Zebrafish Workshop: Water Quality Parameters and Fish Disease

Transcription

1 Zebrafish Workshop: Water Quality Parameters and Fish Disease World Aquaculture Society Aquaculture America Meeting Seattle, WA, USA February 17, 2009 George E. Sanders, DVM, MS, Certified Fish Pathologist (AFS/FHS) University of Washington Department of Comparative Medicine USGS - Western Fisheries Research Center Seattle, WA.

2 Water Quality Parameters and Fish Taxonomy Water Sources Disease Water Quality Parameter descriptions Average ranges for fish and zebrafish Clinical cases of poor water quality and disease Sub-acute or Chronic Acute

3 Taxonomy of Zebrafish (Danio rerio) Family Cyprinidae (Minnows or carps) Order Cypriniformes (carps) Distribution Asia, Pakistan, India, Bangladesh, Nepal, and Myanmar. Biology Breed all year long, omnivorous, best in groups > 5.

4 Water Sources Municipal Sources tap water (treated sewage) Chlorines and Chloramines, pathogens Protected & Unprotected Sources wells, aquifers, springs, rivers, lakes Degassing of water, wild fauna and flora, agriculture Artificial Sources Reverse Osmosis, Distillation, Desalinization Biofilm development, low to no mineral or salt content

5 Water Quality - Introduction Minimal scientific consensus has been reached about the recommended water quality for zebrafish (Danio rerio). Most current standards are based upon what has been done traditionally and appears successful in the laboratory setting. Minimal numbers of controlled studies have been done to evaluate what water quality is best for captive zebrafish.

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7 Water Quality General Guidelines: Zebrafish Temperature: (24-28 O C) Alkalinity: mg/l (ppm) Hardness: mg/l ph : / Salinity: 0.5-1g/L(ppt) Conductivity: μS Un-ionized ammonia (NH 3 ): < 0.02 mg/l Nitrite (NO - 2): < 1mg/L Nitrate (NO - 3): < 50mg/L Chlorine: 0mg/L DO 2 : > 6 mg/l CO 2 : < 5mg/L

8 Water Quality Parameter Monitoring When in recirculation systems, measure water temperature 1, ph 1, Ammonia 1, Conductivity 1, D.O. 1, Chlorine 1, Nitrate 2, Nitrite 2, Hardness 2, Alkalinity 2, and CO 2 2,. 1 measure and record daily 2 measure and record at least weekly Flow through systems: measure temperature daily, ph and ammonia at least weekly. Static systems: measure temperature daily; testing other values depends on frequency and amount of water exchange. NOTE: Acceptable ranges must be posted on animal care sheet, in log books or in the animal housing area for immediate reference.

9 Alkalinity (Carbonate Hardness KH) An aggregate measurement of the capacity of water to neutralize acids. Primarily attributable to the presence of bicarbonate (HCO 3- ) or carbonate (CO 3 2- ) in the water or watershed. Wide range possible: mg/l CaCO 3 Dissolved metals (copper, zinc, and aluminum) are more toxic to fish in water of low alkalinity.

10 Total Hardness (General Hardness GH) The sum concentrations of calcium, magnesium, and other divalent cations. This value depends upon the geology of the watershed of the source. Wide range possible: mg/l CaCO 3 Increasing hardness: Decreases osmoregulatory stress. Decreases the toxicity of dissolved metals like copper and zinc.

11 ph Measurement of basic, acidic, or neutral qualities of a solution. Ranges from or greater Neutral or slightly basic ( ) Can fluctuate in recirculating systems. As ph increases so does concentration of un-ionized ammonia (toxic form).

12 Salinity/Conductivity Amount of dissolved salt in water. Salinity: 0.5-1g/L (ppt) Capacity of water to conduct an electrical current. Conductivity: μs Can both be modified by addition of balanced salt formulations.

13 The Aquatic Nitrogen Cycle

14 Un-ionized Ammonia (NH 3 ) Un-ionized Ammonia (NH 3 ): <0.02 mg/l Requires ph and temp O C measurements to determine the accurate percentage of total ammonia in the un-ionized form. Primary nitrogenous waste product excreted by fish. Can be modified by increasing biofiltration capacity or increasing water volume changes.

15 Nitrite (NO 2- ) Nitrite (NO - ): < 1mg/L 2 Intermediate oxidation product of NH 3 produced by bacterial degradation (Nitrosomonas spp.). Can be modified by increasing biofiltration capacity or increasing water volume changes.

16 Nitrate (NO 3- ) Nitrate (NO - ): < 50mg/L 3 Final oxidation product of NH 3 produced by bacterial degradation (Nitrospira spp.). Can be modified by increasing water volume or frequency of water volume changes.

