🐟Regular Fish Care SOP and Emergency Plan of Action. 🐟¶

Part I. Fish Care SOP¶

1. Overview¶

1A: Intro¶

This plan has been written to explain proper daily, weekly, and longer-term husbandry tasks that need to be conducted in order to ensure proper fish health and quality of life.

This includes the following for all tanks:

Daily health and feeder checks
Water changes twice a week for the shiners and once for the mummichogs (if water parameters are within the expected range)
Weekly monitoring of ammonia, nitrite, and nitrate levels using API drip tests.
Weekly temperature, pH, conductivity, and salinity (mummichog only) or TDS (shiner only) measurements

1B: Fish colonies¶

The Kao Lab currently has two colonies of fish:

Golden Shiners (Notemigonus crysoleucas) housed within 18 tanks in 5530 and the connected testing room housing the two testing arenas
Mummichogs (Fundulus heteroclitus) housed within 4 tanks in 5521 with a testing arena in the same room

2. Responsibilities¶

All members: should record their actions in the fish husbandry spreadsheet. The fish count in a tank should be adjusted whenever it changes (e.g., when a fish dies or when moving them to and from a testing arena).

Technician: Primarily responsible for normal husbandry and colony upkeep tasks listed in the sections below. Other responsibilities include cleaning the housing room and maintaining records and stock of supplies.
PI: Responsible for support of emergency situations such as active flooding or mass fish death.
Other lab members: Can provide support for tasks they feel are necessary or is requested of them.
Research staff: Responsible for maintenance of their arena(s) and the health of the fish within, including feeding, cleaning, water quality, and filters.

3. Acceptable water quality parameters¶

Shiner water parameters¶

Parameter	Acceptable range	Low Urgency	Moderate Urgency	High Urgency
Ammonia (mg/L)	0-0.5	----	1-2	≥4
Nitrite (mg/L)	0-0.25	----	0.5-1	≥2
Nitrate (mg/L)	0-20	----	40-80	≥160
Dissolved oxygen (mg/L)	>6	6-4	4-2	<2
Water temperature (°C)	17-20	outside of the acceptable range	----	----
pH	6.8-8	----	5.1-6.7 or 8.1-8.9	≤5 or ≥9
Conductivity (uS)	2000-4000	not in that range	----	----
TDS(ppt)	<450000	>450000	----	----
***

Mummichog water parameters¶

Parameter	Acceptable range	Low Urgency	Moderate Urgency	High Urgency
Ammonia (mg/L)	0-0.5	----	1-4	≥8
Nitrite (mg/L)	0-0.25	----	0.5-2	≥5
Nitrate (mg/L)	0-20	----	40-80	≥160
Dissolved oxygen (mg/L)	>6	6-4	4-2	<2
Water temperature (°C)	17-20	outside of the acceptable range	----	----
pH	6.8-8	----	5.1-6.7 or 8.1-8.9	≤5 or ≥9
Conductivity (uS)	7800-15600	not in that range	----	----
Salinity (specific gravity)	1.005-1.010	not in that range	----	----
* * *

4. Daily care¶

4A Fish health check¶

Inspect every tank to make sure fish are clearly visible.
If there are any dead fish, remove them with a clean net and place the carcass in a labeled bag (lab name, species name, "Not For Zoo", and date) , sanitize the net with Virkon, and update the log sheet.
Check for visible signs of fish illness (such as color/texture changes or swimming/buoyancy issues)

If there are any issues found during health checks, proceed to section 8

Make sure the canister filter tubing, sponge filter, and UV filters are correctly situated within the water and are functional.

4B Feeder check¶

Check that the feeder is in place and functional (e.g., the numbered arrows are visible, and time is correctly set).
If the food in the feeding drum is less than 25% full, remove the drum and fill it to 95% full of pellet-flake mixture.
The pellet-flake mixture is a 2:3 (pellet:flake) ratio from the labeled food containers in the housing room.
If either food container is running low, refill it using the larger containers kept in the fridge in ISC-5550. Spare food supply is kept in the freezer compartment in ISC-5550, as well as sealed bags in the fridge in the Revell lab.

Further feeding issues are addressed in section 17.

4C Shiner holding tanks A & B¶

Check that there is sufficient water in holding tanks A and B to perform water changes (~40 gallons per rack for a 25% water change).
If not, use the long hose to fill one of the storage tanks, making sure the corresponding valve between the holding tank and the pump is closed. Secure or clamp the hose to the holding tank. Run the cold faucet with the valve turned upward and monitor the amount of filled water using the faucet reader. Do not fill a single holding tank more than 200 gallons.
For every 100 gallons, treat the water (at least 24 hours before using it in the housing tanks) with 2.5 cups of pond salt, 50 mL of API Stress Coat, and 5 tsp of Seachem neutral buffer. All of these ingredients are found in tubs near the door.
Put items back after use.

