Page 1 of 7 Title: Care and maintenance of sailfin and shortfin mollies of the Genus Poecilia Author(s): Facility Manager: Attending: Veterinarian Margaret Ptacek, Professor, PI Mary K. Ramos-Negrete, Graduate Student John Smink, Lab Manager John Parrish, DVM, Ph.D. 1.0 OBJECTIVE The objective of this Standard Operating Procedure (SOP) is to describe standard operating procedures for the care of sailfin and shortfin mollies of the Genus Poecilia. This SOP is based on standard aquarium trade procedures and care protocols from the labs of Dr. J. Travis FSU and Dr. D. Resnick UC Riverside. Like all SOPs, these procedures are subject to revision as better techniques and products become available. 2.0 HEALTH AND SAFETY Attire appropriate to the task must be worn at all times. All Clemson University personnel must be enrolled in the Clemson University Medical Surveillance Program. 3.0 PERSONNEL/TRAINING/RESPONSIBILITIES Personnel familiar with the equipment and laboratory techniques and trained in this and referenced SOPs may perform this procedure. All Clemson University personnel must have completed the IACUC on line training. Prior to being assigned full responsibility for performing this procedure, personnel must have demonstrated proficiency in the use of the technique in a closely supervised environment. Documentation of training should be kept for each person performing the procedure. 4.0 REQUIRED AND RECOMMENDED MATERIALS 4.1 Required Materials 150 gallon cattle tanks Aquarium heaters Trashcan filters Plexiglas lids Luft pumps 55 gallon barrels Airstones and airlines Buckets (large and small) Large and small nets Coarse scouring pads and brushes Water pumps and PVC fittings Hoses and PVC fittings Ziptop baggies Fresh/Marine Liquid Test Kits
Page 2 of 7 Instant Ocean Salt NovAqua Water Conditioner AmQuel Water Conditioner Marine Buffer Stresscoat Min/Max thermometer 4.2 Recommended Materials StressZyme Aquaria (5 & 10 gallon) Mesh aquarium covers Sharpies, pencils, and label tape Clipboards and S hooks Measuring spoons & cups Exterior tank identification Clothespins Metal shelving units 4.3 Prepared Materials Rubbermaid trashcan with holes drilled in the bottom, outfitted with a PVC spraybar, and filled halfway with bio-balls and topped with a coiled mesh filter. 55 gallon barrel filled with 15ppt salt water as needed Flake food mixture of O.S.I. Fresh Water Flakes, O.S.I. Spirulina Flakes, and O.S.I. shrimp flakes as needed 5.0 GUIDELINES The following procedures were developed to meet or exceed the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. AVMA (American Veterinary Medical Association). 2013. AVMA Guidelines on Euthanasia. 6.0 PROCEDURE 6.1 General Care of Fish A. Check room temperature daily Safe range is 75-92 F, above or below these temperatures requires immediate attention. Optimal temperature is 82-87 F. If summertime temperatures exceed 87 F, a shade can be drawn to prevent over heating. Cold temperatures in the winter will be ameliorated with the use of submersible aquarium heaters. B. Check each tank for mortality daily- Any fish found dead should be removed from the tank immediately. Place the dead fish in a ziplock bag and label the bag with all the names and codes written on the tank and the date. Take the bag to the freezer in room 17 of the Aquatic Animal Research Lab (in the basement of the P&As building) that is approved to house vertebrate mortalities. Record the mortality on the daily mortality log including: the date, species and population of the fish, tank number, and sex. Multiple dead fish from a tank can be placed in the same ziplock bag. Sick or wounded fish should not be removed, but the PI or the lab manager should be contacted immediately. If there are no dead fish, record a zero on the daily mortality log. C. Check each cattle tank for filter operation daily Water should be flowing out of the PVC spraybar evenly, through the spiral filter, and back into the tank. If the filter is not working, several steps should be taken. The tank should be temporarily aerated using small Luft pumps and airstones, and water should be changed using aerated water from
Page 3 of 7 the mixing barrels. The source of the pump problem should be identified; possibilities include: tripping the breaker or replacing a bad pump. If the breaker has been tripped, move that breaker to the off position and then back to the on position. If it continues to trip, identify the source and make sure that all electrical equipment is dry. If the pump is bad, replace it with a new pump from one of the shelving units. Note: if aquaria are set up, check to make sure filters are under the water level and aerating in each tank. Adjust filters as necessary to maintain adequate aeration. See section 6.2Cb for more details of sponge filter maintenance. D. Feed each tank flake food daily Each cattle tank is fed a mixture of O.S.I. Fresh Water Flakes, O.S.I. Spirulina Flakes, and O.S.I. shrimp flakes once a day. The flakes are stored in a plastic container in the freezer that is approved to house vertebrate food located in a store room in the BRC Greenhouse. If flakes are low, you should notify the PI or lab manger. The standard measure of flakes is a tablespoon. The number of nearly level (not heaping) spoonfuls is listed in the bottom right hand corner of each tank sheet. For example: a sheet that has S=2 written in the lower right hand corner would receive two full tablespoons of flake food daily. Tanks that appear very cloudy or have sickly fish should not be fed and should be reported immediately to the PI or lab manager. After feeding, the feeder should sign the Daily Feed Sheet and record the daily min and max room temperature ( F) then reset the min/max thermometer. Note: if