Frequently Asked Questions

Zebrafish Tanks
Because the standard Aquaneering tanks are polycarbonate, they can be cage washed up to 250°F (121°C).
If you do not have a cage wash, a general guideline for cleaning tanks is to hand wash the tank with a sponge in hot water, and never use detergents. Although many labs do use detergents, it is important to remember that they are toxic to fish. Detergents should only be used with a proper rinse protocol to check for residual detergent as standard operation procedure for cleaning fish tanks.
Many labs use a high temperature dishwasher to wash and disinfect the tanks. It is important to use a dishwasher that has a reverse osmosis (RO) final rinse cycle to remove traces of chlorine.

Xenopus Tanks
These tanks require a low temperature (below 140°F) for cleaning. Rinse only; do not use detergents.

DC-96 tanks are made of polycarbonate, so you may wash the DC 96 tanks, lids, and dividers in a rack washer or dishwasher. All of the dividers and lids are removable. Do not autoclave.

1. Turn off pumps and UV Sterilizer, and pull the sump out from the rack.2a. Use a siphon hose to clean debris from the sump.OR2b. Pump the water out of the sump.

Store this water if you intend to refill the sump with the same water.
Using a wet (shop) vacuum, clean debris out of the sump.
If you wish to do a water exchange, refill the sump with RO water, or you can refill the sump with the original system water.

3. Move sump back in place under the rack, and turn on pumps and UV Sterilizer.

How to test a 1/3 HP chiller:

You will need:

• (1) Five gallon bucket

• (1) Thermometer

• (1) Timer / stopwatch / phone

• 68°F Water

1. Mix hot and cold water in the five gallon bucket until the bucket is full and a thermometer in the water reads approximately 68°F.

2. Put the chiller coil in the bucket and turn it on.

3. Monitor how long it takes the chiller to drop the temperature of the water a full 10 degrees.

4. Once the water is 58°F, stop the timer.

5. Report to Aquaneering how long it took the chiller to drop the temperature.

6. NOTE: The manufacturer's baseline time to drop the temperature is 7.5 minutes.

7. Depending on the results of this simple test, Aquaneering may look into how the chiller was installed and/or how the tanks on the rack are used.

How to test a 1/2 HP chiller:

You will need:

• (1) Five gallon bucket

• (1) Thermometer

• (1) Timer / stopwatch / phone

• 68°F Water

1. Mix hot and cold water in the five gallon bucket until the bucket is full and a thermometer in the water reads approximately 68°F.

2. Put the chiller coil in the bucket and turn it on.

3. Monitor how long it takes the chiller to drop the temperature of the water a full 10 degrees.

4. Once the water is 58°F, stop the timer.

5. Report to Aquaneering how long it took the chiller to drop the temperature.

6. NOTE: The manufacturer's baseline time to drop the temperature is 5 minutes.

7. Depending on the results of this simple test, Aquaneering may look into how the chiller was installed and/or how the tanks on the rack are used.

• DO NOT UNPLUG OR PLUG IN THE LAMP WHILE THE BALLAST IS POWERED.

• DO NOT TOUCH THE UV LAMP. Always wear rubber gloves when handling the UV lamp. Touching the lamp with bare skin may damage the lamp.

• DO NOT LOOK AT THE UV LIGHT. Protect eyes and skin from exposure to UV light. The UV sterilizer emits ultraviolet energy which can cause serious eye injury and severe sunburn.

• DO NOT RUN THE LAMP DRY. Water must always be running through the UV when it is operating or it will be damaged.

• UNPLUG THE STERILIZER BEFORE PERFORMING ANY MAINTENANCE.

• DO NOT USE ANY TOOLS TO TIGHTEN UV COMPONENTS. Hand tighten only.

• UV LAMP DISPOSAL: UV lamps contain mercury, which is considered a hazardous waste. Disposal of mercury must be made in accordance with local, state, and federal regulations. Consult your local waste authority for proper lamp disposal in your area.

• Check to see that all electrical connections are securely connected to the UV unit and proper outlets.

