The error bars in figures were defined.

Data availability: authors should provide raw data for measurements of clams and water properties. The data provided in figures are based on a series of calculations.

→ We added morphometric information of clams in Table 2 and water property data was provided in supplement.

Line 19: manila clam should be Manila clam, the same changes should be made on line 89.

→ It was corrected.

Lines 28 and 30: give more details for the latter and former;

→ We couldn’t understand what you mean exactly.

Line 51: In addition, the…. What is purpose to show this sentence at this paragraph?

→ We intended to describe the important function of bivalve in the ecosystem.

Line 78-80 this part should be rewritten to show more significant purposes for this study.

→ We think that this sentence implies the purpose of this study well.

Line 174-175 How can you measure the dissolved oxygen, shell length and body weight?

→ The dissolved oxygen was measured by Fibox-3 oxygen meter. It was described in material section (L 165). The shell length was measured with calipers and the body weight was obtained by weighing.

Line 175 body length should be shell length.

→ It was corrected.

Line 229-253 provide more environmental factors in this part, such as salinity, ammonia, nitrogen and phosphate, because these factors are also effected the filtration rate on Manila clam.

→ We did not measure additional environmental factors such as salinity, ammonia, nitrogen and phosphate.

Line 452-455 this part should be rewritten to conclude the main results for this manuscript, and explain how to use the findings to do the further study.

→ It was corrected as follows: This study is significant because it evaluated differences in the filtration rate of R. philippinarum due to differing hydrographic regimes between two tidal flats that has not been studied. Though this study was limited by factors that affect variations in filtration rate, the findings provide important information about the effects of hydrographic regime on the filtration rate of this species.

The English of the discussion should be polished by the English speaker, and more references should be added to support the findings of this study.

→ The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/ASji1i We added more references.

The figures are obscure. The clear figures should be provided for publication.

→ We have uploaded clear figures but we have no idea why that were obscured in PDF file. We think that it is likely due to technical problem.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0228873.r003

Decision Letter 1

Ismael Aaron Kimirei

26 Nov 2019

PONE-D-19-15055R1

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

PLOS ONE

Dear Dr Koo,

==============================

ACADEMIC EDITOR: pleased respond to all the comments by reviewer #1 on the validity of your treatments and the statistics. The comments are explicit and understandable. It should be possible to follow them and make necessary corrections or revisions to your MS and submit for further consideration or provide a rebuttal accordingly. I would consider this MS one more time.

==============================

We would appreciate receiving your revised manuscript by Jan 10 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: As I wrote before, it is not appropriate to combine measurements of three different components of the seston (POC, PON, chla), all collected in the same chamber at the same time, to calculate a mean filtration rate. Mean filtration rate could be based on the filtration of the same component of the water across the two experimental chambers at each site, after each is adjusted for the change in the companion reference chamber. A t-test could then be done using two samples at each site, since there were two experimental chambers (containing clams) at each site. (Note: I am actually uncertain whether the “four chambers” on line 155 refer to four chambers per tidal flat, or four chambers in total. If the latter, then there is not a possibility to compare the two tidal flats statistically, as there is only one filtration value for each flat. But in their response the authors write “two experimental chambers represent replicates” so I think they should be able to do statistics with n=2 at each tidal flat.)

Line 230: How were the control chambers used in the calculations of filtration rate? The authors write: “the filtration rate was calculated for the period in which the maximum slope of the decreasing indicator concentration was observed.” There are three issues to address: 1) The maximum slope at Sihwa (Fig 5) occurs in the first time interval, but is equivalent for clam and reference chambers, such that no net filtration would be indicated IF the values were properly adjusted by comparison with reference; 2) the maximum slope seems to indicate the steepest portion of Figures 5 and 6, rather than the portion with the largest exponent in the equation for filtration; 3) selecting a time of maximum filtration from among the time periods creates potential bias if clam filtration at one site is more variable than at the other. The authors should do the following: 1) determine which reference chamber to pair with each clam chamber, 2) adjust Ct in the clam chamber so that it accounts for change in the reference chamber (add C0-Ct in the reference chamber to Ct in the clam chamber), 3) determine Z for each time interval, 4) select maximum Z to calculate FR (since the authors indicate that they think maximum filtration is the best metric of filtration rate), 5) calculate Z for the entire 4 hours as another way to compare between the two tidal flats. After this process has been done, then the authors can carry out t-tests on n=2 samples per site, for each of the components in the water, and for maximum and 4-hour filtration rates.

Table 2: Fresh weight is always a wet weight. So Fresh dry weight does not make sense. Are these values the Meat dry weights? Also on line 189.

