Fish Health & RAS. The Freshwater Institute. Main System Freshwater Institute Replicated Recirculating Aquaculture Systems (RAS) Replicated RAS

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1 Fish Health & RAS Evaluating Rainbow Trout Performance, Health, and Welfare at The Freshwater Institute Christoher Good1, John Davidson1, Carla Welsh1, Kevin Snekvik2, and Steven Summerfelt1 2Washington The Freshwater Institute 1The Conservation Fund Freshwater Institute, Sheherdstown, WV Animal Disease Diagnostic Laboratory, Washington State State University, Pullman, WA Main System Freshwater Institute Relicated Recirculating Aquaculture Systems (RAS) 150 m3 culture tank 4700 L/min recycle flow 30 min tank HRT kg feed/day Main System Scaled down Relicated RAS Previous Observations at The Freshwater Institute CO2 striing unit stacked over a LHO & sum tank fluidizedsand biofilter 60% flow backwash water 40% flow 3 drum filter reuse ums um sum 5.3 m dualdrain culture tank During low flushing (1(12% water exchange) and high feeding (1.3( kg/d er m3/d makeu flow): 15% flow frequent discharge of backwash makeu water system overflow suernatant settling unit Elevated morbidity and mortality Unknown causation No infectious causes susected Water quality arameters within safe limits intermittent (12 times/day) discharge of solids from base of cone 1

2 Study # 1 High vs. Low Exchange Investigate this otential barrier for RAS managed with high feeding rates; secifically: i. ii. 3. Identical Recirculating Aquaculture Systems (6) Assess fish erformance in high vs. low makeu water exchange systems Assess fish health and welfare in these conditions through organ histoathological evaluation fin condition assessments Monitor changes and differences in water quality between the two treatments 6300 rainbow trout raised for 6 months Stocked at 6 months of age (arox. 135g) 1000 er RAS lus 100 er flowflowthrough (initial #s) 40(min) 80(max) kg/m3 density 3 RAS with high makeu H2O (2.6%) Randomized 3 RAS with low makeu (0.26%) Mean feed loadings of 0.39 and 4.1kg/day er m3/day makeu flow, resectively } Small FlowThrough Comarison Tanks (3) High Makeu H2O Exchange vs. Low Makeu H2O Exchange High Makeu H2O Exchange vs. Low Makeu H2O Exchange 2

3 Data Collection Monthly length/weight samling Daily mortalities Tissue samling every 2 months for histoathological evaluation (5 fish er tank) Washington Animal Disease Diagnostic Laboratory Skin, gill, heart, liver, sleen, swim bladder, anterior and osterior kidney Endof ofstudy lasma collection (5 fish er tank) for small animal chemistry anel Animal Health Diagnostic Center, Cornell University Endof ofstudy fin quality assessment Water quality analysis times/week Parameter TAN (mg/l) * Unionized Ammonia (mg/l) * Nitrite (mg/l) * Nitrate (mg/l) * Alkalinity (mg/l) CO 2 (mg/l) cbod 5 (mg/l) * True Color (PtCo units) * UV Transmittance (%) * Phoshorous (mg/l) * TSS (mg/l) * Temerature ( o C) H DO (mg/l) Results: Water Quality High Makeu 0.29 ± ± ± ± ± 1 9 ± 0 2 ± 0 11 ± 1 89 ± ± ± ± ± ± 0.0 Low Makeu 0.48 ± ± ± ± ± 4 9 ± 0 6 ± 1 74 ± 9 53 ± ± ± ± ± ± 0.1 Flowthrough 0.22 ± ± ± ± ± 0 13 ± 0 1 ± 0 1 ± 0 97 ± ± ± ± ± ± 0.0 Significant difference <0.05 between high and low makeu grous Results: Mortality Mean cumulative mortality risk rates: High Makeu = 0.74% } = Low Makeu = 1.69% Flowthrough = 1.12% } No significant differences Weight (grams) Results: Performance Growth Rate with High and Low Makeu Water and Flow Through (Comarison) Treatments 500 High Makeu Low Makeu 300 Comarison 100 1/20/07 2/9/07 3/1/07 3/21/07 4/10/07 4/30/07 5/20/07 6/9/07 6/29/07 7/19/07 Day Results: Performance Mean Final Weight: High makeu: 1401g Low makeu: 1366g Flowthrough: 1253g Mean TGC: High makeu: 2.64 Low makeu: 2.62 Flowthrough: 2.56 No significant differences Results: Histoathology Tissue Odds 95% Low (vs. High) makeu Ratio Conf. Int. H 2 O (overall results) Gill 0.48 (0.16, 1.41) Bivariate logistic regression models comaring resence/absence of Liver Heart Sleen Anterior kidney (0.61, 4.44) (0.45, 2.21) (1.02, 2.42) (0.17, 1.34) Posterior kidney 0.34 (0.21, 0.57) histoathological lesions Swim bladder in fish reared in high vs. Skin 2.92 (1.76, 4.85) low makeu RAS (all > 1 tissue affected 1.23 (0.64, 2.36) samling data combined) > 2 tissues affected 1.12 (0.58, 2.18) value <0.000 <

