Ferskvannsavlusing small-scale tests: Short-term exposure tests to elucidate handling effects.

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1 Ferskvnnsvlusing smll-scle tests: Short-term exposure tests to elucidte hndling effects. The use of freshwter to control infesttions of the se louse Lepeophtheirus slmonis K on Atlntic slmon Slmo slr L. July 2014 FHF Project number:

2 Prefce This study is prt of the FHF funded project Bruken v ferskvnn for å kontrollere infeksjoner v lkselus Lepeophtheirus slmonis K på tlntisk lks Slmo slr. Steering group: Kjell Lorentsen, GIFAS - Gildeskål forskningsstsjon AS 6. Leder styringsgruppe Finn Wilhelm Sinkberg, Sinkberg-Hnsen AS 3. Styringsgruppe Jonny Hnsen, Norsk Fisketrnsport AS 3. Styringsgruppe Previous studies undertken t Gifs hve shown tht exposing Atlntic slmon to freshwter resulted in reductions of ll infectious stges of L. slmonis, however, the studies lso highlighted the need to fully elucidte the primry cusl fctors which contribute to these reductions. Acute chnges in wter qulity prmeters such s temperture, slinity nd ph my ply crucil role in the removl of lice from the slmon. In ddition, there is need for more detiled nlysis of wter qulity prmeters before nd during tretments. The use of freshwter will be limited due to supply thus studies will be undertken to ssess the potentil for reducing the time slmon need to be exposed to this medium without compromising the effects. Reusing freshwter for treting lrger biomsses of fish will llow for the method to become more prcticl commercilly. Further elucidtion of the mechnicl/hndling effects will lso be undertken in n ttempt to mximise the potentil tretment effects when fish re exposed to freshwter. This study is one in series which re imed t ttempting to fully elucidte the effects of freshwter s biologicl control method in removing infectious stges of Lepeophtheirus slmonis from Atlntic slmon (Slmo slr L.). The study imed to fully elucidte the effects on physicl removl of se lice s fish re pumped/trnsferred from cge to well contining freshwter nd bck to the cge. If se lice levels cn be reduced by up to 40% due to the effects of physicl contct before exposure to freshwter then short-term exposure to freshwter my be sufficient if the fish re pumped bck into the cge fter exposure using the sme method. In ddition, the study ttempted to determine these physicl effects over rnge of exposure times from 15 minutes up to one hour. 2

3 TITLE Ferskvnnsvlusing I brønnbt WRITTEN BY Ptrick Reynolds PROJECT LEADER PROJECT MANAGER GIFAS DATE 30 th July 2014 PROJECT PERIODE Project number FHF: GRADED Open Abstrct/Summry Ptrick Reynolds Kjell Lorentsen June to December Atlntic slmon with n verge initil weight of 2200g were used in the study. The study ws conducted t GIFAS smll-scle fcility Lngholmen. A 2x2x1 m tnk ws filled with freshwter. For ech exposure test, lice counts were undertken on ten fish fter first hndling, fter exposure to freshwter nd fter secondry hndling. This process ws repeted over rnge of three different exposure times (15, 30 nd 60 minutes). All exposure tests were performed in duplicte. The results from this study show cler reductions of ll present infectious stges of L.slmonis fter first nd secondry hndling. The results showed tht the percentge reductions ttined fter the first hndling nd before exposure to freshwter were on verge 45.2% for pre dults (mture mles nd pre-dults), 45.5% for mture femles nd 44.3% for ll stges of L.slmonis. The percentge reductions ttined fter the second hndling nd fter exposure to freshwter were on verge 88.5% for pre dults (mture mles nd pre-dults), 95.5% for mture femles nd 89.1% for ll stges of L.slmonis present on the fish compred to pre-tretment counts. Reductions in the number of ttched stges of L.slmonis pper to be enhnced with dditionl hndling fter exposure to freshwter. Results my indicte tht fish cn be exposed to freshwter under commercil-scle conditions for shorter time periods thn previous studies hd shown s long s the fish re pssed over grder systems before nd fter exposure to freshwter. Lrger biomsses of fish my be treted with freshwter s result to reductions in the time exposed to freshwter s wter qulity my be mintined for longer time periods. The clernce rtes recorded for ll tests re not ttributed to cute chnges in wter temperture or ph. Results from blood nlysis showed tht hndling in freshwter resulted in minor physiologicl disturbnces consistent with stress response with n elevtion in blood glucose, CO2 nd reduction in blood ph. Further hndling nd replcement of fish bck into sewter resulted in n increse in blood sodium concentrtions consistent with cute hyperosmollity stress. Neither the effect of cute freshwter hndling nor the cute osmotic stresses re severe to conclude tht the fish would not be ble to recover without dverse effects Contct informtion: GIFAS, N-8140 INNDYR, NORWAY Office phone: E-mil: 3

4 Tble of Contents 1.0 Introduction Methods Fish Experimentl design Registrtion of se lice levels Fish welfre Wter Qulity Sttistics Results Freshwter exposure tests: Pre-dult lice Freshwter exposure tests: Mture femle lice Freshwter exposure tests: Mture mle lice Freshwter exposure tests: Combined pre-dult nd mture mle lice Freshwter exposure tests: All stges of L.slmonis Percentge reductions of L.slmonis Fish welfre Wter qulity Temperture, Oxygen, ph nd slinity Wter chemistry Discussion Freshwter exposure tests Fish welfre Wter qulity Conclusions References

5 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 List of Figures Digrmmtic representtion of experimentl set-up. Red numbers indicte when lice counts were undertken. 1: bseline count prior to the experiment; 2: Ten fish removed nd se lice counted before they entered the freshwter tnk; 3: Ten fish counted for se lice fter exposure to freshwter nd 4: ten fish counted for se lice fter pssing through the grding system for second time. Figure 2 Averge number of Pre-dult stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hmdling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. Averge number of mture femle stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. Averge number of mture mle stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. Averge number of mture mle nd pre-dult stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. Averge number of ll stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. Pge

6 List of Pictures Pge Picture 1 The tnk used in the study which ws filled with freshwter. 9 Picture 2 The holding tnk used to trnsfer the fish from the cge to the freshwter tnk. The pipe hs slits t the bottom to prevent sweter entering the holding tnk. 10 6