Error bars on the figures: Thank you for specifying that the error bars represent 95% CI, but that does not address the whole issue I brought up last review. Is this the 95% CI based on n=2 (number of chambers), and if not, what is considered a sample?

Line 29: Rewrite – Clams were smaller at Sihwa than Geunso sites, but the filtration rate of 50 clams per chamber was higher at Sihwa than Geunso. This difference was probably not due to the immediate environmental conditions, since the enclosed chambers experienced no net current or tidal exchange during the 4-hour monitoring interval, and the initial seston and chlorophyll concentrations in the chambers were similar. Instead, these filtration rates may differ due to the hydrographic regime, since Sihwa tides are limited by sluice gates. Generally, at Geunso relative to Sihwa, current speeds are faster, and submergence times are shorter. Sihwa typically has higher chlorophyll a concentrations, as well as better food quality based on C/N ratio of POM (Sihwa 12.8, Geunso 15.6). The endogenous circatidal rhythm…

I am not necessarily convinced that the sentence about endogenous circatidal rhythm follows from the data, since there are no data showing that filtration rate changes over time more at Geunso than at Sihwa.

Line 39: There is no information about the effects of hydrographic regime, which implies that causality has been tested. This sentence should be rewritten: These findings suggest that hydrographic regime could be important in understanding in situ filtration rates.

These issues in the abstract also apply to the discussion.

Reviewer #2: The manuscript has been revised according to the comments. The questions have been answered well. The manuscript is recommended to be published in this journal.

**********

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Reviewer #1: Yes: Jennifer Ruesink

Reviewer #2: No

PLoS One. 2020 Feb 10;15(2):e0228873. doi: 10.1371/journal.pone.0228873.r004

Author response to Decision Letter 1

7 Jan 2020

→ The mean filtration rate (three different components of seston) was deleted according to your suggestion. The four chambers represent two experimental chambers (with clams) and two control chamber (without clams) at each site. Thus, we have done t-test using two samples at each site.

→ The filtration was recalculated according to your suggestion (Table 3). The experimental chambers (with clams) were paired up with each control chamber (C1-R1/C2-R2, Fig. 2) and then Ct value of experimental chambers was adjusted by each control chamber value (C0-Ct). The maximum Z among time periods was selected and then filtration rate was calculated. A t-test was carried out based on two samples at each site as well as each component (POC, PON and Chl a). The filtration rate during entire experimental period was described in Table S2.

Table 2: Fresh weight is always a wet weight. So Fresh dry weight does not make sense. Are these values the Meat dry weights? Also on line 189.

→ It was corrected to flesh dry weight in Table 2 and manuscript.

→ The statistical analysis was conducted based on two samples as we noted above.

→ It was corrected as follows : The filtration rate of clams for POM at Sihwa tidal flat (2.86 for POC, 2.29 for PON and 5.46 L h-1 gDW-1 for Chl a) was higher than that at Geunso tidal flat (0.61 for POC, 0.89 for PON and 2.54 L h-1 gDW-1 for Chl a) which resulted from differences in the hydrographic regime, including tide characteristics, current speed and submergence time, and food quantity and quality (L-29). The description of endogenous circatidal rhythm was deleted in the manuscript.

→ It was corrected according to your suggestion as follows : These findings suggest that hydrographic regime could be important in understanding in situ filtration rates of R. philippinarum.

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PLoS One. doi: 10.1371/journal.pone.0228873.r005

Decision Letter 2

Ismael Aaron Kimirei

16 Jan 2020

PONE-D-19-15055R2

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

PLOS ONE

Dear Dr Koo,

==============================

ACADEMIC EDITOR: I am pleased with how the MS has taken shape for the better and would like to invite you to submit minor edits and clarifications as presented in the annotated pdf copy of the revised MS.

==============================

We would appreciate receiving your revised manuscript by Mar 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Ismael Aaron Kimirei, Ph.D.

Academic Editor

PLOS ONE

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PLoS One. 2020 Feb 10;15(2):e0228873. doi: 10.1371/journal.pone.0228873.r006

Author response to Decision Letter 2

18 Jan 2020

→ It was corrected as follows : Dissolved oxygen was monitored to prevent a decrease in metabolic activity due to depletion of dissolved oxygen in the chamber, and when the concentration decreased below 80%, oxygen was supplied using an aerator installed inside the chamber (L141-143).

→ 500 ml is correct volume. We did not use all water samples (750 ml except 250 ml for chlorophyll a analysis) for SS, POC and PON analysis (L 205).

→ It was corrected as follows : Thereafter, no significant changes in the chlorophyll a concentration in the Ruditapes chamber occurred, while that in the control chamber tended to increase (Table S1, L359-361).