• Verify that the harness inside the ballast unit is securely in place.

• Lamp is burnt out. Instructions for changing the lamp can be found in your system user manual.

• Breakage/cracks in the quartz sleeve.

• Endcaps are not tight enough. When reassembling the UV Sterilizer, be sure the endcaps are completely tightened. However, handtighten only; do not use tools.

• O-rings have been reused. Replace the o-rings each time the quartz sleeve is cleaned or replaced.

• O-ring is not seated correctly in the endcap. Make sure the o-ring inside the end cap is properly seated just past the threads.

All instructions on performing maintenance on your UV Sterilizer can be found in your system user manual.

• On a UV Sterilizer with plastic housing, the sight glass may be cloudy.

• The ballast may need to replaced soon.

• The UV lamp has not warmed up completely. This takes about a day.

Possible causes:

• Acclimation process

  • It is normal for the pH to be high at the beginning of the acclimation process. If the ammonia level is below 2 ppm and nitrite level is below 1 ppm, add more fish to the system to lower the pH level.

• Alkalinity of water

  • Water with low alkalinity is very susceptible to changes in pH, while water with high alkalinity is able to resist major shifts in pH. Program the pH transmitter to lower the pH using an acid solution of 60:1 (water : acid). Contact Aquaneering for instructions on dosing acid in your system.

A Fluidized Bed Biofilter is a biological filter in which the upwelling of a fine media in the water allows the growth of beneficial bacteria that aid in the nitrification process: the conversion of ammonia to nitrite to nitrate. Among the 100 - 200 kinds of bacteria found in the FBB, the most common bacteria required in the nitrification process are Nitrosomonas, which convert ammonia to nitrite, and Nitrobacter, which convert nitrite to nitrate.

See our Fluidized Bed Biofilter page for more information and product specifications.

Like all biofilters, the FBB requires an initial acclimation period for the bacteria to grow and convert the ammonia to nitrite to non-toxic nitrate.
The quickest method to establish a bacterial colony in an FBB is to siphon several buckets of media from an operating FBB and add it to the new FBB.

On a new system, it will take 6 to 8 weeks to establish the bacteria in the FBB.

1. Fill the system with water and allow it to run for a day. The water temperature should be between 26˚ to 28˚ C.

2. Add approximately 10% of the system’s capacity of animals. On a 5-Shelf Stand Alone System, 10% would equal about 100 adult zebrafish. Do not feed the animals for the first 1 - 2 days to slowly allow the ammonia to build up in the system.

3. After 1 - 2 days, begin feeding the animals a reduced diet, once per day. Feed ONLY what they will eat in a minute or less. Measure the ammonia levels daily.

4. In 3 - 4 weeks, the measured levels of ammonia will start to decline. At this time, add another 100 zebrafish to the system.

5. If ammonia levels begin to climb above 1.5-2 ppm:
   a. Perform a water exchange. A 50% water change out will reduce the ammonia level by 50%.
   b. Stop feeding the animals for 2-3 days, and then resume feeding at a reduced rate. Less food equals less waste produced, which will lower the ammonia levels and allow the bacteria to multiply to meet the demand.
   c. Stop adding animals to the system, and let the system stabilize.
   d. Make sure the filter pads are being changed 2 to 3 hours after feeding.

6. The ammonia levels will start to drop to undetectable levels in approximately 6 - 8 weeks. Until the ammonia levels drop below 0.5 ppm, only feed the animals once per day.

7. Once the ammonia level is undetectable, the animals can now be fed 2-3 times per day. Only feed what the animals will eat in 1-2 minutes.

8. Make sure to maintain the pH above 7. If the pH is below 7, add sodium bicarbonate in increments of 2 grams per day (on a Stand Alone System).

9. The acclimation process is finished when the ammonia and nitrite levels are undetectable, and the nitrate level is less than 50 ppm.
   NOTE: Additional biomass may increase ammonia concentration for a short period of time.

The FBB is self-cleaning and never requires changing. If you do notice any accumulated debris on the top of the FBB, you can siphon it off.

Test the system water for ammonia and pH. If there is less than 0.5 ppm of ammonia, and the pH is above 6.5, the FBB is filtering properly. In addition, the media should be evenly distributed and upwelling. If the media is compacted, the pump may have shut down or a FBB probe is clogged.

Two indications that a probe is clogged are:

• The media in the FBB is compacted.

• The FBB pressure reads above 5 psi or outside normal operating range.

To clean a clogged probe:

1. Dislodge the clog by briskly sliding the probe up and down.

2. Shut off the FBB pump, open the ball valve on the top of the probe, and slide a hose down the probe. Turn the hose on at full pressure. Slowly work the hose to the bottom of the probe. After cleaning it out, turn off the hose, close the ball valve, and restart the pump.

3. On a Stand Alone System, you can remove the probe and rinse it out in a sink. Be sure to turn off the FBB pump before removing the probe, and turn it back on when you are done.

This is a normal occurrence while the system is acclimating.  You may observe smaller particles of media in the fish tanks for approximately two weeks after the system has been started.  Siphon these particles out of the tanks.  As the FBB starts to acclimate, a sticky film will form on the media, and it will become less likely to “blow out” into the system and tanks.

Adding media to the FBB is known as "charging the FBB".

1. Fill the system with water and turn on the FBB pump.

2. As the pump is running, slowly add FBB media. The amount of media for your system is listed below. Stop adding media when it reaches 75mm (3”) below the FBB outlet.

3. Follow the acclimation process before adding fish to the system.

Amount of media to add by system type:

• Tabletop

  • 10 kg or 25 lbs

• Single-sided Stand Alone System

  • 20 kg or 50 lbs

• Double-sided Stand Alone System

  • 40 kg or 100 lbs

24 hours or more

1. If the fluidized bed is shut down and the glass bead bed collapses for more than 24 hours, it reverts to an anaerobic state, and may produce small amounts of hydrogen sulfide (H2S).

2. Therefore, when the bed is restarted, the rack pump should be shut off and the filter should be operated first until the water in the sump is clear.

3. If there is any black discharge from the fluidized bed or smell of H2S, the filter should be operated for 1-2 hours (until the water in the sump is clear) prior to turning on the rack pump. Check water quality and perform a water exchange if needed.

3 to 4 days

1. If the fluidized bed has been shut down for 3-4 days, the bacteria may have to be re-established.

2. The quickest method to revive the bacterial colony is to siphon a small amount of media from a
   fluidized bed that is operating and pour it into the bed that is being restarted.

Table Top System: 220 liters/58 gallons
Five Shelf System: 320 - 490 liters/85 - 130 gallons
Six Shelf System: 340 - 510 liters/90 - 135 gallons
Sumps:
Single sump = 130 liters/35 gallons
Double sump = 300 liters/80 gallons
Zebrafish Tanks:
0.8L = 0.21 gallons
1.4L = 0.37 gallons
1.8L = 0.48 gallons
2.8L = 0.74 gallons
6.0L = 1.59 gallons
9.5L = 2.51 gallons
Xenopus Tanks:
16L = 4.23 gallons
21L = 5.55 gallons
50L = 13.21 gallons

The overflow (white or gray PVC pipe / elbow) should be adjusted by gently twisting to maintain the water at a height of 12-14 inches. Make sure the float valve is set at a lower height than the overflow.

In order to regulate the water pressure, you will need to perform a "10 Minute Flow Test":

1. Adjust the tank supply valve on a tank on the bottom shelf so the tank will fill completely in 10 minutes.
   NOTE: If any tank fills faster than 10 minutes, there is not enough water pressure to fill the upper tanks.

2. Proceed up the shelves, adjusting the valve on each tank, until every tank fills completely in 10 minutes.

Check your carbon filters; you may need to change the filter sleeves and carbon. You can find instructions for doing so in your system user manual.

When the water level in the sump drops too low, the float switch triggers the control box to power off, which shuts down the system. To restart the system, add water to the sump and lift the float switch.