7 List of Tbles Pge Tble 1 Percentge reduction of ll stges of L. slmonis fter first hndling, exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling 18 Tble 2 Men (SEM) blood prmeters s mesured by istat demonstrting the effects of hndling nd freshwter bth tretments on Atlntic slmon. Vlues with differing letters indicting significnt differences from pre- 19 tretment vlues. Tble 3 Wter qulity prmeters recorded during sixty minute exposure tests 20 Tble 4 Wter qulity prmeters recorded during thirty minute exposure tests 21 Tble 5 Wter qulity prmeters recorded during fifteen minute exposure tests 21 Tble 6 Freshwter chemistry prmeters mesured prior to nd fter fish hve been exposed for one hour. 22 7

8 1.0 Introduction A series of studies hve been undertken t Gifs to ssess the potentil for using freshwter to remove ttched se lice from infected Atlntic slmon. The studies strted in 2011 nd four studies hve now been completed. The first study (report title: The use of freshwter to control infesttions of the se louse Lepeophtheirus slmonis K on Atlntic slmon Slmo slr L. September 2011) initilly ssessed the potentil for using freshwter to remove ttched se lice from infected Atlntic slmon. The study showed tht exposing infected slmon to freshwter resulted in significnt reduction of both mture mle nd femle lice fter three hours nd results from freshwter biossys undertken t the sme time during the first study showed tht fter 1 hour exposure to freshwter, 10% of mture femles were found to be ded whilst 90.9% of mture mles hd died s result to exposure to freshwter. These initil smll-scle studies showed tht there is potentil in using freshwter to delouse infected Atlntic slmon. The second nd third studies (report tilte; Ferskvnnsvlusing i brønnbåt. The use of freshwter to control infesttions of the se louse Lepeophtheirus slmonis K on Atlntic slmon Slmo slr L. April 2013 & October 2013) were undertken under more relistic commercil conditions. These studies showed tht significnt biomss of Atlntic slmon (up to 110 T) could be successfully deloused with freshwter. However, the studies lso highlighted the need to mintin wter qulity prmeters such s dissolved oxygen nd prticulrly ph in order for the ttched se lice to be exposed to freshwter for sufficient time in order to be ffected. During the studies undertken in October 2013, it ws found tht super oxygention system cn mintin sfe levels of dissolved oxygen. Sturtion levels decresed from 124.0% t the strt of the exposure study to 84.0% t which point oxygen ws dded nd levels incresed to 101.0% quickly therefter. However, ph levels stedily decresed to 6.08 ppt during the exposure period At this point the fish were showing signs of cute stress nd it ws decided t this point to strt pumping in sewter to sfegurd the lrge biomss of fish nd to ensure the welfre of the fish. Crbon dioxide redings on bord the well bot (rnging from 19.1 to 68.4ppt) were bsed on ph levels nd were not mesured in rel time. Redings from hnd-held instruments mesured CO2 between 16.0 nd 17.0 ppt t the lter end of the study. For crbon dioxide the sfe criterion used for the Norwegin production of Atlntic slmon smolts is 15 mg L 1 (Fivelstd, S. 2013) provided dissolved oxygen concentrtions re high. However, constnt fish respirtion cn rise crbon dioxide levels high enough to interfere with oxygen intke by fish, in ddition to lowering the ph of the wter. If the cuse of the stress noted in the fish ws ttributed to lowering of ph nd/or n incresed crbon dioxide concentrtion then some form of buffering gent my llevite this problem. A potentil option to prevent swings in ph is to dd Sodium hydroxide (NOH), lso known s custic sod, lye/lut solution or Sodium Hydrte solution. It is highly custic metllic bse nd lkli slt which is vilble s prepred solution t number of different concentrtions. Sodium hydroxide forms n pproximte 50% (by weight) sturted solution with wter. It is commonly used t smolt fcilities which use recircultion systems to help mintin sfe ph levels throughout production. The fourth study (report title: Ferskvnnsvlusing i brønnbåt: Study 4. Wter qulity. December 2013) hd the im of ssessing the potentil of using buffering gent (NOH) to mintin sfe levels of ph when treting lrge biomss of slmon in freshwter for define period of time. Results from the study show tht initilly there ws stedy but smll decrese in ph in both wells once fish trnsfer hd been complete nd prior to the ddition of NOH. The ddition of NOH commenced pproximtely I hr. nd 30 minutes fter the fish hd been trnsferred to both wells t 8

9 rte of 0.25 l/hr. The decline in ph slowed fter the ddition nd in the well contining freshwter even incresed slightly fter 10 minutes post-ddition. The decrese in ph levels continued however, s the rte t which NOH ws incresed there were corresponding smll increses in ph in both wells. This present study showed tht there is potentil for NOH to be used s buffering gent to control ph in wells filled with freshwter. However, further reserch is required to elucidte flow rtes nd how much to dd to mintin sfe levels throughout desired tretment period of pproximtely three hours. The present studies hve shown cler reductions in ll infectious stges of L. slmonis from Atlntic slmon. However, the studies hve lso highlighted the need to fully elucidte the primry cusl fctors which contribute to these reductions. To this end studies will be implemented to identify these fctors. Acute chnges in wter qulity prmeters such s temperture, slinity nd ph my ply crucil role in the removl of lice from the slmon. In ddition, there is need for more detiled nlysis of wter qulity prmeters before nd during tretments. The use of freshwter will be limited due to supply thus studies will be undertken to ssess the potentil for reducing the time slmon need to be exposed to this medium without compromising the effects. Reusing freshwter for treting lrger biomsses of fish will llow for the method to become more prcticl commercilly. Further elucidtion of the mechnicl/hndling effects will lso be undertken in n ttempt to mximise the potentil tretment effects when fish re exposed to freshwter. Aims nd Objectives Primry objective: The im of this study is to fully elucidte the effects on physicl removl of se lice s fish re pumped/trnsferred from cge to well contining freshwter nd bck to the cge. If se lice levels cn be reduced by up to 40% due to the effects of physicl contct before exposure to freshwter then short-term exposure to freshwter my be sufficient if the fish re pumped bck into the cge fter exposure using the sme method. The im of this study is to determine these physicl effects over rnge of exposure times from 15 minutes up to one hour. The study will be performed t Gifs smll-scle fcilities using holding tnks contining freshwter exposing smll groups of Atlntic slmon for ech test. Se lice infesttion levels will be recorded prior to ech test nd immeditely fter trnsfer, fter exposure nd immeditely fter trnsfer bck to the cge. Secondry objectives: Wter smples will be tken from the freshwter tnk before nd during tretment for full chemicl nlysis in order to elucidte sttus nd/or ltertions in wter qulity over time. In ddition, blood smples will be drwn from fish exposed to freshwter nd immeditely before nd fter tretment to investigte issues relting to fish welfre. 2.0 Methods 2.1 Fish Atlntic slmon with n verge initil weight of 2200g were used in the study. All fish originte from the sme group of fish nd shre the sme genetic nd environmentl bckground. These fish hve not been used in ny previous trils. The fish were mintined in one cge of 125 m 3 (5x5x5m). 9