4 Results: Histoathology Results: Caudal Fin Assessment Recirc. (vs. FlowThrough) (overall results) Bivariate logistic regression models comaring resence/absence of histoathological lesions in fish reared in recirculating vs. flowthrough systems (all samling data combined) Tissue Gill Liver Heart Sleen Anterior kidney Posterior kidney Swim bladder Skin > 1 tissue affected > 2 tissues affected Odds Ratio % Conf. Int. (0.70, 3.84) (0.23, 1.16) (1.44, 3.10) (1.56, 3.90) (0.20, 2.73) (0.22, 2.60) (0.24, 17.56) (0.60, 6.15) (0.71, 2.65) (0.85, 3.05) Value <0.000 < Good Condition Moderate Condition Poor Condition High Makeu Low Makeu Significance Mean Prevalence at EndofStudy Good 96/111 (87%) 70/111 (63%) <0.05 Moderate 9/111 (8%) 21/111 (19%) <0.05 Poor 6/111 (5%) 20/111 (18%) <0.05 Conclusions Unable to relicate revious observations Fish erformed surrisingly well under high feed and low flushing conditions Some statistically significant subclinical (histoathological lesions) and clinical (caudal fin condition, mortality) differences noted between treatments Did not affect fish erformance during study eriod Dissolved Carbon Dioxide? Greater tank hydraulic retention time in main system: aroximately 20 mg/l CO 2, versus aroximately 10 mg/l CO 2 in relicated RAS Elevated Carbon Dioxide Observations in revious research: Reduced feed intake Reduced growth Nehrocalcinosis Presumably due to: Stress Hyercania (acidosis) Imaired oxygenhemoglobin binding Increased toxicity of metals at lower H Economic Considerations Puming more water flow (Q)( ) through a culture tank to decrease dissolved CO 2 levels in the tank (C( out ) will increase the variable cost in inverse roortion to the dro in dissolved CO 2 concentration ( C( C = [C out C in ])) that is required 4

5 Study #2 High vs. Low CO 2 in Low Exchange RAS Investigate the effects of high vs. low dissolved carbon dioxide on rainbow trout reared in high feeding, low flushing RAS: Growth Survival Tissue evaluation via histoathology Assess treatment effects on water quality arameters 5100 rainbow trout raised for 6 months in 6 RAS Stocked at 62 ± 1 grams in size 800 er RAS lus 100 er flowthrough 40(min) 80(max) kg/m 3 density 3 RAS with high CO 2 (25 mg/l) 3 RAS with low CO 2 (10 mg/l) } Randomized All 6 RAS with low (0.26%) exchange, high feed loading (4.1 kg/day er m 3 /day makeu flow) Data Collection Monthly length/weight samling Daily mortalities Tissue samling at study s s end for histoathological evaluation (5 fish er tank) Skin, gill, heart, liver, sleen, swim bladder, anterior and osterior kidney Water quality samling times/week Parameters TAN (mg/l) Unionized Ammonia (mg/l) Nitrite Nitrogen (mg/l) Nitrate Nitrogen (mg/l) Alkalinity (mg/l) CO 2 (mg/l) cbod 5 (mg/l) True Color (PtCo units) UV Transmittance (%) TSS (mg/l) Total Particles (060 µm) Temerature ( o C) H DO (mg/l) Results: Water Quality High CO ± ± ± ± ± 2 24 ± 1 5 ± 0 42 ± 1 70 ± ± x ± ± ± 0.0 Low CO ± ± ± ± ± 2 8 ± 1 6 ± 1 48 ± 3 67 ± ± x ± ± ± 0.0 Flowthrough 0.18 ± ± ± ± ± 2 9 ± 1 1 ± 0 3 ± 0 98 ± ± x ± ± ± 0.0 Significant difference <0.05 between high and low CO 2 grous Results: Growth Results: Performance Comarison of Rainbow Trout Growth Rates in Low Exchange Recirculating Systems with High and Low Carbon Dioxide Mean Final Weight: High CO 2 : 849 ± 7 g Low CO 2 : 817 ± 43 g (Flowthrough: 938 ± 17 g) g Weight (grams) High CO2 Low CO2 Control Mean TGC: High CO 2 : 2.56 ± 15 Low CO 2 : 2.55 ± 12 Mean FCR: High CO 2 : 1.25 ± 0.10 Low CO 2 : 1.25 ± /17/07 8/6/07 8/26/07 9/15/07 10/5/07 10/25/0711/14/07 12/4/07 12/24/07 1/13/08 2/2/08 Day No significant (<0.05) differences between high and low CO 2 grous 5

6 Results: Survival Results: Histoathology High vs. Low CO 2 High CO 2 = 98.3% ± 0.2 Low CO 2 = 96.5% ± 1.8 (Flowthrough = 100%) } > 0.05 SKIN (all samled fish) Lymhocytic dermatitis Lymhocytic eidermitis GILL (all samled fish) Lymhocytic branchitis Eithelial hyerlasia Eithelial hyertrohy Caillary thrombosis HEART (all samled fish) Lymhocytic eicarditis Lymhocytic / Histiocytic myocarditis Lesion Prevalence High CO /15 Low CO /15 3/15 Odds Ratio 1.10 (0.21, 5.86) 0.93 (0.07, 12.7) 1.64 (0.26, 10.4) 0.31 (0.11, 0.89) 1.00 () 0.91 (0.42, 1.97) Results: Histoathology, con t High vs. Low CO 2 Results: Histoathology RAS vs. Flowthrough Lesion Prevalence Odds Ratio LIVER (all samled fish) Lymhocytic heatitis, ortal Lymhocytic heatitis, random Heatic liidosis KIDNEY (all samled fish) Tubular hydroic degeneration Increased melanomacrohages Lesion Prevalence High CO 2 Low CO 2 9/15 8/15 9/15 5/15 3/15 5/15 3/15 2/15 Odds Ratio 1.31 (0.50, 3.45) 3.00 (1.11, 8.11) (0.05, 3.49) 0.93 (0.07, 13.2) SKIN (all lesions) Lymhocytic dermatitis Lymhocytic eidermitis GILL (all lesions) Lymhocytic branchitis Eithelial hyerlasia Eithelial hyertrohy Caillary ectasia Caillary thrombosis HEART (all lesions) Lymhocytic eicarditis Lymhocytic myocarditis RAS 21/30 2/30 21/30 29/30 29/30 14/30 3/30 0/30 1/30 8/30 8/30 1/30 Flowthrough 2/15 6/15 5/ (0.03, 1.04) 2.47 (1.03, 5.95) 1.64 (0.22, 12.0) 0.22 (0.02, 2.01) 0.55 (0.22, 1.36) 0.77 (0.20, 2.94) 0.51 (0.03, 7.42) Results: Histoathology, con t RAS vs. Flowthrough Lesion Prevalence Odds Ratio RAS Flowthrough LIVER (all lesions) 17/30 6/ (0.76, 5.05) Lymhocytic heatitis, ortal 14/30 5/ (0.52, 5.84) Lymhocytic heatitis, random 1/ (0.03, 7.01) Heatic liidosis 2/ (0.17, 12.2) KIDNEY (all lesions) 8/30 6/ (0.14, 2.12) Tubular hydroic degeneration 6/30 6/ (0.11, 1.23) Increased melanomacrohages 1/ Conclusions Unable to relicate revious fish health decline No significant differences in growth and survival between the two treatments Some statistically significant subclinical (histoathological) differences noted between treatments Results demonstrate that rainbow trout can be reared in high CO 2 RAS to market size without adverse effects 6

7 Study #3 Ozonation vs. No Ozonation in Low Exchange RAS Reroduce high feed, low flushing conditions with and without ozonation, and investigate fish health through: Growth Survival Tissue evaluation via histoathology Assess effects of ozonation on water quality arameters Recirculating Aquaculture Systems (6) All 6 RAS with low (0.26%) exchange, high feed loading (4.1 kg/day er m 3 /day makeu flow) 3 RAS with ozonation, 3 without (randomized) ORP set oint = 250 mv 6000 rainbow trout raised for 7 months Stocked at 107 ± 1 grams in size 1000 er RAS Ozonation began at 2 months following biofilter acclimation eriod 296 ± 1 g at treatment initiation 40(min) 80(max) kg/m 3 density 24hour hotoeriod Feeding every other hour Data Collection Results: Water Quality Monthly length/weight samling Daily mortalities Tissue samling at study s s end for histoathological evaluation (5 fish er tank) Skin, gill, heart, liver, sleen, swim bladder, anterior and osterior kidney Water quality samling during week of maximum feed loading and fish density Parameters TAN (mg/l) Nitrite Nitrogen (mg/l) Nitrate Nitrogen (mg/l) CO 2 (mg/l) cbod 5 (mg/l) True Color (PtCo units) TSS (mg/l) Total Particles (0200 µm) Heterotrohic bacteria (counts/ml) No 0.59 ± ± ± 1 12 ± 0 5 ± 1 52 ± 3 10 ± x x ± ± ± 1 12 ± 0 2 ± 0 2 ± 1 5 ± x x

8 Results: Water Quality Results: Growth No 1000 Weight (grams) Ozonation begins 200 No 0 Feb08 Ar08 Jun08 Aug08 Date Results: Performance Mean Final Weight : 1161 ± 6 g No : 993 ± 24 g =0.001 Mean TGC : 2.38 No : 2.10 Results: Mortality = 0.7% ± 0.2 No = 1.7% ± 0.5 } = Skin Gill Heart Results: Histoathology Lymhocytic dermatitis Lymhocytic eidermitis Goblet cell hyerlasia Lymhocytic branchitis Eithelial hyerlasia Eithelial hyertrohy Caillary thrombosis Lymhocytic eicarditis Lymhocytic / Histiocytic myocarditis Lesion Prevalence 3/15 7/15 8/15 6/15 No 1 11/14 2/14 1/14 1/14 7/15 Odds Ratio 9.17 (0.67, 125) 0.54 (0.09, 3.11) 5.25 (1.23, 22.3) 15.6 (1.15, 211) 0.93 (0.07, 12.7) 0.69 (0.14, 3.34) 0.20 (0.02, 1.82) Results: Histoathology, con t Liver Lymhocytic heatitis, ortal Lymhocytic heatitis, random Heatic liidosis Kidney Tubular hydroic degeneration Lymhohistiocytic interstitial nehritis Increased melanomacrohages Lesion Prevalence 1 13/15 No 3/15 7/15 8/15 Odds Ratio 5.23 (0.75, 36.5) 1.45 (0.80, 2.64) 3.25 (1.06, 9.98) 2.17 (0.81, 5.84) 1.00 (0.07, 14.2)

9 Conclusions Unable to relicate revious fish health decline significantly imroves water quality in low exchange RAS significantly imroved growth, and resulted in better survival, when raising rainbow trout to market size Some statistically significant subclinical (histoathological) differences noted between treatments Acknowledgements Dr. Kevin Snekvik Washington Animal Disease Diagnostic Laboratory, Washington State University All research was suorted by the Agriculture Research Service of the United States Deartment of Agriculture, under Agreement No Oinions, conclusions, and recommendations are of the authors and do not necessarily reflect the view of the USDA. All exerimental rotocols involving live animals were in comliance with Animal Welfare Act (9CFR) and have been aroved by the Freshwater Institute Animal Care and Use Committee. Questions?? 9