Possible reasons include:

• The carbon filters may need to be serviced. To determine if this is the cause, remove the carbon filters. If water starts flowing, you will need to change the carbon and the filter sleeves. You can find instructions for doing so in your system user manual.

• The pump impeller may be clogged or the pump is defective. Instructions for cleaning the pump impeller can be found in your system user manual.

It is not necessary to pre-filter the water if the system is connected to a building reverse osmosis (RO) or deionized (DI) line. However, if you are using well or city water, you may need a carbon filter to remove chlorine, chloramines, heavy metals, or other impurities in the water. Test your water supply for chlorine. If you find detectable levels of chlorine in the water, you will need a carbon filter. For assistance or other solutions, contact Aquaneering.

The water may also need to be particulate filtered. Many zebrafish researchers use an RO Water Maker, which has both carbon and particulate filters.

See our Carbon Filter page for more information and product specifications.

Zebrafish: 15 adult fish/liter.
Xenopus laevis: 1 adult frog/3 liters

Parameters and acceptable range:

• pH

  • 6.8 - 8.5

• Conductivity

  • 300 - 2000μs

• Temperature

  • 24 - 28°C

• Dissolved oxygen

  • >4 ppm

• Total gas pressure

  • 99.9%

Reference: Harper, Claudia, and Christian Lawrence. The Laboratory Zebrafish. Boca Raton, FL: CRC Press, 2011. Print.
You can purchase The Laboratory Zebrafish through CRC Press.

Aquaneering recommends a water exchange rate of 5-10% per day. This may vary depending on species and population load. For both Stand Alone and Central Filtration Systems, you should exchange about 10% of the total tank volume per day.

• The bacteria have not been adequately established during initial set-up. There may be too many animals on the system.

• The pH is lower than 6.5. When the pH is too low, the bacteria stop nitrifying ammonia.

• If you have introduced antibiotics into the system, this can suppress or kill the beneficial bacteria in the Fluidized Bed Biofilter which nitrify ammonia.

1. Check and document all water quality parameters, including ammonia, nitrite, nitrate, pH, and water temperature.

2. If no bacteria is present: add 1 tablespoon of sodium bicarbonate directly into biofilter every hour. Check water quality and record data.

3. If bacteria is present: pour 1 tablespoon of sodium bicarbonate onto the filter pad under running water every hour. Check water quality and record data.

4. Stop adding sodium bicarbonate when pH is at 7.2-7.5 and let the system run overnight.

5. In the morning, check water quality and record data.

6. Test and calibrate the water exchange for 10%.

7. Monitor the water quality and water exchange over the next few days.

8. After 2-3 days of consistent water quality data without ammonia or nitrite present, add some sentinel fish to the system.

9. Continue to monitor water quality, water exchange, and sentinel fish for 1 week.

There are several factors that influence algae growth, including nitrate buildup. For more information, check out the article What's It All About ... Algae? on our website.

To prevent algae growth:

• Do not overfeed. Without the adequate clean-up crew found in the wild, leftover feeds decompose and leave behind large quantities of organic phosphates.

• Keep flow rates reasonable so that food is not immediately swept fromthe tank and allowed to decompose in the filtration system.

• Slowly introduce animals to new systems so that the biological filter has time to establish itself andmaintain ammonia and nitrite levels properly.

• Carefully watch nitrogenous waste levels during population fluctuations.

• Do regular daily 5-10% water exchanges to keep nitrate levels down.

• If possible, do not position racks directly under overhead light fixtures.

• Use properly sized UV sterilizers on your system.

For more information and additional solutions, contact Aquaneering.

Possible causes include:

• Temperature controller is set to chill. Verify temperature controller programming is set to heat.
  IMPORTANT: Make sure you select the correct mode for the component (chiller = cooling or heater = heating) that is installed in your system. Otherwise, there is a risk of animal mortality.

• Temperature may be higher than setpoint if room temperature is too high or has increased. Recommended room temperature is 24 - 32ºC.

• Not enough tanks on the system.