10 2.2 Experimentl design The study ws conducted t GIFAS smll-scle fcility Lngholmen on 9 th nd 10 th July A 2x2x1 m tnk ws used in the study (picture 1). The tnk ws plced on the pltform nd filled with freshwter. The tnk hd flow-through system deployed which llowed for continul replenishment of the freshwter throughout ll exposure tests. For ech exposure test, 30 fish were lifted from the cge with the id of wet net nd deposited into trnsfer tnk used in the smllscle fcility for grding nd trnsferring fish from cge to cge (picture 2). The first ten fish (n= 10) entered the pipe system individully nd were netted before they entered the freshwter tnk. These fish were sedted nd ny lice present were recorded. After lice counting ws completed these fish were removed. The remining twenty fish were then pssed from the trnsfer system through the pipe system nd into the freshwter tnk. After the desired exposure time to freshwter hd been ttined, ten fish were immeditely removed (n= 10) by netting them into seprte contined for sedtion nd lice counting. After lice counting hd been completed, these fish were removed. The remining ten fish (n=10) were then netted from the tnk nd plced through the trnsfer system for second time. These fish were then sedted nd ny lice present registered. The pipe system ws fitted with grid to remove sewter. Thus, ll fish were trnsferred without sewter entering the cges nd were subjected to the sme hndling nd mechnicl perturbtion. This process ws repeted over rnge of three different exposure times (15, 30 nd 60 minutes). All exposure tests were performed in duplicte Picture 1 The tnk used in the study which ws filled with freshwter. 10

11 Picture 2 The holding tnk used to trnsfer the fish from the cge to the freshwter tnk. The pipe hs slits t the bottom to prevent sweter entering the holding tnk. Fish pumped from cge nd over grding system into holding tnk filled with freshwter. Fish pumped from holding tnk over grder into cge fter exposure Grder FW holding tnk Grder Fish exposed to freshwter for defined short-term periods. 1 5x5x5 m cge Figure 1 Digrmmtic representtion of experimentl set-up. Red numbers indicte when lice counts were undertken. 1: bseline count prior to the experiment; 2: Ten fish removed nd se lice counted before they entered the freshwter tnk; 3: Ten fish counted for se lice fter exposure to freshwter nd 4: ten fish counted for se lice fter pssing through the grding system for second time. 11

12 2.3 Registrtion of se lice levels A lice count ws undertken the sme dy to ssess the lice burden prior to tretment with freshwter. From the cge being used for the study, 30 fish were sedted with Benzok t concentrtion of ctive substnce of between 30-40mg / l (15-20ml Benzok/100 liter) nd ny lice present were recorded. After counting hs been complete, ny lice remining in the continer ws lso recorded. In ddition for ech exposure test, se lice infesttion levels were recorded from 10 fish immeditely fter the fish hve pssed over the grder before exposed to freshwter, these fish were trnsferred to the holding cge fter counting. A lice count ws tken fter the fish hve been exposed to freshwter for the defined period of time (10 fish), gin these fish will be trnsferred to the holding cge fter counting. The remining 10 fish were ssessed for lice sttus fter they hve been pumped from the holding tnk nd hd pssed over the grder or the second time before being trnsferred into the holding cge. All slmon counted for se lice infesttion levels during the tests were netted nd plced into tnk contining sewter nd sedted with Benzok 20mg/l. Lice were registered in 4 ctegories for ll lice counts: Lepeophtheirus slmonis: Adult femle Lepeophtheirus slmonis: Predult nd mles Lepeophtheirus slmonis: Chlimus Cligus elongtus 2.4 Fish welfre To determine the level of stress on fish during tretment blood smples were drwn from rndomly selected fish t different exposure times nd t different stges of tretmet. Ten fish were rndomly netted from the holding cge nd euthnized by n overdose of Benzok nd blood smples were drwn prior to the tretments (n = 10) to estblish bseline stress nd osmoregultion levels. A further five fish (n = 5) were euthnized nd blood smples drwn fter the fish hd pssed over the grder nd exposed to freshwter for 60 minutes. Five more fish were selected fter exposure for 60 minutes nd hving pssed over the grder for second time (n = 5). This process ws repeted for the fish exposed to freshwter for 15 minutes (n = 5) giving totl of thirty fish smpled (N = 30). Cudl blood smples were withdrwn from euthnized fish vi 21 G heprinized needle (Sodium heprin). Blood smples were immeditely injected into n EC8+ crtridge nd nlyzed using n i-stat 300 series Portble Clinicl Anlyzer (Abbott Lbortories. Abbott Prk, Illinois, USA). ph, glucose, sodium (N+), potssium (K+), totl CO2 (TCO2), prtil pressure of CO2 (PCO2), bicrbonte (HCO3), hemtocrit (Hct) nd hemoglobin (Hb) were mesured s potentil indictors of stress nd disturbed osmoregultion. Remining blood ws centrifuged t 5000 xg for 5 minutes nd the plsm decnted into Eppendorf vils nd frozen t -20 o C for potentil further nlysis. 2.5 Wter Qulity Oxygen sturtion (%), slinity (ppt), nd ph within the freshwter tnk ws monitored routinely throughout for ech exposure study. In ddition, wter smples (1 L) were drwn immeditely prior to nd fter the fish hd been exposed to the freshwter tretment for one hour to compre differences to wter chemistry fter short-term exposure. Both smples were stored frozen t 20 0 C for lter nlysis by NIVA. 2.6 Sttistics For lice counts sttisticl significnce of differences were computed from one-wy or two-wy nlysis of vrince (ANOVA) using Minitb TM sttisticl softwre ( Ryn & Joiner, 1994). The normlity nd homogeneity of the vrince of ll dt sets ws tested prior to prmetric sttisticl 12