Water conversions: 378.5 L = 100 gal, 567.8 L = 150 gal, 662.4 L = 175 gal, 757 L = 200 gal.

4D Housing room¶

Before leaving:

Make sure the lights and fan are functional.
Make sure the faucet is off and the water pump is unplugged, if it was used.
Make sure all items and tools are replaced in their proper location.
Log all performed activities in both the physical and the online husbandry sheets.

5. Weekly care¶

5A Water testing¶

See section 3 for acceptable value ranges and section 9-10 for how to deal with water testing issues.

5A-1 Test kits¶

Ammonia, nitrite and nitrate measures

Done once a week unless values are above acceptable ranges from section 3.

API drip test kit¶

For each fish tank rack:

Lay out 3 test tube racks (one for each test), each holding 6 test tubes (one test tube for each housing tank). Using an eyedropper, fill each tube in a rack with 5 mL of tank water being tested; repeat for the other 2 test tube racks.
Ammonia: Add 8 drops of ammonia test solution #1, followed by 8 drops of ammonia test solution #2. Cap and shake for 5 seconds. Wait 5 minutes for the color to change and compare with the reference sheet.
Nitrite: Add 5 drops of nitrite test solution. Cap and shake for 5 seconds. Wait 5 minutes for the color to change and compare with the reference sheet.
Nitrate: Add 10 drops of nitrate test solution #1 and invert the test tube several times. Vigorously shake the nitrate test solution #2 for at least 30 seconds. Now add 10 drops from nitrate test solution #2. Cap and shake for 1 minute. Wait 5 minutes for the color to change and compare with the reference sheet.
Rinse test tubes after use. Allow them to dry after rinsing. The water quality tests for the mummichog tanks are performed the same way; however, 3 racks of 4 tubes should be set up, instead of 3 racks of 6. The test kits are interchangeable for both fresh and salt water, but the appropriate reference card must be used (freshwater for shiners and saltwater for mummichogs).

5A-2 Probes¶

Each done once a week unless values are above or below acceptable ranges from section 3.

Hanna combo probe¶

Water temperature, pH, conductivity, TDS

Open the probe cover and rinse with clean water and press the power button to turn on the probe.
Press the mode button until the pH mode is on and dip the probe into the tank and stir. Wait until the clock symbol (stability tag) disappears. Record the value for pH and water temperature.
Press the mode button until the conductivity mode is on, and once the stability tag disappears again, record the conductivity by multiplying 1000 to convert from mS to uS.
Press the mode button one more time and record TDS.

TDS for shiners only

Repeat the probe readings for each tank, rinsing the probe each time. Fill the cap with a few drops of storage solution and close cap on the probe.

Hanna combo probe manual

Hanna oxygen probe¶

Dissolved oxygen

Tip: if seeing O2 reading is difficult, there is a lightbulb button that turns on a backlight.

Open the carrying case and take out the probe.
Remove the black protective cage from the probe, then remove the clear cap from the probe (which prevents desiccation) and re-attach the cage by screwing it in at the threaded end.
Press the power button and wait for probe conditioning to finish.
Put the probe into the tank water sitting near the bottom of the tank and wait for the hourglass (stability) icon to disappear.
Record the O2 value, and repeat this process for each tank.
When finished, press the power button to turn off the probe. Unscrew the protective cage, replace the clear cap, and slide on the protective cage before placing the probe back in the carrying case.

Hanna DO probe manual

Refractometer¶

Salinity

For mummichogs only

Take the probe from the case, open the plate, and put a few drops of DI water from the small squeeze bottle by the sink. Look through the eyepiece in light and make sure that the blue area is in line with the zero marker.
To measure salinity, either use a pipette or dip the slanted surface into the tank. The surface should be coated with water, and the plate should be lowered with no air bubbles visible.
Look through the eyepiece into the light and use the specific gravity scale to record the value at the blue line.
Repeat steps 2 & 3 for each tank you want to measure.
Rinse the probe again with DI water and return to its case.

5B Water changes¶

5B-1 Shiner water changes¶

Make sure enough treated water is available in the water holding tanks before conducting water changes.
Use the valves in front of each tank to drain the water 25% using each rack's drainage pipe to channel the water into the floor drain.
If needed, more water can be emptied from a housing tank using a siphon (5gal = ~25% water volume).
Close individual tank drainage valves, and open or close the larger valves to guide the water from the holding tank through the water pump and into the corresponding fish rack that needs to be filled.
Plug in the water pump (using the cord near the fan) and make sure if any of the housing tanks are overfilled, the excess water can still flow into the floor drain. Unplug the cord when done.
Repeat for each of the 3 racks and record as an X for water change in the water change column of the husbandry log sheet.