Although there is not a physical barrier between the clean and dirty water zones in the system sump, the two zones are divided by hydraulic separation.

• Feed your zebrafish high protein foods, such as the Zebrafish Select Diet offered by Aquaneering which contains at least 52% protein. Feed the fish only as much as they can consume in 1 - 2 minutes.

• Spawn the fish on a regular basis. Every two weeks is generally considered the normal egg laying cycle.

• Zebrafish spawn in response to light. The recommended light cycle is 14 hours on / 10 hours off, which simulates optimal natural breeding conditions.

• The best male to female ratio is 1 - 2 females to 1 male. Optimal breeding ages are between 3 – 18 months for females and up to 1 year for males.

• Zebrafish spawn naturally in shallow water and vegetation. Optimal water quality parameters include:

  • pH: 7.2 - 7.4

  • Conductivity: 800 – 2000μs

  • Temperature: approximately 28°C

• After the zebrafish lay their eggs, they will tend to eat them. Protect the eggs by providing a substrate in the tank for the eggs to drop into.

Please check out the Aquaneering AquaSpawner and crossing tanks for zebrafish breeding options.

There may be several different reasons, but one possibility is that the larvae are using up their yolk sac and are subsequently starving. Zebrafish are born with yolk sacs with provide them with nutrients until they inflate their swim bladders (approximately 5 days postfertilization). The swim bladder enables the larval fish to swim up to the surface of the water to feed. Although there is some overlap between the periods of yolk sac absorption and external feeding, the yolk sac is usually depleted by approximately 7 dpf. If the larvae are not provided with any feed before this time, they will starve to death by 10 dpf. Another reason your larval fish are dying may be due to an infestation of coleps in the fish water or paramecia cultures. Coleps are freshwater protozoa that will attack and completely devour the larval fish. For more information, please read “Coleps, Scourge of the Baby Zebrafish” (http://zfin.org/zf_info/monitor/vol5.1/vol5.1.html#Coleps, Scourge of the Baby Zebrafish).

Recommended density is 40 – 50 larval fish per liter.

Larval fish should be raised in a shallow level of water (e.g. a petri dish) until about 4 - 5 days post-fertilization (dpf) so their swim bladders will inflate properly.  After 4 - 5 dpf, the larval fish can be transferred to a nursery tank containing 300 ml of static system water.  Be sure to install a fry screen in the tank.  Many Aquaneering customers start a slow drip 6-7 dpf.  At approximately 15 dpf, you can turn on a slow, constant (24-hour) water flow to the tanks.

Generally, once you have begun feeding the larval fish a more complex diet such as Artemia (brine shrimp) or a processed diet (approximately 15 – 20 dpf) that necessitates more frequent water changes, you can put the nursery tanks on a recirculating filtration system. However, be sure that the flow rate into the nursery tanks is not too high.

Optimal light levels for zebrafish breeding are 5-30 foot-candles (about 54 - 323 lux) at the tank's surface. The recommended light cycle for breeding zebrafish is fourteen hours on, ten hours off.

• Feed your zebrafish high protein foods, such as the Zebrafish Select Diet offered by Aquaneering which contains at least 52% protein. Feed the fish only as much as they can consume in 1 - 2 minutes.

• Spawn the fish on a regular basis. Every two weeks is generally considered the normal egg laying cycle.

• Zebrafish spawn in response to light. The recommended light cycle is 14 hours on / 10 hours off, which simulates optimal natural breeding conditions.

• The best male to female ratio is 1 - 2 females to 1 male. Optimal breeding ages are between 3 – 18 months for females and up to 1 year for males.

• Zebrafish spawn naturally in shallow water and vegetation. Optimal water quality parameters include:

  • pH: 7.2 - 7.4

  • Conductivity: 800 – 2000μs

  • Temperature: approximately 28°C

• After the zebrafish lay their eggs, they will tend to eat them. Protect the eggs by providing a substrate in the tank for the eggs to drop into.

Please check out the Aquaneering AquaSpawner and crossing tanks for zebrafish breeding options.