13 Averge number of pre-dult lice per fish nlysis. Normlity ws tested by grphic exmintion of probbility plots nd the Anderson- Drling test. Significnt differences between tretments were determined by Tukey s multiple rnge test (p < 0. 05). Differences in men bundnce of ttched se lice were detected fter log trnsformtion of the dt. For blood smples, dt ws nlysed using one wy nlysis of vrince with bonferroni plnned contrst ginst the pre-tretment vlues. Where ssumptions of normlity were not met by the dt, Kuskl Wllis non-prmeteric ANOVA on rnked dt ws performed. 3.0 Results 3.1 Freshwter exposure tests: Pre-dult lice The results from the freshwter exposure study for pre dult lice cn be seen in figure 2. There ws significnt reduction in ll stges of se lice counted fter the fish hd pssed over the grder nd before exposure to freshwter. Reductions in the verge number of pre-dult lice decresed from 1,1 per fish (pre-count) to 0,45 per fish for both the sixty nd thirty minute tretments (F1, 2 169,1; p < 0.01 nd F1, 2 169,2; p < 0.01 respectively) whilst the fifteen minute tretment hndling effect ws reduction to 0.35 per fish (F1, ; p < 0.001). The levels of pre-dult lice recorded fter direct exposure to freshwter were similr to those recorded fter the first hndling. The number of pre-dult stges recorded on the fish exposed to freshwter for sixty minutes ws 0.4 per fish which ws significntly lower compred to the pre count vlue (F1, ; p < 0.001) but not significntly lower thn the counts recorded fter first hndling. A similr trend ws recorded for fish exposed to freshwter for thirty nd fifteen minutes (F1, 2 9.0; p > 0.05 nd F1, ; p < respectively). After exposure to freshwter for sixty minutes nd the fish hd pssed over the grder for second time, there ws significntly less pre dult stges ( 0.5 per fish) compred to the pre count (F1, p < 0.001). For fish pssing over the grder for second time fter thirty minutes exposure to freshwter there ws n verge of 0.25 pre-dult lice per fish which ws significntly lower compred to the pre count vlue (F1, p < 0.01 whilst fish exposed to freshwter for fifteen minutes nd hving pssed over the grder were found to hve no ttched pre dult stges present. 1,2 1 0,8 I hour exposure 30 minutes exposure 15 minutes exposure 0,6 0,4 0,2 0 pre count 1st hndling exposure 2nd hndling Figure 2 Averge number of Pre-dult stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hmdling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. 13

14 Averge number of mture femle lice per fish 3.2 Freshwter exposure tests: Mture femle lice The results from the freshwter exposure study for mture femle lice cn be seen in figure 3. There were reductions in femle stges of se lice counted fter the fish hd pssed over the grder nd before exposure to freshwter. The verge number of mture femle lice decresed from 0.37 per fish (pre-count) to 0.45 nd 0.20 per fish for the sixty nd thirty minute tests fter the first hndling but not significntly so (p > 0.05). The first hndling of fish for the fifteen minute tretments resulted in significnt reduction to 0.15 per fish (F1, p < 0.001). The levels of mture femle lice recorded fter direct exposure to freshwter were similr to those recorded fter the first hndling. The number of mture femle stges recorded on the fish exposed to freshwter for sixty nd thirty minutes (0.2 per fish for both tretments) ws lower thn the pre count vlue but not significntly so (p > 0.05). There ws further significnt reduction of mture femle lice fter exposure to freshwter for thirty minutes nd fter hndling (F1, ; p < 0.05) After exposure to freshwter for ll tretments nd the fish hd pssed over the grder for second time, there ws significntly less mture femle stges (0.05 per fish) for the sixty minute exposure tests (F1, p < 0.05) whilst for both the thirty nd fifteen minutes tests there were no mture femle lice recorded on ny of the fish (F1, p < 0.001). 0,6 0,5 0,4 I hour exposure 30 minutes exposure 15 minutes exposure 0,3 0,2 0,1 0 pre count 1st hndling exposure 2nd hndling Figure 3 Averge number of mture femle stges of L. slmonis per fish recorded prior to tretment (precount), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. 3.3 Freshwter exposure tests: Mture mle lice The results from the freshwter exposure study for mture mle lice cn be seen in figure 4. There were reductions in mle stges (from 0.63 per fish for the pre-count) of se lice counted fter the fish hd pssed over the grder nd before exposure to freshwter for both the sixty (0.55; p > 0.05) nd fifteen minute (0.4 per fish; F1, p < 0.001) tretments. There ws no reduction in the verge number of mture mle lice fter first hndling for the thirty minute exposure tests (p > 0.05). The levels of mture mle lice recorded fter direct exposure to freshwter were lower to those recorded fter the first hndling for the sixty nd thirty minute tretments (0.45 nd 0.35 respectively) but not significntly so (p > 0.05). 14

