Effect of yellow clay on respiration and phytoplankton uptake of bivalves

Authors


*Tel: 82-51-720-2522. Fax: 82-51-720-2266. Email: cklee@nfrdi.re.kr

Abstract

ABSTRACT:  Yellow clay dispersion has been applied to mitigate red tides in Korea since 1995. The present study documents the effect on physiology of two marine bivalves by examinations of clay uptake, respiration, and clearance rate (CR) of animals after the treatment of yellow clay. The amount of clay in gills and inside cavities of shellfish was increased with time, and the inside of the shellfish filled with clay in 2 h. The ejection of the accumulated clay was evident from as early as 30 min after removal from clay pools. Animals removed from the clay pool discharged all observable clay by 6 h. Within 30 min, CR decreased quickly and returned to the level of the untreated control groups within 2 h.

INTRODUCTION

Red tides, or harmful algal blooms, have caused enormous economic and environmental damage in the world.1,2 Among several mitigation techniques that have been applied,3 yellow clay dispersion is recognized as the best practical method in Korea,4–6 as it is cheap and easy to apply in the field without notable effects on water quality.7–9 The removal efficiency with clay can be as high as 80%.10

The effect of clay should be considered when investigating abnormalities in the ecosystem, even after the clay washed out of the area, because it can result in delayed or remote animal exposure. Several biological indices have been developed and used to assess the ecologic status of marine environments.11 Mortality as an indication of the effect on animals has turned out to depend on the kinds of animals and the test conditions.12 Benthic species are sensitive indicators of temporal as well as chronic disturbances in the marine environment because of their relatively sessile habit, wide distribution, inability to avoid the clay, relatively long life and their integration with water and sediment quality conditions.13–16

Changes in the ingestion and respiration of animals would be a good indicator for the ecologic effect on the clay dispersion. The clearance rate, respiration rate and growth rate had proven to be meaningful to evaluate sublethal effects on benthic organisms.17–19 Some of these studies revealed the potential effects on the benthic ecosystem by application of clay (1–10 g/L),18,19 while the low concentration of clay may enhance digestion processes in benthic animals including cucumbers and scallops.20 In studies like the above it is very important to use local yellow clay collected for the mitigation practice in the field, to sample the blooming phytoplankton from the fisheries area, and to consider the local benthic animals. This study aimed to examine the effect of concentration of yellow clay on the physiology of two important fisheries animals: mussels and oysters in the southern coast of Korea, by the examination of the internal organs, measurement of oxygen consumption, and the grazing ability of the animals after yellow clay treatments.

MATERIALS AND METHODS

Experimental organisms and acclimation

Mussel Mytilus galloprovincialis and oyster Crassostrea gigas were provided by the National Shellfish Research Center, Namhae, Korea. To remove basiphytes, approximately 100 individuals of each species measuring 5–7 cm shell length were gently cleaned with a brush and washed in running sea water and filtered on a 10-μm filter. The animals were then transferred into 500-L flow-through aquaria containing 400 L of filtered sea water at 21°C. Shellfish were acclimated and starved for 24 h. Experiments were conducted at a low light intensity (<5 μmol/s per m2).

Clay uptake experiment

Yellow clay used for this experiment was collected from Tongyeong, Korea. The clay consists of quartz and elements including Si (211.7 mg/kg), Al (842.2 mg/kg), Fe (62.3 mg/kg) and Mg (337.3  mg/kg).12 After all discernible lumps and friable particles had been broken, yellow clay was ground and sieved with a 850-μm sieve. The clay was then placed in a drying oven (60 ± 5°C) for two days. After cooling and weighing, powdered clay was mixed well with filtered sea water in 25-L vessels to make suspensions of 0.01, 0.05, 0.25, 1.25 and 6% (w/w) concentration. After the 24-h acclimation, animals were taken from the aquaria. Magnetic stirrers were used to assure uniform mixing of clay suspensions during the whole experiment. Twelve animals were placed in each 25-L vessel to be tested for yellow clay uptake. Including a vessel without yellow clay as a control group, a total of six vessels were filled with 12 animals each. Each two individuals from each vessel were harvested at time (t) = 0.5, 1, 2, 4 and 6 h, weighed, and dissected to look for the accumulation of yellow clay in their organs.