aquaria are set up, each aquarium should be fed according to the directions on the tank using teaspoons. S=1/2 written in the bottom corner of the aquaria indicates that it should be fed ½ teaspoon of flake food. E. Chemical testing in tanks weekly Water quality in each cattle tank should be measured weekly. Use the YSI unit to measure:, salinity, and temperature. Measure ammonia, nitrate, nitrite levels, and ph using Fresh/Marine Liquid Test Kits (Aqua. Pharm.). Data should be written down on the Water Quality Sheet. Safe ranges for each water quality parameter are listed on the top of the Water Quality Sheet. If levels for ammonia or nitrate are high, 50% of the water in the cattle tank should be changed and record on the Tank Sheet. Note: if aquaria are set up, pick one tank per row and measure: ph, ammonia, and nitrite levels using Fresh/Marine Liquid Test Kits (Aqua. Pharm.). Record all data on the Water Quality data sheet. Safe ranges for each water quality parameter are listed on the top of the Water Quality data sheet. If levels for ammonia or nitrate are high, 33% of the water in all aquaria should be changed and record on the Water Change Sheet. F. Water level adjustment weekly Each week, cattle tanks should be topped off with fresh water. Allow the water to run into the drain for 3-5 minutes before using to make sure the line is clean of rust and stagnant water. Fill cattle tanks so that the water level is a couple of inches above the first rung inside the cattle tank. Note: if aquaria are set up, each tank should be filled to the black line weekly with fresh water. The black line is marked on the upper right corner of the tank. G. Water changes according to water quality Water changes should be done for each cattle tank as needed. Need should be determined by water quality and the amount of algae growing inside the cattle tank. If the inside of the cattle tank has become so dark in color that it is hard to see the fish, it should be cleaned. Unplug the heater and pump. Use a scrub brush to scrape the algae off of the sides and bottom of the cattle tank. Allow the water to settle. Then using a simmer pump, 50-80% of the water should be
Page 4 of 7 drained and replaced with water from the mixing barrel. While draining water use the hose to vacuum up any loose particles that have accumulated on the bottom. Always flush the hoses before filling any tank with mixed saltwater. The cleaning date and amount of water changed should be recorded on the Tank Sheet. Plug the heater and pump back in and make sure the filter is running properly before leaving. H. Mix saltwater barrels when emptied The 55-gallon barrels are to hold the prepared 15 ppt salt water for the aquaria. In filling the barrel, the tap water should be allowed to run for 3-5 minutes to clear any dissolved metals from the water pipes and hose. Add two tablespoons NovAqua, two tablespoons of AmQuel, and 1 tablespoon of Marine Buffer to the barrel. Add 10 cups (each full measuring cup holds 2 cups) of Instant Ocean salt to a small white bucket and dissolve it in fresh water before adding it slowly to the mixing barrel. Place an airstone in the barrel. Water should be made 24 hours before it is needed to make sure it has time to age and come to room temperature. 6.2 Specialized Care of Fish (Ptacek Research Staff) A. Mortality or cloudy water a. Identify the source of mortality as quickly as possible. First, examine the remaining live fish for signs of distress. Look carefully at their fins, scales, eyes, and gills. White slime, white spots, blood streaks, or ragged fin edges are signs of disease. Look carefully at their swimming posture. Fish that seem to float, sink, or lean to one side are also showing signs of disease. Contact the PI or lab manger immediately if any of these symptoms are observed. Fish that have an elevated respiration rate, inflamed gills or are breathing from the surface may be suffering from ammonia toxicity. Again, contact the PI or lab manger immediately! See Diseases and Ammonia Toxicity below for more information. An immediate change of water might be necessary. b. Check tank water chemistry Any tank with abnormal mortalities or with cloudy water should have a full water chemistry check (temperature, ph, salinity, ammonia, nitrite, and nitrate). If conditions are out of range, perform a 50% water change and replace with mixed saltwater. Use the simmer pump to drain and refill tanks. Always flush the hoses before filling any tank with mixed saltwater. B. Treatments a. Diseased tanks Any tank diagnosed with diseased fish should receive their daily dose of medication as noted on the tank. Tanks with new fish or those recovering from ammonia toxicity or medication should be treated with StressCoat (5mL/10 gallons). See diagnosis and treatment of diseases below. C. Maintenance a. Clean the room daily Coil all hoses and place on the clamps on the cattle tanks in the center of the room; put away all pencils, Sharpies, tape, nets, and buckets before leaving. NEVER clean floors with any type of soap or disinfectant containing ammonia or an antibacterial agent. Toxic fumes could be transferred to the water in the aquaria. b. Sponge filter adjustment For aquaria, proper sponge filter operation is very important to the health of the fish. The sponge filters provide a
Page 5 of 7 substrate for helpful bacteria to breed and a source of water oxygenation. Make sure the standpipe is resting on the bottom of the tank and that the bubbles are making contact with the surface of the water (allowing gas exchange to take place). If the filter is not running properly, make sure that the water level is to the indicator line in the upper right hand corner of the tank. The tank may need to be topped off with fresh water. Remove the sponge and rinse it in the sink under warm (never hot) water. Second, check to see if the filter standpipe is clogged by removing the airline. If bubbles are coming from the airline, clean or replace the filter standpipe. It is often necessary to gently pick the little hole that the airline attaches to with a dissecting probe to clear clogs. Third, if no air comes from the airline check to make sure that there aren t any kinks in the air hose from the filter to the gage valve, and all the way to the whisper pump. Make sure the correct valves of the gage valve are opened and closed. Finally check that the whisper pump is working properly. c. Mixing barrels To keep a barrel from laden with algae and hard water deposits, rinse it with tap water while scrubbing the sides with the scouring pad each time the barrel is empty. This seems to keep the sides cleaner, longer; however calcium deposits will form. Occasionally it is necessary to soak the barrels in vinegar water (to get rid of hard water deposits) or bleach water (for excess algae growth). After a vinegar soak, it is necessary to use the scouring pad around the sides. Two days may be necessary to soften the calcium. After a soak, rinse the barrels well and then rinse them once again. d. Supplies Extra mesh (aquarium covers), aquaria, airline, air stones, etc. are located in the AARL in the basement of the P&As building. Ask the PI or lab manager about needed supplies. Please let the PI or lab manager know when supplies are getting LOW so new supplies can be ordered so that we do not run out of anything. D. Water Quality a. Ammonia is the most critical water quality issue It can kill fish if the concentration in water gets too high. Luckily, the mollies tanks do not seem to have an ammonia problem. However, if the fish do start to act sluggishly or do not behave as normal, check the ammonia concentration in the tank. Inflammation of the gills, surface breathing and shimming are all signs of ammonia stress. There is a test kit in the room, and if it registers high, then a water change is needed. b. Cloudiness can also be due to good bacteria searching for a home The bacteria are the kind that break down ammonia, but can take some days to settle into the sponge filter and start doing their job. Why the bacteria take so much time to settle is not known, but it s what the experts say. E. Disease and Toxicity a. Diseases and parasites are relatively uncommon, especially once fish have been in captivity for more than one generation. However, recently collected fish should be isolated and watched carefully for signs of disease. If
Page 6 of 7 you suspect disease, use the diagnosis chart provided in any Aquarium Pharmaceuticals product. Isolate any fish to be treated into a clearly marked tank and write the date and dosage given on the tank. Diseased fish should be checked twice a day and should be treated with stresscoat during the recovery. b. Ammonia toxicity The most common source of fish mortality is ammonia toxicity. Ammonia is the nitrogen waste product that fish urinate and can also be given off by decaying bacteria and uneaten food. When a new tank is set up, the Nitrosomas bacteria that break down ammonia are not yet present in sufficient quantities to keep up with ammonia production. As ammonia concentration rises, fish begin to show signs of respiratory stress. They breathe faster, their gills become red and inflamed, and they may attempt to breathe at the surface of the water. They may also appear listless and just lie on the bottom of the tank or shimmy. High ammonia levels are easily detected with an ammonia test kit. Just follow the instructions on the kit to determine the ammonia level. During the first seven days after a tank is set up, ammonia levels will usually rise from 0.25ppm to 6.0ppm. An ammonia level above 3.0 requires some preventative action. StressZyme is a concentrate of Nitrosomas bacteria that is useful in establishing a normal biological filter, but it takes several days after inoculations to bring down high ammonia levels. AmQuel is a chemical chelating agent that binds to free ammonia and reduces its toxicity. However, often the best treatment of ammonia toxicity is a water change. 33%-67% of the water can be changed depending on the severity of the case. If half of the fish have died and the other half are lying on the bottom of the tank, I would recommend a 100% water change. Ammonia is colorless and odorless so don t expect the tanks to look unhealthy. Just pay attention to how many days it has been since the tank was set up. Days 7-14 should be the peak of ammonia. Days 14-21 should be the peak of nitrite. After day 21, the biological filters of the tanks (the sponges) should be converting ammonia to nitrite and nitrite to nitrate. You know when nitrate is being produced as algae will begin to grow in the tank. The presence of algae is a good sign, for it means that the toxic forms of nitrogen are now being converted to the harmless form of nitrate. The algae have the added benefit of providing food for the fish. F. Emergency a. Power Outage This facility is equipped with a backup generator that will provide power in case of a power outage. We have our pumps plugged into the generator so that the fish will continue to receive aeration, which is most critical to their survival. Tanks should be monitored closely for temperature and water quality in the unlikely event of a long term power outage. If necessary, emergency heat should be supplied. 7.0 QUALITY CONTROL CHECKS AND ACCEPTANCE CRITERIA All procedures are subject to periodic review by the Attending Veterinarian(s) and the Institutional Animal Care and Use Committee.
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