15 Averge number of Mture mle lice per fish There ws no significnt reduction of mture mle lice fter exposure to freshwter for fifteen minutes nd fter hndling (p > 0.05). After exposure to freshwter for ll tretments nd the fish hd pssed over the grder for second time, there were less mture mle stges (0.05 nd 0.25 per fish) for the sixty nd thirty minute exposure tests (F1, p < 0.01nd p > 0.05 respectively) whilst for the fifteen minutes tests there were no mture mle lice recorded on ny of the fish (F1, p < 0.001). 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 I hour exposure 30 minutes exposure 15 minutes exposure pre count 1st hndling exposure 2nd hndling Figure 4 Averge number of mture mle stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. 3.4 Freshwter exposure tests: Combined pre-dult nd mture mle lice The results from the freshwter exposure study for pre-dult nd mture mle lice cn be seen in figure 5. There were significnt reductions (from 1.73 per fish for the pre-count) of combined mture mle nd pre-dult lice counted fter the fish hd pssed over the grder nd before exposure to freshwter for both the sixty (1.0 per fish; F1, p < 0.001) nd fifteen minute (0.75 per fish; F1, p < 0.01) tretments. There ws reduction of pre-dult nd mture mle lice for the thirty minute exposure tests (1.1 per fish) compred to the pre count vlue but not significntly so (p > 0.05). The levels of combined pre dult nd mture mle lice recorded fter direct exposure to freshwter were lower to those recorded for the pre count nd fter the first hndling for the sixty nd thirty minute tretments (0.85 per fish; F1, p < 0.01 nd 0.85 per fish p > 0.05 respectively). Exposure for fifteen minutes resulted in significntly lower pre-dult nd mture mle lice (0.9 per fish) (F1, p < 0.05) compred to the pre-count but there were no further reductions fter first hndling. After exposure to freshwter for ll tretments nd the fish hd pssed over the grder for second time, there were less pre-dult nd mture mle stges (0.1 nd 0.5 per fish) for the sixty nd thirty minute exposure tests (F1, p < 0.01nd p > 0.05 respectively) whilst for the fifteen minutes tests there were no pre dult nd mture mle lice recorded on ny of the fish (F1, p < 0.001). 15

16 Averge number of mle nd pre dult lice per fish 2 1,8 1,6 1,4 I hour exposure 30 minutes exposure 15 minutes exposure 1,2 1 0,8 0,6 0,4 0,2 0 pre count 1st hndling exposure 2nd hndling Figure 5 Averge number of mture mle nd pre-dult stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. 3.5 Freshwter exposure tests: All stges of L.slmonis The results from the freshwter exposure study for ll stges of L. slmonis cn be seen in figure 6. There were significnt reductions (from 3.83 per fish for the pre-count) of ll lice counted fter the fish hd pssed over the grder nd before exposure to freshwter for both the sixty (2.35 per fish; F1, p < 0.001) nd fifteen minute (1.65 per fish; F1, ,1 p < 0.001) tretments. There ws reduction in the thirty minute tretments of ll stges of lice (2.40 per fish) compred to the pre count vlue but not significntly so (p > 0.05). The levels of lice recorded fter direct exposure to freshwter were lower to those recorded for the pre count nd fter the first hndling for the sixty nd thirty minute tretments (1.9 per fish; F1, p < 0.01 nd 1.75per fish p > 0.05 respectively). Exposure for fifteen minutes resulted in significntly lower lice (2.0 per fish) (F1, p < 0.05) compred to the pre-count but there were no further reductions fter first hndling. After exposure to freshwter for ll tretments nd the fish hd pssed over the grder for second time, there were significntly less lice (0.25 per fish; F1, p < 0.01 nd 1.0 per fish; F1, p < 0.05) whilst for the fifteen minutes tests there were no lice recorded on ny of the fish (F1, p < 0.001). 16

17 Averge number of ll stges of lice per fish 4,5 4 3,5 I hour exposure 30 minutes exposure 15 minutes exposure 3 2,5 2 1,5 1 0,5 0 pre count 1st hndling exposure 2nd hndling Figure 6 Averge number of ll stges of L. slmonis per fish recorded prior to tretment (pre-count), fter first hndling, fter exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Vlues represent mens ± S.D. Men vlues which do NOT shre letter were found to be significntly different by ANOVA nd by Tukey`s multiple rnge test. 3.6 Percentge reductions of L.slmonis The percentge reductions for ech stge of L. slmonis before, during nd fter exposure to freshwter cn be seen in tble1. For pre-dult stges, the percentge reductions ttined fter first hndling during freshwter exposure nd t second hndling were between 59.1 nd 68.2%. After exposure to freshwter, reductions were between 54.5 nd 63.6%. After second hndling, percentge reductions were 77.3 nd 95.5% for thirty minutes nd sixty minutes tretments respectively whilst there ws 100% reduction for the fifteen minute tretment fter second hndling. For mture femle stges, the percentge reduction fter the first hndling nd before exposure to freshwter ws between 31.8 nd 59.1%. After freshwter exposure vlues rnged between 45.5 nd 86.4% After second hnding fish exposed to freshwter for sixty minutes hd totl reduction of 86.4% whilst for fish exposed for thirty nd fifteen minutes hd 100% reduction of mture femle lice. For mture mle stges, there ws 13.2% reduction for the fish for the sixty minute tretment nd 36.8% for the fifteen minute tretment whilst there ws no percentge reduction recorded for the fish during the thirty minute tretment. The percentge reduction recorded fter freshwter exposure rnged between 21.1% for the fish exposed to freshwter for fifteen minutes nd 44.7%. for fish exposed for thirty minutes. After exposure nd second hndling, there ws 60.5% reduction for the thirty minute tretment group nd 92.1 nd 100.0% reductions for the sixty nd fifteen minute groups respectively. For combined mture mles nd pre-dult stges, there were reductions rnging from 36.5 nd 56.7% fter first hndling. After freshwter tretment, percentge reductions rnged from 48.1 nd 51.0% for the three tretments. After second hndling, there ws percentge reduction of 71.0% for the thirty minute tretment group whilst the sixty minute tretment groups ttined n overll reduction of 94.2% nd the fifteen minute group ttined 100.0% reduction. For ll stges of L.slmonis, the percentge reduction ttined fter first hndling for ll three tretments rnged from 37.4 to 57.0%. After freshwter exposure, the percentge reductions rnged between 47.8 nd 54.3% whilst fter second hndling, percentge reductions for fish treted for 17