Oxygen consumption

The change in dissolved oxygen (DO) level was measured as an indication of the oxygen consumption by the shellfish using an oxygen and temperature meter (YSI-55, YSI Environmental, Yellow Springs, OH, USA). Filtered sea water was used in all of the experiments. The shellfish were maintained in air-sealed respiration chambers for approximately 2 h before the tests to eliminate ‘handling stress.’ The amount of DO (mg/L) was measured every 10 min and was compared after addition of a series of yellow clay concentrations of 0.25, 0.75 and 1.25% (w/w). Background oxygen consumption was measured by running blanks (i.e. no yellow clay) for the control. Respiration ratio (RR) was calculated as oxygen consumption rate (mg O2/mg wet weight per h):

image

where V is the volume of the chamber (1 L), n is the weight of a shellfish (mg wet weight), t is the duration of the experiment (hours), DO0 is the DO at t = 0 and DOt is the DO at time t in vessels with animals. The DO at different yellow clay concentrations were tested and compared by regression analysis and one-way analysis of variance (anova).21

Recovery experiment

To test the recovery of grazing activity of the two shellfishes, phytoplankton from local sea water was used to imitate the field conditions before the clay dispersion. Local sea water was pumped at the sea along the coastline of Namhae, Korea and transported to the laboratory. Within one hour, the local sea water was filtered through a 200-μm mesh net to remove the zooplankton and was called the 100% plankton pool. The composition and the amount of phytoplankton species were determined under light microscopy at 400× magnification using a Sedgwick–Rafter slide. The concentration of phytoplankton in the 100% plankton pool was 43 100 cells/mL.

The main component of the phytoplankton pool was diatoms with a small amount of chlorophytic species (Table 1). To find the optimal concentration of phytoplankton for the grazing experiment, the series of 10, 20, 60 and 100% of phytoplankton mixture from the phytoplankton pool was prepared and clearance rate (CR) was calculated according to time after addition of animals.

Table 1.  Composition of phytoplanktons used for grazing experiments
PhytoplanktonNumber/mLProportion (%)
Skeletonema costatum (Diatom)290 00067
Chaetoceros pseudocurvisetus (Diatom)56 00014
Leptocyindrus danicus (Diatom)36 0008
Pseudo-nitzschia pungens (Diatom)31 0007
Asteronellopsis gracialis (Diatom)12 0003
Thalassiosira rotula (Diatom)5 5001
Dunaliella sp. (Chlorophyta)500
Total431 000100

As a control, a grazing experiment with graded mixtures of plankton (0, 10, 20, 60 and 100%) was conducted with or without animals to find the best concentration of phytoplankton for the uptake experiment. First, the shellfish were transferred after 2-h treatment with different concentrations of clay. A 2-hour treatment was used because it was the required interval for full clay uptake from the clay uptake experiment. Second, 12 individuals from different clay concentrations were placed into phytoplankton containers. Third, at t = 0.5, 1, 2, 4 and 6 h, two individuals from the pool were harvested and the changes in phytoplankton concentration were measured by fluorometry (see below). Harvested animals were weighed and then dissected to examine the amount of clay remaining inside their organs. The relative amount of clay found in shellfish organs was categorized into three levels that were marked using a ‘+’ symbol. Wet weight (wet wt) was determined to allow the expression of phytoplankton uptake on a weight-specific basis (μg chlorophyll/mg wet wt).

Pulse amplitude-modulated fluorometry (PAM) has been shown to be a useful tool for grazing experiments with mixtures of different phytoplankton species.22 This method allows for quantitative differentiation between cyanobacteria, green algae and diatoms using chlorophyll fluorescence of specific pigments.23,24 During the grazing experiment after the animals were taken, 5 mL of sea water was sampled at t = 0, 0.5, 1, 2, 4 and 6 h to measure the concentration of phytoplankton using a pulse amplitude modulation phytoplankton analyzer (PAM ED-101US, Walz, Effeltrich, Germany), controlled by a personal computer and Phyto-Win software v1.45 (Walz) to calculate specific clearance rate.

During the depuration of clay, the temporal change of the phytoplankton concentration by addition of animals was measured. CR is a measure of grazing activity by shellfish and is calculated as the volume of water (mL) from which the animal (mg, wet wt) has removed all of the food particles of given size per unit time. Specific clearance rate (mL/mg wet wt per h) was calculated according to Coughlan25 as:

image

where V is the volume of the phytoplankton suspension (3 L), n is the weight of an animal (mg wet wt), t is the duration of the experiment (h), C0 is the phytoplankton concentration (number/mL) at t = 0, Ct is the phytoplankton concentration at time t in vessels with shellfish. The concentration of phytoplankton in the control vessels at time t were not considered in our calculation, because the cell concentration in the controls did not differ significantly from zero and within the variation level in our short-term experiment.

CR of shellfish taken from the different clay concentration were tested by one-way anova, and in the grazing experiments a two-way anova was used to test clearance rates between yellow clay groups and between harvesting times, using the probability P = 0.01 as an index for significance.21

RESULTS

Clay uptake

Mussels took in much more clay earlier and retained the clay longer than oysters (Table 2). No clay was found inside oysters treated with 0.01% yellow clay over 6 h.

Table 2.  Yellow clay uptake by Crassostrea gigas and Mytilus galloprovincialis with time
Time (h)Yellow clay concentrations (%)
00.010.050.251.256
  1. –, not detected; +, low; ++, medium; +++, high.

Crassostrea gigas
0.5++++++
1+++++
2+++++++
4+++++
6+++++
Weight of oysters (g)130.0 ± 20.097.0 ± 15.692.0 ± 15.697.0 ± 17.687.0 ± 17.6112.0 ± 14.4
Mytilus galloprovincialis
0.5++++
1++++++
2++++++++++
4+++++++++++
6+++++++++
Weight of mussels (g)110.0 ± 0.090.0 ± 6.091.0 ± 23.293.0 ± 5.694.0 ± 15.285.0 ± 8.0

At 0.01% clay concentration, clay was found inside the mussels after 2 h. In the 0.05% treatment, an indication of clay appeared in oysters after 2 h. At 0.25% clay, oysters had a significant amount of clay within 30 min. By 2 h of treatment, gills and the digestive tract of tested shellfish were filled with a great quantity of clay (Fig. 1).

Figure 1.

Accumulation of clay (arrows) for (a,b) 2 h and (c,d) clay depuration for 1 h in phytoplankton chamber of Crassostrea gigas (a,c), and Mytilus galloprovincialis (b,d). Scale bars, 0.5 cm.

Respiration in clay suspension

DO of the control (i.e. no yellow clay) was 6.6 ±  0.3 mg/L. Change of DO by the addition of yellow clay was not significant (DO = 6.6 ± 0.1).

The effect of clay on the respiration of shellfishes was indirectly estimated by measuring the temporal variation in DO. Compared to the untreated organisms, the RR of shellfishes showed a tendency to decrease by the function of the interval (Fig. 2). Respiration was higher in relatively low concentrations of clay (0.25 and 0.75%) in both species. For oysters, if respiration was inhibited, initial consumption of dissolved oxygen was low by high concentration of clay. However, a clear statistical relationship between RR and clay concentration increase was not found (P > 0.023).

Figure 2.

Respiration ratio (RR) of (a) Crassostrea gigas and (b) Mytilus galloprovincialis in clay suspensions at 0.25% (●), 0.75% (▴), 1.25% (▪) and no clay (○) w/w concentration.

Clay discharge and grazing ability after clay treatment

Shellfish were removed from clay containers after 2 h. In all tested animals, the level of the internal clay decreased as early as 30 min after being taken from clay containers (Table 3). By 6 h, clay was not found inside harvested animals, as if all of it was discharged from tested animals.

Table 3.  Yellow clay discharge from Crassostrea gigas and Mytilus galloprovincialis with time after 2-h yellow clay exposure
Time (h)Yellow clay concentrations (%)
0.010.050.251.256
  1. –, not detected; +, detected.

Crassostrea gigas
0.5+
1++
2++
4+
6
Weight of oysters (g)77.0 ± 19.674.0 ± 14.8112.0 ± 11.682.0 ± 18.4102.0 ± 6.4
Mytilus galloprovincialis
0.5++++
1+
2++
4++
6
Weight of mussels (g)84.0 ± 10.893.0 ± 9.690.0 ± 6.099.0 ± 13.290.0 ± 12.0

The trend of CR was similar in both oyster and mussel; higher CR was in lower amounts of phytoplankton mixture (Fig. 3).

Figure 3.

Clearance rate (CR) of (a) Crassostrea gigas and (b) Mytilus galloprovincialis in concentrations of phytoplankton 100% (●), 60% (○), 20% (▴) and 10% (▵).

CR was calculated to compare the temporal effects on the grazing of shellfishes by clay treatment (Fig. 4). Within 2 h, most of internal clay had disappeared (Fig. 1a and b), and CR became very similar to that of untreated control groups (Fig. 4). Therefore, the trend in CR suggests that the feeding physiology of shellfishes returned to normal right after they were rescued from clay suspensions, although clay particles were still inside their organs after 2 h (Table 3).

Figure 4.

Clearance rate (CR) of (a–f) Crassostrea gigas and (g–l) Mytilus galloprovincialis in clay suspensions: no clay (a,g), 0.01% (b,h), 0.05% (c,i), 0.25% (d,j), 1.25% (e,k) and 6% (f,l). Error bars, standard deviation.

For oysters, the amount of clay contributed to increase the variation of the amount of phytoplankton consumed (Fig. 5), which was in agreement with results from paired comparisons that showed a statistical difference of CR among clay groups (P = 0.003). However, anova indicated that the influence of clay treatment was temporary (P = 0.053). After 30 min, the strength of phytoplankton uptake by oysters became similar in all clay-treated oysters (P = 0.260).

Figure 5.

Phytoplankton consumption by (a) Crassostrea gigas and (b) Mytilus galloprovincialis in clay suspensions at 0% (●), 0.01% (▪), 0.05% (▴), 0.25% (○), 1.25% (□) and 6% (▵) w/w concentrations.

However, CR of mussels was constant without correspondence with the clay concentration: P = 0.184 in a paired comparison test and P > 0.052 in anova test. The correlation coefficient between CR and the concentration of clay suspension was −0.270 (n = 122) in oysters and −0.304 (n = 150) in mussels.

DISCUSSION

Testing on animal physiology would be useful to evaluate sublethal effects of clay. Lethality tests of clay on marine animals gave various results.26 For example, flatfish showed a strong resistance to yellow clay and no significant effect was found with a treatment at 0.8% clay concentration, whereas abalone was sensitive to turbidity and the viability of abalone decreased after 10 h.12

In our results, the gill and digestive duct of marine shellfish were full of clay within 2 h of treatment. Depuration, ejection of clay, started immediately after the animals were removed from the suspension of clay. The clay in the gill and digestive duct were gone 6 h after rescue. It is known that ingestible particles may enter directly into the mouth and unwanted particles (e.g. with respect to size or food quality) are sorted on the gills and labial palps, embedded in mucus and then expelled as pseudofeces in the surrounding water, where they can easily disperse.27,28

Presumably additional stress by clay, such as anoxia, may induce the animals to require extra energy. The effects of clay on the RR of animals are a function of both the concentration of clay and the duration of exposure.11 The low respiration rate of shellfish in this experiment was probably due to a decrease in opening shells, which led to a slower metabolism and lower oxygen demand. However, when either exposed to toxicants or subjected to natural stress, marine benthos show increased metabolism compared to unstressed animals, followed by an increase in oxygen demand.29,30 In our experiments, respiration by shellfishes was higher at relatively lower clay concentrations (0.25 and 0.75%), whereas respiration at high clay concentration was very similar to that of the control group without clay application.

For the clearance experiment, we used a mixture of phytoplankton sampled from the local bloom site to mimic practical conditions. In addition, the highest clearance rate of phytoplankton by marine filter feeders was found when they were feeding on a mixture of phytoplanktons.22 Cell clearance of oyster and mussel was not correlated with the number of phytoplankton cells added, as demonstrated before.31

Shellfish can be classified as either rapid or slow feeders. For example, mussels and scallops feed very rapidly but accumulation rates for oysters were low.31 A rapid feeder like mussel has been shown to take only few week to depurate, but slow feeders typically take several months to one year.32 Our current results, however, showed the CR changes of two species were similar. The reason for this result may be that the rate of depuration strongly depends on the environmental conditions such as competition33 and on the amount of chemicals present during the accumulation period, rather than on intrinsic, species-specific causes.34 According to previous studies, some mussels show CR reduction by these kinds of treatments35–37 and others show opposite results.38–40 The reason for the diverse results would come from the concerted regulation of filtration and oxygen consumption by filter feeders.41,42

In Korea, so far, clay dispersion has been used as the prime mitigation technique for phytoplankton blooms.4,5 The harmful blooming phytoplanktons are removed by means of the coagulation, sedimentation and destruction of the organisms.3,8 Acute or chronic exposure to the clay dispersion may not be lethal to juvenile fish, shellfishes or invertebrates: the toxicity of a mixture of the settled clay and red-tide organism was not significantly different from that of red-tide organism alone.43 Specifically, the effects of clay on the respiration of animals are a function of both the concentration of clay and duration of exposure.11 For the practical treatment of the red tide, the use of 200–400 g/m2 of clay has been suggested by Shirota.7,8 For many years, Korea has dispersed approximately 100–400 g/m2 of yellow clay on the red tide invading farming areas. If it is assumed that most target areas for clay dispersion are approximately 10 m in depth, the amount of dispersed clay in the area will be lower than 0.04%. Scattered clay in the open sea might minimize the effects on animals in the local area even more.8

CONCLUSION

Concerning the dependence on clearance rate of the strength of clay treatment, our data did not reveal obvious effects of clay concentration on the clearance rate of oyster and mussel, except for during the interval just after the clay treatment. More intensive study is required. For example, the possible toxicity of flocculation of clay mixed with blooming organism should be clarified.43 Experiments flocculating local blooming organisms are necessary.

ACKNOWLEDGMENTS

This study was supported by a grant from the National Fisheries Research and Development Institute (RP-2007-ME-034). Thanks to Dr Y.K. Shin, Mr H.S. Park and Ms B.L. Choi for assistance during experiment and to Dr M.R. Sengco for helpful comments in the preparation of this manuscript.

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