5B-2 Mummichog water changes¶

For the mummichogs, water is normally treated on demand. Connect the extra hose (red end to black end) to the faucet under the sink to fill the holding bin to the line that indicates 20 gallons. Treat the water with 2 drops per gallon of FRITZ Complete water conditioner and ~1.5 cups of marine salt per 2 gallons of water. Stir with a net until the salt has dissolved, and the specific gravity reads 1.005 (ideal) or in the acceptable range before adding it to the tanks.
Close each intake and outflow valve on each canister filter to prevent filters from taking up air and un-priming.
Use a siphon and 5-gallon bucket to drain at least 25% of the water in a tank (marked on the tank).
Plug in the green sump pump in the holding bin and pump water into the tanks (tank 1 holds ~30 gallons, tanks 2 and 3 hold ~20 gallons, and tank 4 holds 45 gallons).
Repeat step 1 as necessary until water changes for all of the tanks are complete and then reopen all of the canister filter valves.

More detailed water change instructions can be found in the lab documentation ¶

6. Monthly and as needed care¶

6A Monthly care¶

6A-1 Probe calibration and maintenance¶

Once per month the probes should be calibrated. Calibration is done at the calibration area inside the Kao Lab.

Hanna combo probe¶

manual for detailed instructions

pH: Turn on the probe and place it in pH mode, then hold the power button until it says Cal. Place the probe in the appropriate pH standards as the probe displays them. If the calibration is successful, the probe will have the “cal” icon displayed on the left side when in pH mode.
Conductivity: Turn on the probe and place it in conductivity mode, then hold the power button until it says Cal. Place the probe in the conductivity standard when displayed. If the calibration is successful, the probe will have the “cal” icon displayed on the left side when in conductivity mode.
TDS: is not calibrated and left on stock calibration

Note that one of the combo probes is not able to calibrate its conductivity and should be used with stock calibration by hitting the reset button during calibration.

After calibration, turn the probe off and rinse. Add storage solution and recap the probe.

Hanna oxygen probe¶

Manual for more information

Before calibration, take out the probe and remove both the protective cage and the clear cap to make sure that there are no air bubbles in the membrane. If there are bubbles, unscrew the membrane cap and replace the electrolyte fluid inside using the small blue electrolyte bottle. Then make sure the zero-oxygen solution has not expired (it lasts for a month from the mixing date). If expired, more can be created by opening a new box of zero-oxygen solution, and mixing the part I powder into the part II liquid.
To calibrate, turn on the probe and wait for it to finish conditioning. Place the probe into the zero-oxygen solution, then press the "Cal" button and then the DO calibration button displayed on the screen. Automatic one-point calibration will start once the reading becomes stable and the stability marker disappears. Press the "CFM" button on screen to confirm and then hit the "ESC" button to leave calibration.
Rinse probe and replace clear cap and protective cage. Return the probe back in case.

6A-2 Virkon change¶

Once a month Virkon from labeled buckets in husbandry rooms should be refreshed this in done by:

Pour Virkon solution from the bucket into the sink. Rinse the bucket with fresh water.
Wear gloves. Add 3-4 gallons of water into the labeled bucket and add 1 scoop of Virkon Aquatic powder per gallon of water into the bucket to make a solution. Stir to dissolve the powder.
The solution is ready to use! Make sure the bucket is covered with the labelled lid when not actively in use.

6A-3 Stock check¶

Stock of items should be monitored to determine which items are running low within the lab or the husbandry rooms. Aim to have enough essential items such as consumables to not be used up within 6 months.

Examples of supplies:

pH buffers
water conditioners
medications
fish food
API test kits
calibration standards if expiring

6B As needed¶

6B-1 Canister filter maintenance¶

If flow is not as strong as expected

check for clogs in the pipes and clear clogs if necessary
clean intake cage
rinse out biological filter media
replace carbon media if needed

6B-2 Fish census¶

If the fish count estimated is doubted, the fish can be scooped into a bucket, filled with treated water, and moved to another filled bucket with a net to count them. When done, return fish gently to their tank with a net.

6B-3 UV filter¶

Every 6 months or longer based on powered-on hours, the UV bulb within the filter needs to be replaced.

This can be done by:

unplugging the filter.
removing the large cartridge from the filter. Do not open to remove the bulb inside!
unbox and attach a new bulb cartridge and reconnect to the filter.
if needed, the sponge in the filter intake can be cleaned or replaced.

6B-3 Housing room cleaning¶

The floor can be swept and mopped to remove salt build up.
The sides and lids of the tanks can be wiped with a clean cloth to remove salt and/or food build up.
If a deeper clean is necessary, the shelving can be cleaned as well.