18 sixty minutes ws clculted to be 93.5% whilst for the thirty minute tretments, reduction of 73.9% ws ttined. For fish treted for fifteen minutes percentge reduction of 100% ws recorded. Tble 1 Percentge reduction of ll stges of L. slmonis fter first hndling, exposure to freshwter for 60, 30 nd 15 minutes nd fter second hndling. Stges Pre-dult stges Mture femle stges Mture mle stges Combined mles nd predults Totl lice Tretment time Percentge reduction 1st hndling exposure 2nd hndling 1 hour 59,1 63,6 95,5 30 minutes 59,1 54,5 77,3 15 minutes 68,2 63,6 100,0 1 hour 31,8 45,5 86,4 30 minutes 45,5 86,4 100,0 15 minutes 59,1 45,5 100,0 1 hour 13,2 28,9 92,1 30 minutes 0,0 44,7 60,5 15 minutes 36,8 21,1 100,0 1 hour 42,3 51,0 94,2 30 minutes 36,5 51,0 71,2 15 minutes 56,7 48,1 100,0 1 hour 38,7 50,4 93,5 30 minutes 37,4 54,3 73,9 15 minutes 57,0 47,8 100,0 3.6 Fish welfre Blood prmeters mesured by istat nlysis re summrised in tble 2. Bseline blood N + levels were mm prior to tretment. For the sixty minute tretments, fter first hndling nd before exposure to freshwter, N + levels decresed to mm nd incresed to mm fter exposure to freshwter nd fter second hndling (p < 0.05). For fish exposed to freshwter for fifteen minutes, blood N + levels incresed to mm (p < 0.05). Bseline blood K + levels were 4.06 mm prior to tretment. Levels incresed to 4.16 mm fter exposure to freshwter for sixty minutes nd fter second hndling whilst for fish exposed for fifteen minutes, blood K + concentrtions incresed to 4.24 mm fter exposure nd second hndling. For totl CO2, bseline levels were found to be 9.6 mm, this incresed to 10.8 mm for fish exposed for sixty minutes nd fter second hndling whilst for fish exposed to freshwter for fifteen minutes totl CO2 levels were found to be 9.0 fter second hndling. Beeline glucose levels were 78.9 mg/l nd fter second hndling of fish exposed to freshwter for sixty nd fifteen minutes, levels significntly incresed to 96.6 nd 89.0 mg/l (p < 0.05). Bseline percentge hemtocrit levels were 26.9%, this incresed significntly to 27.6 nd 31.0% fter sixty nd fifteen minutes exposure nd fter second hndling (p < 0.05). Blood ph levels decresed from 7.53 to 7.2 nd 7.14 fter second hndling for both groups (p < 0.05). 18

19 Tble 2 Men (SEM) blood prmeters s mesured by istat demonstrting the effects of hndling nd freshwter bth tretments on Atlntic slmon. Vlues with differing letters indicting significnt differences from pre- tretment vlues. N + K + - TCO 2 Glucose Hct PCO 2 HCO 3 Hb Tretment ph mm mm mm mg/l % mmhg mm g/100ml Pre , (0.7) (0.24) (0.4) (2.2) (1.0) (0.033) (1.22) (0.37) (0.34) 1h 1x hndling b b 24.12b (0.6) (0.18) (0.9) (3.8) (0.9) (0.045) (1.80) (0.81) (0.30) 1h 2xhndling 162.4b b b 25.12b (2.5) (0.30) (0.8) (4.4) (1.9) (0.018) (0.99) (0.70) (0.63) 15 min 1x hndling b 31.6b 7.119b 28.32b (0.7) (0.37) (0.5) (3.0) (0.8) (0.019) (1.06) (0.38) (0.28) 15 min 2x hndling 166.4b b 23.7b (1.3) (0.72) (0.7) (3.5) (0.8) (0.038) (2.01) (0.61) (0.27) Normlity Yes No No Yes Yes Yes Yes Yes Yes ANOVA F H H 4.44 F F F F F F df 4, , P vlue < < <0.001 <

20 Prtil CO2 pressure significntly incresed from bseline levels of mmhg to nd 23.7 mmhg fter sixty nd fifteen minute exposure tests nd fter second hndling (p < 0.05). Bseline blood HCO3 - levels were 9.11 mm. This incresed to 9.94 mm fter sixty minutes exposure nd second hndling whilst blood HCO3 - levels were found to be 8.14 mm fter fifteen minutes exposure nd second hndling. Bseline hemoglobin levels were found to be 9.14 g/100 ml. This incresed to 9.42 g/100 ml fter second hndling of fish exposed to freshwter for sixty minutes nd to g/100 ml fter second hndling of fish exposed to freshwter for fifteen minutes. 3.6 Wter qulity Temperture, Oxygen, ph nd slinity For the sixty minute exposure tests, wter qulity prmeters re summrised in tble 3. Oxygen concentrtions rnged between 74.2% nd 89.3% for test 1 nd 84.2 nd 101.4% for test 2. ph levels for test 1 decresed from 8.1 to 7.6 t the end during test 1 nd rnged between 7.89 nd 8.23 for test 2. Slinity ws mintined between 0.16 nd 0.17 ppt for test 1 nd between 0.16 nd 0.18 ppt for test 2. Temperture vried slightly for both tests Tble 3 Wter qulity prmeters recorded during sixty minute exposure tests Sixty minute freshwter exposure: Test 1 Time Oxygen (%) ph Slinity (ppt) Temperture ( 0 C) 0 89,10 8,10 0,16 13, ,30 8,20 0,16 13, ,90 8,10 0,17 13, ,90 8,40 0,19 13, ,20 7,90 0,19 12, ,50 7,80 0,18 12, ,10 7,70 0,18 12, ,10 7,60 0,17 13,1 Test 2: 0 84,20 7,89 0,16 13, ,20 8,10 0,16 13, ,20 8,23 0,17 13, ,70 8,08 0,17 13, ,20 8,03 0,16 13, ,30 8,01 0,17 13, ,40 8,03 0,18 13, ,40 8,01 0,18 13,4 For the thirty minute exposure tests, wter qulity prmeters re summrised in tble 4. Oxygen concentrtions rnged between 95.1% nd 101.2% for test 1 nd 92.6 nd 101.1% for test 2. ph levels for test 1 decresed from 8.17 to 7.9 t the end during test 1 nd rnged between 8.1 nd 7.9 for test 2. Slinity ws mintined between 0.19 nd 0.23 ppt for test 1 nd between 0.2 nd 0.23 ppt for test 2. Temperture vried slightly for both tests For the fifteen minute exposure tests, wter qulity prmeters re summrised in tble 5. Oxygen concentrtions rnged between 92.1% nd 94.5% for test 1 nd 92.5 nd 94.5% for test 2. ph levels for test 1 vried between 8.12 to 8.16 t the end during test 1 nd rnged between 7.9 nd 8.1 for

21 test 2. Slinity ws mintined between 0.2 nd 0.22 ppt for test 1 nd between 0.19 nd 0.21 ppt for test 2. Temperture vried slightly for both tests. Tble 4 Wter qulity prmeters recorded during thirty minute exposure tests Thirty minute freshwter exposure: Test 1 Time Oxygen (%) ph Slinity (ppt) Temperture ( 0 C) 0 95,10 8,17 0,20 13, ,20 8,15 0,21 13, ,10 8,10 0,20 13, ,20 8,20 0,19 13, ,40 8,10 0,21 13, ,80 7,90 0,23 13,40 Test 2: 0 92,60 8,10 0,21 13, ,30 8,20 0,22 13, ,10 8,10 0,20 13, ,90 8,10 0,21 13, ,50 8,00 0,20 13, ,40 7,90 0,23 13,20 Tble 5 Wter qulity prmeters recorded during fifteen minute exposure tests Fifteen minute freshwter exposure: Test 1 Time Oxygen (%) ph Slinity (ppt) Temperture ( 0 C) 0 94,50 8,16 0,21 13,3 5 93,50 8,16 0,22 13, ,40 8,14 0,20 13, ,10 8,12 0,21 13,4 Test 2: 0 94,50 8,10 0,19 13,1 5 94,20 8,10 0,19 13, ,90 8,00 0,21 13, ,50 7,90 0,20 13, Wter chemistry Wter qulity prmeters mesured before nd fter one hour exposure to freshwter cn be seen in tble 6. The ph remined similr fter one hour exposure (7.91) compred to the pre-tretment smple (7.87).There were smll increses in conductivity, lklinity nd turbidity fter the one hour tretment. Totl N incresed from 345 to 500 µg N/L fter the tretment. Nitrte (NO3-N) nd totl orgnic crbon (TOC) concentrtions remined little unchnged fter exposure compred to the pre-smple. There were smll elevtions in chlorine (Cl); sulphte (SO4) nd CO2 fter exposure whilst levels of rective Aluminium (Al) decresed slightly fter one hour exposure. Non-lbile Al concentrtions incresed from < 5 µg/l to 13 µg/l whilst lbile Al levels decresed from > 24 µg/l to 7 µg/l fter exposure. 21

22 Clcium (C), copper (Cu) nd Iron (Fe) levels remined little unchined fter one hour exposure whilst there were smll elevtions in Potssium (K), Mgnesium (Mg) nd Mngnese (Mn) concentrtions fter one hour exposure. Sodium (N) levels incresed from 12.7 mg/l to 17.2 mg/l fter exposure. Tble 6 Freshwter chemistry prmeters mesured prior to nd fter fish hve been exposed for one hour. Prmeter Mesurement Pre-smple 1hour exposure ph ph 7,87 7,91 Konduktivitet ms/m 29,2 32,8 Alklinitet mmol/l 1,998 2,049 Turbiditet (TURB860) FNU 12,5 15,3 Totl N µg N/L Nitrt NO3-N µg N/L TOC mg C/L 2,3 2,4 Cl mg/l 26,2 34,6 Sulft (SO4) mg/l 4,21 5,37 CO2 mg/l 1,9 2,5 Al - rektivt µg/l Al - ikke lbilt µg/l <5 13 Al/ICP mg/l 0,052 0,056 Al - lbilt * µg/l >24 7 Klsium (C) mg/l 48,4 49,5 Kobber (Cu) ICP mg/l <0,002 <0,002 Jern (Fe) ICP mg/l 0,0424 0,0486 Klium (K) mg/l 1,15 1,53 Mgnesium (Mg) mg/l 3,16 3,79 Mngn (Mn) ICP mg/l 0,0076 0,0086 Ntrium (N) mg/l 12,7 17,2 4.0 Discussion 4.1 Freshwter exposure tests The results from this study show cler reductions of ll present infectious stges of L.slmonis fter first nd secondry hndling. The results showed tht the percentge reductions ttined fter the first hndling nd before exposure to freshwter were on verge 45.2% for pre dults (mture mles nd pre-dults), 45.5% for mture femles nd 44.3% for ll stges of L.slmonis present on the fish. There were no chlimus stges present on ny of the fish exmined for se lice throughout ll tests. These reductions due to physicl perturbtion were similr with results from previous studies undertken t Gifs. It hs been shown from previous studies tht s the fish re being pumped from polr circle cges to well bots tht there ws reduction in the verge number of infectious stges immeditely fter pumping nd before the fish were exposed to freshwter. The percentge reductions recorded for chlimus, pre-dult nd mture femle stges (Reynolds 22

23 October 2013) were 77%, 30% nd 14% respectively, giving totl reduction for ll stges of 39%. Similr percentge reductions were observed from previous studies undertken t Gifs where it ws shown tht trnsferring fish from one cge to nother or crowding the fish resulted in reduction of up to 40% compred to pre-count levels of infesttion (Reynolds 2011). The reductions in ttched stges recorded immeditely fter the fish were pumped from the holding cge nd before exposure to freshwter from these tests cn be ttributed to mechnicl perturbtion.: physicl contct from crowding, contct with the inner surfce of the pipes used to pump the fish, netting nd contct with the grding system. Previous studies undertken t Gifs ssessing the potentil for freshwter to be used s n effective delousing tretment, hve minly focussed on using well-bots under commercil-scle conditions. During these studies, efficcy of tretment ws ssessed by counting ny remining lice on the fish immeditely fter finishing freshwter exposure nd no lice counting hd been undertken t the point when the fish were being pumped bck into the polr circle cges. These studies were imitted primrily to ssess this secondry hndling effect. Results from these studies showed tht the percentge reductions ttined fter the second hndling nd fter exposure to freshwter were on verge 88.5% for pre dults (mture mles nd pre-dults), 95.5% for mture femles nd 89.1% for ll stges of L.slmonis present on the fish compred to pre-tretment counts. The percentge reduction ttined for ll infective stges of se lice found on Atlntic slmon exposed to freshwter (89.1%) would be considered to be successful tretment outcome nd infection levels would be below tretment thresholds imposed under Norwegin legisltion (0.5 sexully mture femles per fish). The reductions recorded fter secondry hndling my be ttributed to exposing the se lice to freshwter for short time periods. Direct exposure to freshwter my hve ffected the lice ttched to the fish nd wekened them llowing for the physicl effects of n dditionl hndling to remove them from the fish s they pssed over the grder system. Previous studies hve shown lower reductions of ttched stges of L.slmonis when exposed to freshwter for short time periods nd ll well-bot studies hve shown tht fish hd to exposed to freshwter for up to three hours to chieve effective clernce rtes. The reductions recorded during these tests my be ttributed to the stress effects of freshwter on ttched stges of L.slmonis. However, the reductions my lso be purely ttributed to the physicl effects of hndling the fish two times with no contributing effects from freshwter exposure. A repet of these tests using sewter would elucidte these effects fully. The clernce rtes recorded fter the second hndling were generlly higher for fish exposed to freshwter for the shortest time period (fifteen minutes) For pre-dult stges, sixty minute exposure nd secondry hndling resulted in reductions of 94.2% overll whilst for fish exposed to thirty minutes, reduction of 71.2% ws ttined fter second hndling. However, for fish exposed to fifteen minutes, there were no lice present on the fish fter they hd pssed over the grder for the second time, constituting 100% reduction of ll ttched stges. A similr trend ws recorded for mture femle stges with both the 30 minute nd fifteen minute exposures resulting in totl clernce of 100% whilst for fish exposed for sixty minutes, totl clernce ws clculted to be 86.4%. For ll stges of L. slmonis, fish exposed for fifteen minutes chieved clernce rte of 100% compred to fish exposed for sixty nd thirty minutes (93.5 nd 73.9% respectively). If the results re prtilly ttributed to the stress of freshwter exposure on se lice physiology then shorter exposure times my be used commercilly with well-bots. This would hve the dded dvntge of potentilly treting lrger biomsses of fish with the sme wter thn hs been done in previous commercil scle tests. Fish cn be exposed for short time to freshwter nd s long s they re pssed over the grder system on the well-bot for second tme prior to being returned to the cge then the freshwter my be used over longer time period without compromising wter qulity. Hnding Atlntic slmon for two times during freshwter tretment my lso prove chllenging in tht excessive physicl contct with grders nd/or pipes my result in surfce dmge to scles nd excess removl of mucus from the surfce of the fish. If such system is used commercilly, cre 23

24 would be needed s to when fish re treted nd t wht size. Delousing with freshwter in lte Autumn/erly winter my result in wounds ssocited with lower wter tempertures if cre is not tken. 4.2 Fish welfre A principl concern of ny tretment is its effect on the welfre of the fish. Freshwter delousing my be considered less stressful method compred to current chemicl methods used in the industry t present. Results from blood nlysis showed tht hndling in freshwter resulted in minor physiologicl disturbnces consistent with stress response with n elevtion in blood glucose, CO2 nd reduction in blood ph. Further hndling nd replcement of fish bck into sewter resulted in n increse in blood sodium concentrtions consistent with cute hyperosmollity stress. Neither the effect of cute freshwter hndling nor the cute osmotic stresses re severe to conclude tht the fish would not be ble to recover without dverse effects (Powell, pers comm.) 4.3 Wter qulity Previous studies undertken t Gifs performed under commercil-scle conditions showed tht there were cute differences between the temperture of the sewter ( C) nd the freshwter contined within the well-bot (5.5 0 C) (Reynolds October 2013). In ddition, during these studies, lowering ph levels in the wells contining freshwter resulted in the fish showing signs of cute stress. The clernce rtes ttined for ttched se lice durig these studies constituted tretment success nd it hs been proposed tht these cute chnges in wter qulity my hve contributed to the reductions of ttched se lice. There is currently reserch underwy to elucidte tht if different stges of se lice re exposed to brupt chnges in wter temperture nd/or decresing ph levels then survivl is compromised. Wter qulity prmeters recorded during these tests showed tht freshwter ph levels were mintined throughout ll test regimes nd t no time did ph levels drop to levels recorded during previous well bot tests. In ddition, the temperture in the tnk contining freshwter ws comprble to tht of the sewter (13 0 C for freshwter compred to C for sewter). Thus, the reductions recorded during these tests re not result of cute chnges in wter temperture or ph nd re directly s result of hndling effects nd/or freshwter exposure. Wter chemistry chnged little fter the fish were exposed for one hour to the freshwter bth tretment. Most prmeters mesured fter exposure were similr to those prior to tretment. In ddition, the prmeters mesured could be considered to hve no long lsting hrmful effects to the fish fter short-term exposure. For exmple, ph below 6.0 hs been shown to impir osmoregultory bilities nd sewter tolernce of S. slr smolts (Sturnes et l., 1993). The ph levels recorded before nd fter one hour exposure were within rnge tht would unlikely cuse stress to the fish. The sfe levels of CO2 for Atlntic slmon prr nd smolt is 15 mg/l (Fivelstd, 2013). With regrd to toxicity in fish, the two sttes of mmoni, ionized mmoni (NH4 + ) nd un-ionized mmoni (NH3), should be indicted. TAN expresses totl mmoni nitrogen (NH3-N + NH4-N). Generlly, sfe levels hve been stted s mg/l NH3-N since NH3 esily crosses the gill membrne by diffusion. For freshwter fish, n overll sfe level held for mny yers ws 25 µg/lnh3-n (Albster nd Lloyd, 1980), while the sfe limit for Atlntic slmon cn be s low s < 2 µg/l. C nd N levels were slightly higher thn recommended for freshwter tretment of AGD (< 10 mg /L respectively) (Powell nd Kristensen 2014), however, it would be unlikely tht there would be ny long-lsting effects ffrom such short exposure time. 24

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