Aftereffects of Hypoxia Exposure
on Ruditapes philippinarum
in Omaehama beach, Japan
Yasunori Kozuki1, Ryoichi Yamanaka1, Maya Matsushige2,
Azusa Saitoh2, Sosuke Otani3 and Tatsunori Ishida1
(The University of Tokushima, Japan)
Osaka Bay
・ Sea area ： 14,000km2
・Population ：12,000,000
Tokyo Bay
Ise Bay
Osaka Bay
Seto Inland Sea
The coast line in the innermost of Osaka bay is covered with
artificial structures and only two natural beaches are left there.
Omaehama is one of them
1
Omaehama beach
Investigation station
400m
10km
Enclosed coasted area in the innermost of the bay.
Eutrophication happens especially in summer.
Omaehama beach functions as a very important natural beach.
2
Omaehama beach
Blue tide
"Blue tide"
sometimes occurs in summer
3
Omaehama beach
Blue tide
Hypoxic condition
“Are survived clam healthy or damaged?”
4
Past studies
Past studies
•Clam dies when it is exposed to blue
tide.( Hagita,1985)
•Clam has tolerance to hypoxia.(Kakino,1982)
•The number of dead individual increases when
clam is exposed to hypoxia repeatedly. (Uzaki,2003)
It has never being examined how frequent
hypoxia condition affects active and filterfeeding function of survived clams.
5
Objective
Objective
Influence of hypoxia exposure to the purification
function of the beach.
The change of active and filter feeding
speed of survived clams before and after
exposure to frequent hypoxia.
Experiment item
・ Mortality
・Filter feeding speed
・Glycogen
6
What is glycogen ?
Glycogen is one of the index
for the nutritious condition of clam.
Past studies
・ When clam is exposed to hypoxia, it closes its shell and
tries to survive by using glycogen which is produced in
anaerobic metabolisms in its body.（P.W.Hochachka,1965）
・ Glycogen contents in clam may decides the mortality by
hypoxia and its health condition. （Kuroda,1998）
Method
Anthrone-sulfuric acid method
（Yoshikawa,1955）
7
Experimental procedure
First exposure
hypoxia(DO 0.0～0.5mg/L)
0 day
exposure
In hypoxia
for 3 day
7day・・・
DO
Recover
Second exposure
hypoxia(DO 0.0～0.5mg/L)
0 day
exposure
In hypoxia
for 3 day
4 day
Recovery
tendency
4 day
7day
14day
DO
Recover
Control case
Initial
Temperature ：25℃ Salinity ：28psu.
7day
21day
28day
42day
8
Exposure procedure
Clams were kept in a water tank which was
maintained similar to natural condition.
90cm
Tidal range
:60cm
60cm
Sediment
depth:20cm
Shell size ： 30 to 35 mm
Shell condition：had never been exposed to hypoxia
9
Filter-feeding procedure
①Skeletonema costatum were put artificial seawater（500ml）
②A clam was placed in ①.
③Experiments were started when clam extended its siphon.
（artificial seawater+plankton+Clam）
C0
Control
case
…
10mints,20mints,30mints,40mints,50
mints. We measured plankton.
Ct
…
10
Formula
Filter-feeding speed
ｍ：filter-feeding speed of a clam
Ｍ：water volume to filter-feeding
ｎ： number of individuals
C0： suspended solids (starting value)
Ct： suspended solids (after t hour later)
We did not use those clams for our experiments that didn’t
extend their siphons for 2 hour.
11
Initial
Thick
Expose
Thick and black
Clam closes its shell
0 day
Siphon and open it
widely for breathing
DO
Recovery
Finer
4 day
Thinner
7day
The siphon black
ends dropped
Few
12
20
2
1.5
N =10
N =10
N =7
1
N =7
N =7
0.5
0
initial
7
21
28
42 (day)
Filter-feeding speed
Average was 0.72～1.23L/h
【past studies】
Healthy clam of filter-feeding
speed is 0.73～1.24L/h
(Fisheries Agency,2006)
G lycogen
(% on dry basis)
Filter Feedin Speed
(L/h/ind.)
Result of “Control case”
15
N =5
10
N =5
N =5
N =5
N =5
28
42 (day)
5
0
inital
7
21
Glycogen
Average was 5.0~10.02%
【past studies】
Other clams in the same period of the
experiments from June to August and it
was from 5 to12%.
(Siraishi,1995)
13
Mortality of control clam after six weeks was 6%.
・ Reduced to 50% after
four days
・ Recovery tendency in
the second week
2
1.5
N =8
N =10
1
50%
N =10
N =9
N =9
N =9
0.5
Filter Feeding Speed
(L/h/ind.)
First exposure
Filter Feeding Speed
(L/h/ind.)
Result of Filter-feeding speed
0
inital 0
4
7
14
21 (day)
2
1.5
Second exposure
・Decreased to 70% after
seven days.
•No recovery.
1
N =40
70%
N =16
0.5
0
inital
0
N =5
3
N =7
N =6
7
14 (day)
14
Result of Glycogen concentration
・ Reduced to 77% after
seven days.
・ Recovery tendency
after three week.
15
N =5
N =5
N =5
77%
10
0
N =5
N =5
N =2
5
intial 0
4
7
14
21 (day)
20
G lycogen
(% on dry basis)
First exposure
G lycogen
(% on dry basis)
20
15
Second exposure
・ Never recover.
・Glycogen stayed low level.
10
N =5
N =5
5
0
70%
inital
0
N =3
7
N =3
14 (day)
15
Result of Glycogen concentration of dead clam
G lycogen
(% on dry basis)
20
15
N =6
10
5
N =6
0
intial
dead
(day)
・ Glycogen content of dead clams
were less than 1%.
16
First exposure
・ Mortality of clam
after three days was 16%.
・ Clams did not die
after two weeks.
％）
M ortality（
Result of Mortality
100
80
60
16%
40
20
0
initial 1
2
3
Second exposure
・ Mortality of clam
after 3 days： 50%
after 14 days：76％
・The number of dead
individual continued to
increase .
％）
M ortality（
Hypoxia
1
2
3
7
（
day）
normoxia
100
8～20 21
76%
80
50%
60
40
20
0
initial 1
2
Hypoxia
3
1
2
3
normoxia
7
8～13 14
（
day）
17
Impact assessment
The effects of hypoxia water exposure on the water
purification of Omaehama beach in Osaka Bay.
DL：0.0ｍ
DO：0.4mg/L
2000
1500
1000
3
2
(m /day/m )
Filter-Feeding volume
2009.9.1
500
0
Non-exposed clam
Exposed clam
The water purification function will drop to about 1/3.
18
Conclusion
We revealed even if clam survives from the
exposure to hypoxia, it will have residual disabilities
such as the disfunction of Filter-feeding speed.
Clam dies when its glycogen content reduces to less
than 1%.
When we evaluate the water purification of the
beach, we need to consider the record of hypoxia
exposure there.
19
Thank you for
your attention!
3. Glycogen procedure
anthrone-sulfuric acid method
（Haruki Yoshikawa,1955）
① Whole meat of Clam was freeze-dried for over 24 hours.
② The freeze-dried whole meat was placed in a test tube and
30% potassium hydroxide solution was added.
The test tube was placed in a boiling water bath for one hour
③ The test tube was left in a coolant for 20 minutes, and ethanol
was added to remove supernatant liquid.
Sediment was disolved with distilled water appropriately
④ ③ was placed in anthrone-sulfuric acid reagent and measured
by absorbance at 620nm
Glycogen
filter-feeding speed
experimental
Recovery
3 days
7 days
・・
8. Discussion
First exposure
Second exposure
Filter-feeding speed
・Recovery tendency in
the second week
•No recovery.
Glycogen
・Recovery
tendency after
two week.
・ Never recover.
・Glycogen stayed low
level.
Once the filter-feeding function gets damages, clam cannot
take nutrition sufficiently and save glycogen in its body.
2
20
1.5
N =8
N =10
1
N =10
N =9
N =9
N =9
0.5
0
inital 0
4
7
14
G lycogen
(% on dry basis)
Filter Feeding S peed
(L/h/ind.)
8. Discussion
21 (day)
15
N =5
N =5
N =5
10
N =5
N =2
5
0
N =5
intial 0
4
7
14
21 (day)
the influence of hypoxia to clams as follow
 Few individuals after being exposed to hypoxic water for 3days
Filter-Feeding Speed and Glycogen decreased temporarily, it recovered.
some individual died after the second exposure.
the number of individual increased even after the exposure stopped.
Filter-Feeding Speed and Glycogen of survived individuals
did not recover.
17