→ It was corrected as follows : Han et al. (2008) reported that the filtration rate of small Ruditapes philippinarum (0.2 gDW) was about 28% higher than that of large ones (0.4 gDW) at 20 °C. This indicated that the filtration rate of Ruditapes can be changed by size and biomass, but, the difference in filtration rate is too great to be explained by size and biomass in this study (L 404-407)

→ It was corrected as follows : One experimental condition that must be considered carefully when calculating the amount of POM removed from seawater by filter feeders using a closed chamber, as indicated in this study, is that the concentration of POM introduced into the chamber at the beginning of the experiment should be maintained (L408-411).

→ It was corrected as follows : The relatively high filtration rate at Sihwa tidal flat can be attributed to this difference in hydrographic regimes. Another factor affecting the difference in filtration rate is high productivity in the water column at Sihwa tidal flat, which causes a quantitative difference in the food available to Manila clams. The Manila clams at Sihwa tidal flat had a higher growth rate than those at Geunso tidal flat, and the mean chlorophyll a concentration in the water column was significantly higher at the former site than the latter, with values of 3.1 and 52.5 μg L-1, respectively (L435-440) .

→ It was corrected as follows : The filtration rates obtained in this study, especially at Sihwa tidal flat, were higher than those reported in previous studies. While Lee [21] in similar field experimental settings as the current study reported the filtration rate of the Manila clams ranging from 0.13 to 0.97 L h-1 gDW-1, our study reports significantly higher values (2.29 to 5.46 L h-1 gDW-1), which could be related to differences in experiment methods. This value is significantly lower than the experiment results of the present study (L452-456).

→ It was corrected as follows : In another field experiment, Aoyama and Suzuki [23] reported the filtration rate of 3.44 L h-1 gDW-1 (L 464-465).

→ It was corrected as follows : In conclusion, this study evaluated differences in the filtration rate of R. philippinarum due to differing hydrographic regimes between two tidal flats that the influence of differing hydrographic conditions on the filtration rates of Manila clams has not been studied previously. Though this study was limited by factors such as salinity, turbidity and particle size that affect variations in filtration rates, the findings provide important information about the effects of hydrographic regime on the filtration rate of this species (L472-475).

→ We added this sentence : We would like to thank Dr. Jennifer Ruesink and anonymous reviewer for their valuable comments.

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PLoS One. doi: 10.1371/journal.pone.0228873.r007

Decision Letter 3

Ismael Aaron Kimirei

27 Jan 2020

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

PONE-D-19-15055R3

Dear Dr. Koo,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ismael Aaron Kimirei, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

In the discussion, LN 456, please delete this sentence: "This value is significantly lower than the experiment results of the present study" Because it is already captured in the preceding sentence.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0228873.r008

Acceptance letter

Ismael Aaron Kimirei

30 Jan 2020

PONE-D-19-15055R3

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

Dear Dr. Koo:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Variations on mean concentration of SS and POM in two experimental chambers and seawater over the entire study period at Geunso and Sihwa tidal flats.

(DOCX)

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S2 Table. Comparison of filtration rate of Ruditapes philippinarum for POM between Geunso and Sihwa tidal flats over time.

(DOCX)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes

Bon Joo Koo

Jaehwan Seo

Roles

Abstract

Introduction

Study area

Fig 1.

Table 1. Comparison of hydrographic regimes between Geunso and Sihwa tidal flats.

Materials and methods

1) In situ experiments

(1) Experimental equipment

Fig 2. Schematic diagram of the in situ experiment to measure filtration rates of Ruditapes philippinarum.

(2) Installation of chambers and sampling

Fig 3.

2) Sample analysis

(1) Total suspended solids

(2) Particulate organic carbon and particulate organic nitrogen

(3) Chlorophyll a

3) Calculation of POM filtration rate in the water column

Results

1) Water temperature and dissolved oxygen

Fig 4.

2) Suspended solids

Fig 5.

Fig 6.

3) POC and PON

4) Chlorophyll a

5) The filtration rate of R. philippinarum

Table 2. Comparison of morphometric data of Ruditapes philippinarum between Geunso and Sihwa tidal flats.

Table 3. Comparison of filtration rate of Ruditapes philippinarum between Geunso and Sihwa tidal flats.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ismael Aaron Kimirei

Roles

Author response to Decision Letter 0

Decision Letter 1

Ismael Aaron Kimirei

Roles

Author response to Decision Letter 1

Decision Letter 2

Ismael Aaron Kimirei

Roles

Author response to Decision Letter 2

Decision Letter 3

Ismael Aaron Kimirei

Roles

Acceptance letter

Ismael Aaron Kimirei

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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