17 Chlorine (CL 2 )/Chloramine Chlorine: 0 mg/l (ppm) Chloramine: Chlorine +Ammonia Can be modified by use of commercial dechlorination products (sodium thiosulfate), filtration using activated charcoal, or aeration over time*. Zebrafish can tolerate low (0.5-1ppm) levels of CL 2, however chronic exposure can cause skin, eye, and gill irritation.

18 Dissolved Oxygen (D.O.) D.O 2 : > 6 mg/l. Saturation >100% can lead to supersaturation of water, which may result in gas bubble disease. Can be modified by the use of mechanical aeration devises, degassing towers, trickle filters, or by the introduction of oxygen gas.

19 Carbon Dioxide (CO 2 ) CO 2 : < 5mg/L Unless problematic excess dissolved gases, (from water source, component, or configuration of system), is tested and recorded infrequently. Can be modified by use of degassing towers, packed columns, trickle filters, etc.

20 Commercial Test Kits Hach Fish Farming Test Kit (FF-1A) Aquarium Pharmaceuticals Ammonia, Nitrate, and Nitrite test kit Tetratest General and Carbonate Hardness Oaklon Acorn Series Meter conductivity and ph.

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23 Poor Water Quality Induced Disease Sub-acute to chronic, sub-lethal levels of toxic agents can cause: Stress Immunosuppression Disease Death Ammonia Nitrate Nitrite Low water hardness Heavy Metals

24 Nitrite Toxicity Case Investigative group reported mortality in group of 500 Coho salmon (Oncorhynchus kisutch) fry. Mortality began 8 days after system conversion from flow-through to recirculation configuration. Average rate of 7 17 fish per day.

25 Nitrite Toxicity Case: Diagnostics Average Water Quality Parameters: Temperature 11.5 C ph 7.5 D.O Conductivity 181 mg/l CaCO 3 TAN 1.0 * (< 0.25 mg/l) Un-ionized Ammonia mg/l Nitrite 1.65 mg/l (< 0.5 mg/l) Total Hardness 51.3 mg/l CaCO 3 Alkalinity 34.2 mg/l CaCO 3

26 Nitrite Toxicity Case: Diagnostics Necropsy report: Reverse Saddle Back lesions originating at vent. Coelomic distention with clear, straw-colored, to reddish opaque ascites. All internal organs NSL Bacterial Culture report: Aeromonas hydrophila/caviae group Photobacterium (Vibrio) damsela Nitrite toxicity with secondary gram negative bacterial septicemia.

27 Nitrite Toxicity Case: Treatment Reduce Nitrite levels below 0.5 mg/l. Daily water changes. Addition of beneficial nitrification bacteria to biologic filter. Increase chloride or conductivity levels.* Per veterinary consultation, order medicated feed (oxytetracycline 6%) and begin treatment for 14 days consecutively.

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29 Ammonia Toxicity Case Investigator reports low level mortality and ulcerative lesions on new shipment of fish in quarantine facility.

30 University of Washington University of Washington Ulcerative Dermatitis: Bacterial University of Washington Un-ionized ammonia (NH 3 ): > 0.02 mg/l

31 Ammonia Toxicity Case: Treatment Euthanized all severely ulcerated fish. Buffered MS-222 solution. Decreased ammonia levels: Decreased ph of system Water exchanges Addition of beneficial nitrification bacteria to biologic filter Increased conductivity level of system.

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33 Inadequate Hardness Case Investigative group reports adequate spawning rate but poor survival rate to adulthood. Large mortality seen in embryos and larvae Average Water Quality Parameters: ph (with significant NaHCO 3 - use) Temperature 29 C Conductivity 900 μs Alkalinity 17.1 mg/l CaCO 3 Total Hardness mg/l CaCO 3

34 Inadequate Hardness Case: Treatment and Resolution Evaluated and corrected problematic issues of paramecium culture and some husbandry issues. Improved adult fish nutrition feed diversification. Increased alkalinity by the addition of autoclaved crushed coral to the system. Adjusted Water Quality Values: ph (with minimal NaHCO 3 - use) Temperature 29 C Alkalinity mg/l CaCO 3 Total Hardness mg/l CaCO 3 Significant increase in spawning efficiency and fish survival rate.

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36 Poor Water Quality Induced Disease Acute, lethal levels of toxic agents can cause: Stress Immunosuppression Disease Rapid Death Gas Supersaturation Chlorine Chloramine Heavy metals

37 Gas Supersaturation (Gas Bubble Disease - GBD) Likely to occur when water becomes saturated with gas (nitrogen, oxygen, etc.). Fish breathe before water has equilibrated to atmospheric pressure and dissolved gases leave solution into the fish s tissue or blood vessels. Severity of damage depends upon number of emboli formed and which tissues are affected.

38 University of Washington Gas Bubble Disease: Acute Presentation University of Washington

39 Gas Bubble Disease University of Washington University of Washington Sub-acute Presentation

40 System Findings and Problem Resolutions Manipulated system component revealed air pocket and active cavitation within the strainer housing area. Numerous fine bubbles were also noted on the mesh screens of each tank s outflow vent. Pump was shut down & strainer cover was removed and resealed with proper sealant on O-ring. Air was bled off and pump re-started. Increased level of aeration in all tanks. Facilitates more rapid equilibrium of system water to atmospheric pressure Mortalities were removed from tanks. Morbid fish were pooled and euthanized.

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42 Chlorine Toxicity Case Technician performing system daily system cleaning. Within one hour, fish began to exhibit dyspnea and death in other tanks on the main system. Technician contacted facility supervisor ASAP when fish were noticed to be dead or dying. Technician was instructed to turn off the system pumps and turn off each individual tank feed on every tank on main system.

43 Problem Event Time Line Facility manager upon arrival (~15 min) found several dead and dying fish. ½ - ¾ cup ( L) of sodium thiosulfate was added to sump in attempts to neutralize the bleach. Triage was initiated and live fish were removed from main system and transferred to quarantine system.

44 Problem - Event Time Line Additional Treatments Morbid fish were humanely euthanized. Buffered MS-222 solution Feeding was suspended for hrs to reduce stress. Remaining fish were monitored for morbidity and mortality.

45 Ramifications Approximately 3,200 adult zebrafish died. 5 unique strains of transgenic fish lost. 2+ years of active research and many $$$$$ lost. Incident reported to regulatory and funding agencies. Main System Restart: Bleached, cleaned, de-chlorinated, flushed (multiple times), tested for chlorine (in multiple locations), and restarted. All disposable system components were replaced (particulate filters, carbon, and crushed coral).

46 Benaroya Research Institute

47 Chlorine Use & Toxicity Common disinfectant utilized in municipal water systems*, for surface, and clothing disinfection or decontamination, etc. Fish are exceptionally sensitive to chlorine toxicity. From 0.01 ppm to 5 ppm is acutely toxic to most species. Human threshold for detection by smelling is ~ ppm*.

48 Chlorine Toxicity / Neutralization M.O.A. : oxidation at an alkaline ph Causes acute necrosis of branchial tissue which leads to asphyxiation. Is rapidly inactivated by organic matter; with enough contact time. Can be neutralized by sunlight, aeration, activated carbon, or chemical interactions with sodium thiosulfate*, sodium sulfite*, or sulfur dioxide gas.

49 Chlorine Toxicity This Case 1L of bleach solution (6.15% active) in 57L = 1,076 ppm of chlorine was added to the tank. With dilution by sump volume of 246L = 4.38 ppm of chlorine added directly to the system. Dilution (sump & head tank) = 2.0 ppm chlorine. This amount of chlorine rapidly broke through the two particulate and one carbon filters in parallel and lead to fish death within minutes.

50 A FINE BALANCE HOST PATHOGEN ENVIRONMENT Slide Courtesy of: Dr. T. Miller-Morgan

51 Thank You for Your Attention Danio rerio picture (Darer_u2.jpg) by Lehmann, F.

52 Any Questions?

53 References Astrofsky, K.M., R.A. Bullis, and C.G Sagerstrom. Ch 19 - Biology and Management of the Zebrafish, In Laboratory Animal Medicine, 2nd Edition Academic Press. Courtland, S Recirculating Systems for Zebrafish. Lab Animal 31: Stoskopf, M.K., Ch 20 - Biology and Health of Laboratory Fishes, In Laboratory Animal Medicine, 2nd Edition Academic Press. Ostrander, G.K. The Laboratory Fish Academic Press. Fish Base - Hovanec, T.A., et al Nitrospira-Like Bacteria Associated with Nitrite Oxidation in Freshwater Aquaria. Applied & Environmental Microbiology. 64:

54 References Taxonomic Fish Database / Catalog - M. Westerfield. The Zebrafish Book, 5th Edition Wildgoose, W.H. BSAVA Manual of Ornamental Fish, 2nd Edition Casebolt, D.B. et al Care and use of fish as laboratory animals: current state of knowledge. Lab. Anim. Sci.48: Noga, E Fish Disease: Diagnosis and Treatment. Mosby. Stoskopf, M.K Fish Medicine. Saunders.