In recognition of the fact that there are numerous aquatic species from a diverse range of environments, and subsequently a comparably diverse range of captive husbandry requirements, this SOP cannot cover all aquatic species. This document provides guidance on the general issues that must be addressed in an SOP, as well as items such as lab hygiene that are not a variable. Species-specific SOP's need to be developed by the researchers for each species being worked on to ensure the proper housing and care needs of that species are adequately met.
Aquatic Animal Care Standard Operating Procedures
Water Sources and Effluent
- Water Sources
Freshwater is available as tap water. Tap water must be dechlorinated (BBC), and in some areas, chloramines must be addressed as well. Freshwater can further be treated with Reverse Osmosis and deionization as necessary to alter mineral contents and alkalinity. Seawater is available at BBC via seawater wells. Additionally, seawater can be made from commercially available sea water mixes and fresh water.
Wastewater effluent from non chemically contaminated tanks can be drained into the floor drains in the Marine Biology Buildings aquarium rooms or the drain field in the Mesocosms. Chemically contaminated water must be disposed of according to EH&S policy.
- Life Support Systems
Life Support Systems maintain water quality and circulation within an aquatic lab habitat. The level and complexity of an LSS vary with the needs of species, stocking densities, feeding levels, and experimental needs. In all events, the LSS needs to be sufficient to maintain water quality and circulation needs of the fish.
- Nitrogen Compounds
Ammonia is the primary excretory product of aquatic organisms, as well as from the breakdown of feces and excess food. Ammonia is converted by biological filtration into nitrite and finally nitrate. Ammonia is toxic to aquatic organisms. Nitrite is toxic to freshwater fish. Nitrate is not considered toxic, but excessively high levels can have adverse health impacts on some species. The susceptibility to ammonia and nitrite toxicity varies between species and life stages within a species. Ammonia in water exists as both toxic unionized ammonia and ionized ammonium. The concentration of the toxic unionized ammonium varies with pH, salinity, and temperature, as well as the total concentration of ammonia. These factors need to be considered when determining acceptable levels for a specific organism.
Control of these nitrogen compounds is done through a varying combination of filtration and water exchange. Closed systems relying primarily on filtration will need frequent monitoring while the biological filtration is cycling. Large frequent water exchanges will reduce the frequency of testing needed. Testing frequency for a giving life support system set up can be based on knowledge gained from prior experience with comparable systems, or established via weekly testing to gain the necessary knowledge to establish the needs of a given system.
Oxygen concentrations need to be appropriate for the species. Concentration of oxygen in water varies with temperature, salinity, and biological oxygen demand. Oxygen levels can be maintained by circulation and supplementary aeration as needed.
pH needs need be appropriate to the species. In closed systems, the various biological process occurring tend to drive down pH. As a result, pH needs to be monitored and adjusted as necessary. The rate of pH change can vary greatly, depending on the type of life support system used, stocking density, and rate of water exchange. Therefore the frequency of pH monitoring needs to be determined based on the specific needs of the system being set up.
Salinity requirements and tolerances vary widely among species and life stages. The salinity should be appropriate for the species, and any changes made slowly. Generally, a rate of 1ppt/hr is acceptable, with larger changes acceptable for estuarine species.
The temperature should be maintained within normal ranges for the species. Changes of temperature are generally best kept to no more than 1 degree C per hour, although some species may tolerate larger rates of change.
- Animal Density
Animal density is determined based upon species needs and support capacity of the holding system being used. Schooling fish can be raised in high-density systems with adequate LSS, while large territorial fish are not tolerant of high density.
Disease in fish typically occurs in one of 3 forms, parasites, bacterial and fungal. In most cases, bacterial and fungal infections are secondary to injury, from handling, fighting or parasite infections. Animals should be visually inspected daily for signs of disease and stress. If a disease is suspected, the PI should be contacted and closer inspection made in conjunction with an experienced aquarist familiar with the organism and it's potential disease issues. If it's determined there is a disease, the FIU Vet should be informed, and appropriate measures are taken to determine a course of action. To help with identification of fish health issues and determining courses of treatment, an excellent resource is Dr. Edward Noga's "Fish Disease, Diagnosis and Treatment".
Types of feed and frequency of feeding needed are highly variable among differing aquatic species. All efforts should be made to supply proper nutrition at a sufficient rate of feeding to ensure good health of the organism being raised. Feeds should be stored in accordance with the manufactures instructions. Storage areas should be clean, and not shared with chemicals or dead specimens.
- Handling and Restraint
Aquatic organisms should be handled with care. Clean disinfected nets should be prewetted to soften the netting and should be of a mesh size to avoid injury. Clean hands or gloves should be wet. Any surfaces used for procedures, such as measuring boards or work tables used for tagging, should be clean, wet, and disinfected as necessary. Exposure to air should be brief and minimized. For procedures that involve keeping a fish out of water for longer than a minute or two, a hose can be used pump water over the gills to enable to fish to breath.
- Hygiene and Disinfection
The interior of labs, counters, sinks, floors, etc. should be kept clean. General lab Surfaces, tanks and equipment can be disinfected with a bleach solution, potassium permanganate, Virkon or iodine-based disinfectants as required and appropriate. Tanks and associated support equipment should be cleaned and disinfected between uses. Common use equipment, such as nets and siphon hoses, should be disinfected between uses to prevent cross-contamination between tanks.
- Quarantine and acclimation of animals to laboratory conditions
A newly acquired stock should be quarantined, as far as practicable from existing stocks. The quarantine period should be sufficient to monitor for and address potential disease problems, as well as allowing the new stock to acclimate to lab conditions. During the quarantine period, close daily visual checks should be made. Any issues noted and brought to the attention of the PI. If disease or other health issues are noted, consultation should be made with someone knowledgeable in disease and health issues of the species being worked with, as well as the FIU Vet being notified.
Fish should be transported in coolers to minimize temperature fluctuations. For short duration transport, fish can be placed in a cooler 1/3 to 1/2 filled with clean water or individually bagged in appropriate sized fish bags that allow the fish to swim upright with the bag 2/3 filled with air. For long duration transport, over 8 hours, fish should be in aerated coolers or packed in bags with pure oxygen. If transport will be upwards of 12 to 24 hours, fish should be held (if at all possible) without food and allowed to purge for 24 hours prior to transport in order to minimize fouling of the transport water. Various ammonia detoxifiers are available from aquarium and aquaculture suppliers that can be used in transport water to further help maintain transport water quality.
Surgery, Anesthesia, Euthanasia and Animal Disposition
Anesthesia in fish is generally best accomplished with MS-222 or Clove Oil. Levels required vary greatly among species and life stages within a species. MS-222 is acidic, and therefore water should be adequately buffered to prevent a substantial lowering of the pH that could lead to acidosis. Low alkalinity (<50mg/L CaCO3) water needs buffering, while high alkalinity water is naturally well buffered. The working solution, not a stock solution (if a premade stock solution is used), should be buffered as necessary. Sodium Bicarbonate in a 2:1 (weight) ration to MS-222 is adequate. When practicable, anesthesia for be used for any invasive surgery in the lab, and can be useful for minimally invasive procedures in some cases. In the field, animals are typically returned to the wild immediately after the procedure. In this scenario, anesthesia has a negative impact on survival and is, therefore, counter-indicated.
Euthanasia is best accomplished with an overdose of anesthetic. Additionally small tropical and subtropical fish can be euthanized with an ice water slurry. With either method, fish should be left in the anesthetic solution or ice slurry for at least 10 minutes after all movement ceases.
At the conclusion of an experiment, live animals can be transferred out to another protocol, donated to another facility, or euthanized as appropriate and approved by the IACUC. Bio-hazardous materials should be disposed of accordingly to EH&S policy.
Surgery in aquatic organisms is most commonly used for tagging. This can range from simple implantation of dart tags to more complex insertion of acoustical tags into the abdominal cavity of fish. In the lab, these procedures should be done in a clean area of the lab away from foot traffic and other sources of contamination. In field situations, a clean work area should be set up such that any chances of contamination are minimized.
Monitoring and Quality Control
Live animals in the lab should be monitored daily. This daily monitoring should visually examine all animals for health and all LSS for proper function. Daily logs should be maintained for all animals or groups of animals (as appropriate). These logs should include the date, name (initials are OK) of person, and time at a minimum. Any observations should be noted, such as something not looking right, a pump needing repair, dead animals, etc. Additionally, any specific routine activities such as feeding, water changes, tank cleaning, water quality measurements, procedures performed, etc. should all be noted on the daily log as they occur. The log should be readily available in the lab, hanging directly on a tank or rack of tacks is ideal. Such routine monitoring and logging of daily activities help to ensure a high quality of animal care and high-quality experimental results.