6B-4 Tank gravel cleaning¶

If the tank substrate or bottom is covered by a large amount of algae or waste, it can be agitated and cleaned by moving the siphon while performing a manual water change around the bottom of the tank.
Gravel can also be removed from the tank and cleaned by hand if needed.

6B-5 Adjust water change outflow¶

During a water change for the shiners, if the tanks are not filling up at an even rate, there is a valve above the tanks through which the water from the pump enters the tank. By opening/closing the tank valve, flow can be increased/decreased.

7. Record and stock keeping¶

7A Fish husbandry digital sheet¶

The digital sheet can be found here!

Please record on each room summary sheet as well as each individual tanks tab:

Daily

health checks and deaths
feeder checks

Weekly

census summary
probe measures
water changes (2x for the shiner colony)

Monthly

changing Virkon
cleaning
probe calibration
inventory of items

7A-1 Archiving sheet and monthly rollover¶

At the beginning of the month, the digital log sheet needs to be archived and refreshed for the next month.

The digital file should be downloaded as both an .xlsx and .pdf file and named using the template below and placed in the KaoLab Google Drive folder

FishHusbandry_YYYY_MM.pdf or FishHusbandry_YYYY_MM.xlsx

After logging, roll over the document by changing the month listed on each tab, copying over the ending census as the new month's starting census, and clearing all other data

7B Fish husbandry physical sheet¶

The summary tab for each colony must also be copied onto a physical sheet left near the outside of the husbandry room doors. This sheet is replaced every month by the animal facility staff.

Part II. Fish care emergency plan of action ⚠️¶

Emergency procedure introduction¶

This plan exists to give specific instructions on how to deal with unexpected situations. It categorizes issues into four different levels of urgency. * No issue: Parameters are within acceptable range, and equipment is functional. No action beyond normal tasks is required. * Low urgency: Increased monitoring required; extra water changes or other tasks may be required. * Moderate urgency: Actions beyond normal tasks are necessary; the issue must be resolved within seven days. * High urgency: A solution to the issue must be implemented on the same day observed. The problem must be stabilized (not getting worse) or shown to be actively improving before leaving on the day observed.

Escalate or de-escalate urgency levels based on water parameter measures, equipment issues, and fish health. Use later sections as a guide for urgency levels of each potential situation.

Here are the acceptable ranges of the water parameters:

Shiner water parameters¶

Parameter	Acceptable range	Low Urgency	Moderate Urgency	High Urgency
Ammonia (mg/L)	0-0.5	----	1-2	≥4
Nitrite (mg/L)	0-0.25	----	0.5-1	≥2
Nitrate (mg/L)	0-20	----	40-80	≥160
Dissolved oxygen (mg/L)	>6	6-4	4-2	<2
Water temperature (°C)	17-20	outside of the acceptable range	----	----
pH	6.8-8	----	5.1-6.7 or 8.1-8.9	≤5 or ≥9
Conductivity (uS)	2000-4000	not in that range	----	----
TDS(ppt)	<450000	>450000	----	----
***

Mummichog water parameters¶

Parameter	Acceptable range	Low Urgency	Moderate Urgency	High Urgency
Ammonia (mg/L)	0-0.5	----	1-4	≥8
Nitrite (mg/L)	0-0.25	----	0.5-2	≥5
Nitrate (mg/L)	0-20	----	40-80	≥160
Dissolved oxygen (mg/L)	>6	6-4	4-2	<2
Water temperature (°C)	17-25	outside of the acceptable range	----	----
pH	6.8-8	----	5.1-6.7 or 8.1-8.9	≤5 or ≥9
Conductivity (uS)	7800-15600	not in that range	----	----
Salinity (specific gravity)	1.005-1.010	not in that range	----	----
* * *

8: Fish illness & death¶

8A: Signs of illness¶

Signs and symptoms to look out for:

Swimming or buoyancy problems
Injury (e.g, missing fins) or red spots from lack of scales.
White/gold spots, fuzz, or other patches on fish body
Bulging or cloudy eyes
Abnormal social behavior
Bloating or bulging scales

If any fish are visibly ill, they can be removed from the tank and euthanized. If a large portion or all of a tank is infected with a disease, the tank should undergo a treatment regimen as described in the next section, or euthanized.

8B: Treatment & euthanasia procedure¶

Treatment of a tank¶

Treatment of a tank is identical to the medication order when fish are received and quarantined [seen here (https://www.thekaolab.com/docs/site/fish-care/new-fish/). Medicate once filtration (UV and carbon) has been removed.

Day 1:

Medicate tanks using medication trio: FRITZ Maracyn (1 scoop per 10 gallons), FRITZ Paracleanse (1 scoop per 10 gallons), ICK-X (1 capful per 10 gallons)

Day 2: