experiment tank was raised 1 C per day, organisms were fed every 2 days, and
water exchanged every 2 days or everyday if the water looked murky andor smelt bad. Water exchange was done every 2 days in experiment tank to make
the water quality as similar to the control tank, because water exchange in the control tank done every day for approximately 30-60 minutes with through flow
system. Water was exchanged everyday in the experimental tank when the quality of water in the experimental tank decreased because of the mortality or
because organisms produced lots of feces or organic material. The changing temperature and the activity Peck et al., 2008 andor mortality of the organisms
for the thermal limit experiments were noted. The design of the equipment thermocycler tank can be seen in Figure 10
and Appendix 1.
Figure 10. Design of thermocycler tank for temperature limit experiment. For daily thermal limit experiments, separate tanks plastic tank for water
exchange were prepared. First, seawater was added into this plastic tank. Second, the filter, adjustable heater, and aeration were added and put inside the
plastic tank. Third, the filter, aeration, and heater were turned on. Fourth, the temperature on the heater was adjusted to reach the target temperature, same as
in the experimental tank when the exchange was done. 10-20 of each species were observed in this experiment total of
organisms in experiment and control.
Freshwater cooling fluid 31 cm
75 cm
3.3.7.2. Pilot study
The pilot and main experiment were done separately. The purpose of doing the pilot study were: 1 to identify the parameters to indicate “not
responsive” organisms. For the ophiuroids the turning response; and for the asteroids the turning response and attachment capability. 2 Knowing when to
turn the specimen. It was when the specimens look weak and were not attached to the wall already at the bottom of the experimental tank.
3.3.7.3. Performance and control
During the hourly experiment, the organisms were checked every hour and the temperature was checked every 20 minutes. During, daily experiments,
the organisms were checked everyday at the same time as the experiment started. To check the organisms, first activity movement was monitored,
whether their tube feet and tentacles were out, and if the arms and body were in normal condition ophiuroids: arm tips were a little bit elevated and central disk
were not swollen; asteroids: skin was not showing any white spots and the animal was still attached to the ground or wall and not showing any abnormal condition.
If they were showing any abnormalities ophiuroids: arms curling or loosen, body color changing, central disk swelling, not attached to the wall anymore, or others;
asteroids: not attached to the wall anymore or staying on the bottom, with white spots, the organisms were checked ophiuroids and asteroids: turned over, to
see whether they could turn over again. During the experiment, activity of the specimens and the condition was
noted. After the experiment, the weight and size of the specimens were measured, and it was noted if something had happened e.g. any autotomy,
condition of the arms loosen or curling, condition of the body stiff, wounded, etc.
3.3.8. Salinity stress experiment 3.3.8.1. General setup
The general setup for the hourly decrease salinity stress experiments was the same as the temperature limit experiment e.g. thermocycler, refractometer,
filter, camera, checking salinity in control and experiment tank. The salinity stress experiment was done hourly decrease salinity 1 per hour by adding
freshwater into the experiment tank.
Before the experiment, the tank size was measured to plan the seawater level inside the tank. The tank used in the salinity limit stress capacity
experiment was the thermocycler tank, so that it would be comparable to the temperature limit experiments. The size of the tank inside was 40 x 75 x 32 cm.
The water depth was 25 cm. So, the volume of water in the tank was 40 x 75 x 25 cm = 75,000 cm
3
= 75 dm
3
= 75 L. To measure the water level inside the tank, a ruler was put inside the tank and was stuck to the tank wall Figure 11.
3.3.8.2. Pilot study
Before performing the hourly decrease salinity limit experiment, a trial was completed to test the flow of seawater after freshwater was added to the
experiment tank. This trial was done to see the direction of flow of water inside the experimental tank to make sure that the water was mixed evenly during the
experiment. For this trial, the experiment tank was filled with seawater and then the filter was put inside and turned it on. To measure the flow of the water, milk
was used as an indicator. The flow of the water inside the experiment tank can be seen in Figure 12 and Appendix 5. After finishing this trial, the experimental tank
was being cleaned again before being used for the real salinity decrease experiment.
Figure 11. Condition in salinity decrease experiment tank. The ruler is attach to the wall inside the experiment tank.
Ruler
3.3.8.3. Performance and control
When the tank was set up and clean, the seawater was put into the experiment tank until it reached 25 cm height. The water quality ammonia, pH,
nitrite, and nitrate and salinity were measured before the experiment started. The experiment decreased the salinity at 1 per hour. The amount of freshwater
added to experiment tank in 1 hour was noted. The next hour when salinity was decreased again, the amount of water that was added during previous hour was
taken out first before more freshwater was added to experiment tank. The number of animals ophiuroids: Ophiomastix annulosa and
Ophiocoma cf. dentata used in this experiment is 7 organisms in the experiment and 3-5 ophiuroids for the control.
3
6 7
2 1
4 5
Filter
Bottom current Surface current
Figure 12. Sketch of waterflow during trial with milk. Fixed line shows the surface current; dashed line shows the bottom current; point 1-7 are the points
where milk was dropped. The slowest current among 7 points was at point 3. The other points point 1, 2, 4, 5, 6, 7 are faster than point 3.
To check the organisms during experiments, first their activity was monitored moving, whether their tube feet and tentacles were out or not, and
