Aquaculture 362–363 (2012) 193–199

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Salmon lice-induced mortality of Atlantic salmon postsmolts experiencing episodicacidification and recovery in freshwater

B. Finstad a,⁎, F. Kroglund b, P.A. Bjørn c, R. Nilsen c, K. Pettersen a, B.O. Rosseland d, H.-C. Teien d, T.O. Nilsen e,S.O. Stefansson e, B. Salbu d, P. Fiske a, L.O.E. Ebbesson e,f

a Norwegian Institute for Nature Research (NINA), P.O. Box 5685 Sluppen, N-7485 Trondheim, Norwayb Norwegian Institute for Water Research (NIVA), Televeien 3, N-4879 Grimstad, Norwayc Institute of Marine Research, N-5817 Bergen, Norwayd The Norwegian University of Life Sciences, N-1432 Ås, Norwaye Department of Biology, University of Bergen, N-5020 Bergen, Norwayf Unifob Environmental Research, Unifob AS, N-5006 Bergen, Norway

⁎ Corresponding author. Tel.: +47 73 80 14 00; fax:E-mail address: bengt.finstad@nina.no (B. Finstad).

0044-8486/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2010.10.037

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 October 2009Received in revised form 7 October 2010Accepted 26 October 2010Available online 3 November 2010

Keywords:Atlantic salmonSalmo salarSalmon liceLepeophtheirus salmonisSusceptibilityPhysiology

Acid rain has reduced several salmonid stocks in Norway and salmon lice have been identified as a majorpopulation regulation factor. The combination of these two factors has also been seen to reduce postsmoltsurvival. In the present study, we have addressed the effects of an episodic exposure to acidic water and laterrecovery in good freshwater quality (Acid/Recovery groups) followed by salmon lice infestation in seawater inmore detail. The ecological perspective of this can be directly addressed to changes over the last decades fromchronic acidification over Norway and Europe, to more episodic spring acidification of rivers prior to or duringdownstream migration of smolts. The results showed that salmon lice-induced mortality increased in allepisodic Acid/Rec groups. However, the group given the longest recovery period experienced the lowestmortality compared to the other treatment groups. A period of recovery after acid exposure may eventuallyrestore the fish back to normal physiological level, but in the present experiment a period of 14 days ofrecovery after a 2 day exposure to acid water was too short to fully restore the fish back to normal levels. Evenshort-time episodic acidification followed by recovery during springtime and the vulnerable smoltificationprocess, may therefore have negative and often unnoticed effects in wild salmonids until the postsmolts meetother stressors in the marine phase such as salmon lice and other fish diseases.

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1. Introduction

Several reports have been presented of sea lice epizootics onsalmonids in Norway, Scotland, and Ireland, and in more recent yearsalso in Canada (Heuch et al., 2005; Revie et al., 2009). Infestedpostsmolts and adult fish have often been reported to be in poorphysical condition with some showing severely damaged caudal anddorsal fins and skin lesions. It is likely that the increased infestationrate of sea lice on salmonids is correlated with the presence of salmonfarms, which act as focal points for production and dispersal of salmonlice in coastal areas (Heuch et al., 2005; Revie et al., 2009; Finstadet al., 2011) as seen from Norway.

The effects of salmon lice (Lepeophtheirus salmonis Krøyer) on hostphysiology on salmonid species are now well understood (e.g.Grimnes and Jakobsen, 1996; Bjørn and Finstad, 1997; Nolan et al.,1999; Finstad et al., 2000; Bjørn et al., 2001; Wells et al., 2006; Finstad

et al., 2007; Wagner et al., 2008; Jones et al., 2008). Major primary,secondary and tertiary physiological effects (Wendelaar Bonga, 1997;Iwama et al., 1997) – including elevated plasma cortisol (Bjørn andFinstad, 1997; Finstad et al., 2000) and glucose (Wells et al., 2006),and reduced osmoregulatory ability – occur when lice develop intomobile stages (Grimnes and Jakobsen, 1996; Bjørn and Finstad, 1997;Nolan et al., 1999; Finstad et al., 2007). Sublethal tertiary effects, suchas reduced growth, reduced reproduction, reduced swimmingperformance and impaired immune defence have also been reported(Bjørn and Finstad, 1997; Nolan et al., 1999; Finstad et al., 2000; Bjørnet al., 2001; Wagner et al., 2003; Tveiten et al., 2010).

As shown in previous papers (Kroglund et al., 2007, 2008 andreferences therein) high levels of H+ and aluminium (Al) are lethal toAtlantic salmon (Salmo salar L.) smolts. This toxicity is related toincreased concentrations of H+ (reduced pH) and inorganic mono-meric aluminium (Ali) in freshwater. At lethal concentrations, H+ actsprimarily on the permeability of the cell membrane disrupting ion-regulation, whereas aluminium exerts its toxic properties byaccumulation on and in the gill tissue, disrupting ion-regulation andimpairing respiration. At lower concentrations, Al can still affect

194 B. Finstad et al. / Aquaculture 362–363 (2012) 193–199

population traits by affecting growth, swimming performance,immune defence, behaviour and seawater tolerance (Rosseland andStaurnes, 1994; Staurnes et al., 1995, 1996; Kroglund and Staurnes,1999; Ytrestøyl et al., 2001; Kroglund and Finstad, 2003; Finstad et al.,2007; Kroglund et al., 2007, 2008; Monette and McCormick, 2008;Monette et al., 2008).

A previous study (Finstad et al., 2007) has shown that fish thatexperience high acid, moderate acid and episodic acidic water infreshwater are more susceptible to salmon lice attacks and mortalityin seawater compared to fish that experience good freshwater quality.The combined effect of poor water quality in freshwater, and salmonlice attacks in coastal seawater, may therefore explain some of thevariation in Atlantic salmon year-class strength in Norwegian rivers(Finstad et al., 2007). However, while acidification in the past oftenwas chronic, reduced acid rain over Norway and Europe during thelast 20 years (Skjelkvåle et al., 2001), have resulted in more episodicspring acidification of rivers, and often followed by a recovery periodof better water quality (Kroglund et al., 2012). The effect of liming alsomakes an important contribution to the restoration of salmon informerly acidified rivers (Hesthagen et al., 2011). In the present study,we therefore have addressed the combined effects of an episodicexposure to acidic water and later recovery in good freshwater quality(Kroglund et al., 2012) followed by salmon lice infestation inseawater. The ecological perspective of this can be directly addressedto episodic spring acidification of rivers prior to or during down-stream migration of smolts (Hesthagen and Østborg, 2008) and thesubsequent risk of salmon lice epizootics in intensively fish farmedcoastal areas (Finstad et al., 2011).

2. Materials and methods

2.1. Fish stocks and rearing conditions

Offspring of Atlantic salmon were derived from spawning first-generation adult sea-ranched salmon collected from the River Imsa,South-Western Norway, in the fall of 2004. The offspring of thesesalmon were considered native River Imsa stock. The eggs andsubsequent juvenile fish were reared under low intensity hatcheryconditions with a naturally simulated light regime. During this periodthe fish were fed ordinary commercial dry diet, according totemperature and fish size. The fish were one-year old smolts havinga mean length of 15.0±0.9 cm, a mean weight of 31.7±5.7 g and acondition (K) factor of 0.92±0.06 in spring 2006.

2.2. Experimental design

The experiment was performed at the NINA Station at Ims from 27April to 11 May 2006 (freshwater period) and from 12 May to 28 June(seawater period). One group was kept in good water quality(Table 1) for the whole exposure period in freshwater (Control),

Table 1Water chemistry and physiological parameters in the five tanks prior to transfer of fish to sea11May in the afternoon. One group was kept in good water quality for the whole exposure perecover in goodwater for 14 days (Acid2/Rec14), one group exposed to acid water for 7 dayswater for 14 days and allowed to recover in good water for 2 days (Acid14/Rec2) and one gexplained in Section 2.2 and in Fig. 1. For pH and Ali the means are values/concentrations pinorganic monomeric species of Al (Ali) represents the toxic form of Al. Asterisk (*, **) denofrom **.

Group pH Ali(μg l−1)

Gill Al(μg g−1 dry w

Control 6.8±0.2 3±2 7.2±2.0*Acid2/Rec14 5.7±0.1 10±2 16.0±3.4ns

Acid7/Rec7 5.7±0.1 10±2 11.0±2.1Acid14/Rec2 5.7±0.1 10±2 20.5±7.2**Acid 5.7±0.1 10±2 25.1±4.6 ns

one group exposed to episodic acid water for 2 days and allowed torecover in good water for 14 days (Acid2/Rec14), one group exposedto episodic acid water for 7 days and allowed to recover in good waterfor 7 days (Acid7/Rec7), one group exposed to episodic acid water for14 days and allowed to recover in good water for 2 days (Acid14/Rec2) and one group exposed to acid water during the wholefreshwater period (Acid) (Fig. 1). A thorough description of the waterquality is given in Kroglund et al. (2012).

2.3. Fish sampling and analyses

To monitor effects of exposure and recovery, samples were takenfrom the fish after each freshwater experiment (Table 1, Fig. 1). Fromeach freshwater exposure, groups of 150–200 smolts were transferredto tanks fed seawater to monitor long time effects of freshwaterexposure on survival in seawater (5 tanks). A second group of fish(n=110–230) was stocked into another 5 separate tanks. Here, thesmolts were infested with salmon lice on 12 May. The term postinfection (p.i.) was used to describe the time after infection of salmonlice copepodids in the tanks in seawater. The salmon lice challengeprotocol followed Finstad et al. (2007). Copepod density wascalculated to be 2.4 copepods g−1

fish weight. Mortality was recordeddaily. Blood and gill samples were taken after 24 h in seawater tomonitor initial physiological responses (plasma Cl−, gill Na_K_ATPaseactivity (gill NKA)), and thereafter in regular intervals (Fig. 1).Seawater temperature on 12 May was 7.5 °C, it increased graduallyand reached 9.9 °C at the end of the experiment on 28 June (meantemperature 8.4 °C (±0.9)). Salinity was 33.0‰ (±1.4) during theexperimental period.

At each sampling point in seawater, 15 fish were dip netted out ofeach fish tank, anaesthetized separately in buckets by use of clove oil(0.5 ml/l) (Anderson et al., 1997), and killed by a blow to the headbefore blood samples were withdrawn from the caudal vein using 1-ml heparinized syringes. The second gill arch on the right hand side ofeach fish was dissected out and frozen in pre-weighed, acid washedpolyethylene vials for analysis of total gill aluminium (gill Al) content(Kroglund et al., 2007), while the second gill arch on the left hand sideof each fish was dissected out and frozen in 2 ml eppendorf tubes in aSEI buffer solution (Zaugg, 1982) for gill NKA analysis. Fish werethereafter kept separate in plastic bags for later lice analyses. Theblood was transferred to 2-ml eppendorf tubes, and plasma wasseparated by centrifugation (3000 g for 5 min). Blood plasma wasthen stored at −28 °C.

In the laboratory the fish were thawed, salmon lice stages wereexamined according to Schram (1993) and Bjørn and Finstad (1998)and parasitological terms used in accordance with Bush et al. (1997).The fish were measured to the nearest mm (fork length) and weighedto the nearest 0.1 g (UWE, HGS-3000). Plasma chloride was analyzedby use of a Radiometer CMT10 chloride titrator and radioimmunoas-say (RIA) was used to measure blood plasma cortisol according to

water. Exposure to high acid and episodic acid was initiated on 27 April and lasted untilriod in freshwater (Control), one group exposed to acid water for 2 days and allowed toand allowed to recover in goodwater for 7 days (Acid7/Rec7), one group exposed to acidroup exposed to acid water during the whole freshwater period (Acid), The groups areer time±S.D., for gill Al, plasma chloride and gill NKA values are means±S.D. Labile ortes significant differences (pb0.05) between the groups. ns — not significantly different

eight)Plasma chloride(mM)

Gill NKA(μmol ADP mg protein−1h−1).

137.2±1.2* 13.9±4.1126.2±11.9 ns 14.2±3.7130.8±8.2** 14.8±4.0125.3±10.5 ns 15.2±3.3118.5±10.8** 14.4±3.8

Waterageing Acid

Waterageing Contr.

Al+Acidaddition

Acid2/Rec14

Acid7/Rec7

Acid14/Rec2

Freshwater Seawater27 April 11 May 12 May 28 June

Contr.

Acid

Acid2/Rec14

Acid7/Rec7

Acid14/Rec2

Contr.

Acid

Acid2/Rec14

Acid7/Rec7

Acid14/Rec2

Non-licegroups

Licegroups

Fig. 1. Illustration of the experimental setup. All fish were exposed to acid water in a single tank. One group was kept in good water quality for the whole exposure period infreshwater (Control), one group exposed to acid water for 2 days and allowed to recover in good water for 14 days (Acid2/Rec14), one group exposed to acid water for 7 days andallowed to recover in good water for 7 days (Acid7/Rec7), one group exposed to acid water for 14 days and allowed to recover in good water for 2 days (Acid14/Rec2) and one groupexposed to acid water during the whole freshwater period (Acid). From each freshwater exposure, groups of 150–200 smolts were transferred to tanks fed seawater to monitor longtime effects of freshwater exposure on survival in seawater (5 tanks). A second group of fish (n=110–230) was stocked into another 5 separate tanks. Here, the smolts were infestedwith salmon lice on 12 May.

Fig. 2. Total mean number of lice (abundance) from lice infected groups during thewhole experiment. White bars represent Control groups in good water (Control),dotted bars represent Acid2/Rec14 groups, hatched bars represent Acid7/Rec7 groups,grey bars represent Acid14/Rec2 groups and black bars represent Acid water groups.The groups are explained in Table 1 and in Section 2.2 and in Fig. 1. The dates 23 May, 6June, 19 June and 28 June represent 12, 26, 39 and 48 days post infection (p.i.),respectively. Values are given as means±1 S.E. Asterisk (*, **) denotes significantdifferences (pb0.05) between groups.

195B. Finstad et al. / Aquaculture 362–363 (2012) 193–199

Iversen et al. (1998). Gill NKA was analyzed using the method ofMcCormick (1993) as described in Finstad et al. (2007). Gillaluminium content was analyzed according to Kroglund et al.(2001a,b, 2007). Water chemistry was analyzed at the NorwegianInstitute for Water Research's laboratory according to standardprotocols (Kroglund et al., 2012). Aluminium was fractionated usingthe PCV method (see Kroglund and Finstad, 2003; Kroglund et al.,2007). Because of high flow-through of water (low intensity culture),in situ fractionating of Al in the field did not show any significantdifferences between species distribution of Al in and out of the tanks(Teien et al., 2006). Based on this we assume that Al-speciation didnot change within the tanks.

2.4. Statistical analysis

Statistical tests were done using SPSS 17 for Windows. A Shapiro–Wilks test for normality combined with normal plots and detrendednormal plots were used to evaluate departure from normality. Due toa lack of normal distribution, nonparametric tests were chosen for theanalysis of statistical differences in infection parameters andphysiological parameters. A two-tailed Mann–Whitney U-test wasused for testing significant differences between the groups withrespect to lice levels, plasma chloride, plasma cortisol, gill aluminiumand gill NKA activity and binary logistic generalized linear modelswere used to test significant differences among groups with respect tomortality. A level of pb0.05 was considered as significant and valuesin figures and table are means (±1 S.E.).

3. Results

3.1. Water chemistry, gill aluminium and physiological parameters infreshwater

Water chemistry and physiological parameters in the fiveexposure tanks prior to transfer of fish to seawater is given inTable 1. Both pH (6.8), levels of inorganic monomeric aluminium(3 μg l−1) and gill aluminium (7.2 μg g−1 dry weight) were withinthe normal range for the Control group during the treatment phasefrom 27 April to 11 May, representing values for non-acidified waters.Values for plasma chloride and gill NKA were also within the normalrange, indicating fish in physiological homeostasis. Gill aluminium ofthe Acid groups was significantly higher (Mann–Whitney U-test,

pb0.05) compared to the Control group, and plasma chloride in theControl group was significantly higher (Mann–Whitney U-test,pb0.05) compared to the Acid episodic groups. For gill NKA the levelswere not significantly different between groups.

3.2. Susceptibility to lice infestation in seawater

The mean numbers of lice on 23 May (day 12 post infection fromsalmon lice copepodids (p.i.)) were highest in the Acid exposuregroups ranging from a mean of 37 lice in the Acid14/Rec2 group to amean of 33 in the Acid2/Rec14 group (Fig. 2). For the Control groupthe mean lice levels were 24 and significantly lower (Mann–WhitneyU-test, pb0.05) than the Acid groups, except the Acid2/Rec14 group.Lice levels decreased at the second sampling at 6 June (day 26 p.i.).Acid exposure groups had mean lice levels from 31 to 23 and the

a

b

Fig. 4. Seawater values of gill NKA in smolts from the Control groups (open circles),Acid2/Rec14 (filled diamonds), Acid7/Rec7 (filled triangles) and Acid14/Rec2 (filled

196 B. Finstad et al. / Aquaculture 362–363 (2012) 193–199

Control group had the significantly lowest (Mann–Whitney U-test,pb0.05) lice level of 17 lice. At the third sampling (day 39 p.i.), licelevels were also significantly lower (Mann–Whitney U-test, pb0.05)in the Control group— amean of 1 louse compared to the range from 3to 15 lice in the exposure groups. The Acid2/Rec14 group also hadsignificantly lower (Mann–Whitney U-test, pb0.05) lice levels at thisdate compared to the other groups (except the Control group).

On the first sampling on 23May (day 12 p.i.), only chalimus 1 stage(lice larvae) was present. At the second sampling 6 June (day 26 p.i.),18% of the lice were chalimus 3 stages, 39% were female 1 stages, 40%were male 1 stages and the remaining 3% consisted of male 2 stages.At the third sampling on 19 June (day 39 p.i.), 1% of the lice werefemale 1 stages, 3% were male 2 stages, 55% were female 2 stages and41% were adult male stages. At the fourth sampling on 28 July (day 48p.i.), 27% were adult males and 73% of the lice were adult females.

3.3. Fish mortality in seawater

There was a relatively lowmortality (Fig. 3) in the different groups12 days p.i. (23 May). A significant difference (χ2=9.909, df=4,pb0.05) between the Control groups and the Acid treatment groups inthis period was observed. In 28 June (day 48 p.i.), mortality hadincreased severely in all lice infested groups and was significantlyhigher compared to non-infested groups (χ2=375.2, df=1,pb0.001). There was a low mortality in the non-infested groups atthis time and no significant differences between any groups here(χ2=3.293, df=4, p=0.510). For the lice infested groups there weresignificant differences in mortality among the groups (χ2=63.1,df=4, pb0.001). The mortality in lice infested Acid, Acid7/Rec7 andAcid14/R2 was significant higher (χ2=5.127–31.987, df=1, pb0.05)compared to lice infested Acid2/Rec14 and lice infested Controlgroups.

3.4. Gill NKA, hydro-mineral balance and stress response in seawater

Fig. 4a shows the development of gill NKA in non-infected fish inseawater. At 12 and 16 May the non-infected Control group had

turned triangles) and Acid (filled squares). The groups are explained in Table 1 and inSection 2.2 and in Fig. 1. Panel a shows non-lice infected smolts and panel b shows liceinfected smolts. Values are given as means±1 S.E. (n=15). Asterisk (*) denotessignificant differences (pb0.05) between groups. See also text in the Results for furtherstatistical explanations.

Fig. 3. Proportion of dead fish (percentage) in the exposure groups 12 and 48 days p.i.(23 May and 28 June, respectively). White bars represent Control groups in good water(Control), dotted bars represent Acid2/Rec14 groups, hatched bars represent Acid7/Rec7 groups, grey bars represent Acid14/Rec2 groups and black bars represent Acidwater groups. The groups are explained in Table 1 and in Section 2.2 and in Fig. 1. Forthe two dates the first bar on the left for each grouping represents non-lice infested fishwhile the second bar to the right represents lice infested fish. See also text in the Resultsfor further statistical explanations. Asterisk (*, **) denotes significant differences(pb0.05) between groups.

significantly higher gill NKA values compared to all Acid groups(Mann–Whitney U-test, pb0.05). At 6 (day 26 p.i.) and 19 June (day39 p.i.), gill NKA had increased in all groups, but Acid14/Rec2 andAcid had significantly lower values than the others (Mann–WhitneyU-test, pb0.05). At the last sampling, gill NKA in the Control groupwas significantly higher (Mann–Whitney U-test, pb0.05 — exceptAcid7/Rec7) than the other groups. At 6 June (day 26 p.i.), the gillNKA value in lice infested Acid group was significantly higher and at19 June (day 39 p.i.) significantly lower (Mann–Whitney U-test,pb0.05), than the other groups (Fig. 4b). Gill NKA was notsignificantly different (Mann–Whitney U-test, pN0.05) betweengroups at the last sampling point. However, the comparison of thesame groups for non-infested and infested fish, showed asignificantly lower gill NKA level in most infested groups at mostsampling occasions.

Plasma chloride values in non-infested groups (Fig. 5a) weresignificantly lower (Mann–Whitney U-test, pb0.05) in the Controlgroup compared to the other groups on 12 May with no significantdifferences (Mann–Whitney U-test, pN0.05) between groups from 23May (day 11 p.i.) to the last sampling on 28 June (day 48 p.i.). For thelice infested groups (Fig. 5b) the Acid7/Rec7 group had significantlyhigher values (Mann–Whitney U-test, pb0.05) compared to the othergroups (except for the Acid14/Rec2 group) at 19 June (day 39 p.i.).

a

b

Fig. 5. Seawater values of plasma chloride in smolts from the Control groups (opencircles), Acid2/Rec14 (filled diamonds), Acid7/Rec7 (filled triangles) and Acid14/Rec2(filled turned triangles) and Acid (filled squares). The groups are explained in Table 1and in Section 2.2 and in Fig. 1. Panel a shows non-lice infected smolts and panel bshows lice infected smolts. Values are given as means±1 S.E. (n=15). Asterisk (*)denotes significant differences (pb0.05) between groups. See also text in the Results forfurther statistical explanations.

a

b

Fig. 6. Seawater values of plasma cortisol in smolts from the Control groups (whitebars), Acid2/Rec14 (dotted bars), Acid7/Rec7 (hatched bars) and Acid14/Rec2 (greybars) and Acid (black bars). The groups are explained in Table 1 and in Section 2.2 andin Fig. 1. Panel a shows non-lice infected smolts and panel b shows lice infected smolts.Values are given as means±1 S.E. (n=15). Asterisk (*) denotes significant differences(pb0.05) between groups. See also text in the Results for further statisticalexplanations.

197B. Finstad et al. / Aquaculture 362–363 (2012) 193–199

The Acid2/Rec14 group had a significantly lower plasma chloridevalue (Mann–Whitney U-test, pb0.05) at 28 June (day 48 p.i.). Meanchloride values in the Acid7/Rec7 and the Acid14/Rec2 groups weresignificantly higher (Mann–Whitney U-test, pb0.05) in lice infestedcompared to non-infested groups at 19 June (day 39 p.i.).

Plasma cortisol values in the non-infested groups were notsignificantly different (Mann–Whitney U-test, pN0.05) at the twosampling dates (Fig. 6a). There was however a trend towards lowerplasma cortisol values in all groups from the first to the secondsampling point. For the lice infected fish (Fig. 6b), plasma cortisol wassignificantly lower (Mann–Whitney U-test, pb0.05) in Acid7/Rec7and Acid14/Rec2 compared to the other three groups at the firstsampling date. At the last sampling date, values between groups werenot significantly different (Mann–Whitney U-test, pN0.05). However,a strong tendency towards elevated plasma cortisol valueswas seen inall lice infested groups from the first to the second sampling pointwith groups Acid7/Rec7 and Acid14/Rec2 having a significantincrease. There was also a significantly higher cortisol value (Mann–Whitney U-test, pb0.05) in the lice infested Control, Acid2/Rec14 andAcid14/Rec2 groups compared to non-infested groups at 06 June (day26 p.i.). For the other two groups, values were also higher in the liceinfested groups, however not significantly (Mann–Whitney U-test,pN0.05).

4. Discussion

In the present experiment, water chemistry, gill aluminium levelsand physiological parameters in the Control group were within thenormal range for fish in normal rearing freshwater at Ims (Kroglundet al., 2007). For the four Acid groups, water quality was significantlyreduced and Al levels were within the range considered suboptimaland even toxic for Atlantic salmon smolts (Kroglund and Staurnes,1999; Kroglund et al., 2001a,b; Kroglund and Finstad, 2003).Surprisingly, recovery for 2, 7 and 14 days in the Control waterafter episodic exposure for the Acid water did not bring the gill Aldown to levels as seen in the Control water quality. This increased

198 B. Finstad et al. / Aquaculture 362–363 (2012) 193–199

accumulation of aluminium on the gills is known to induce negativephysiological responses on smolts (Kroglund and Finstad, 2003;Kroglund et al., 2007, 2008). There was also an observed decrease inplasma chloride levels in the Acid groups which is in accordancewith the increased gill Al levels on fish (Kroglund et al., 2007). Priorto the transfer to seawater in the present experiment, thephysiological status of the fish was reduced in the following orderControl–Acid/Rec–Acid.

Salmon lice development corresponded to previous reports forAtlantic salmon (Johnson and Albright, 1991). In the abovementionedexperiment, seawater temperature in the experimental tanks washeld at approximately 10 °C. However, in the present experiment, andin Finstad et al. (2007), seawater temperatures were lower (range7.5 °C–9.9 °C) and therefore the development from the attachedchalimus stages 1–4 to the mobile preadult- and adult stages tooklonger than reported in Johnson and Albright (1991). In accordancewith earlier studies (see references in Finstad et al., 2011), the presentexperiment verified that mortality and physiological disturbancesincreased significantly when lice developed into their mobile phase.

Also in accordance with a previous study by Finstad et al. (2007),the mean number of lice was lowest in the Control group comparedto the Acid groups. Lice- and fish mortality led to a decreasednumber of lice on all groups throughout the experimental period. Itis known that stress causes immunosuppression which results inincreased susceptibility for infectious diseases (Iwama et al., 1997;McKinnon, 1998). In the present study the stressor was suboptimalwater quality (Acid water) and even 14 days of recovery in goodwater did not “repair” the fish before transfer to seawater, resultingin the fish being more susceptible for lice infections. Suchinfestations have led to increased cortisol secretion, reducedimmune defence and depressed seawater tolerance (Bjørn andFinstad, 1997; Finstad et al., 2000). Thus, stress caused by the firstsalmon lice infestations (Bjørn and Finstad, 1997; Finstad et al.,2000) will probably again increase the host susceptibility for re-infection by salmon lice in the wild as well as making the fish moresusceptible to secondary infections and diseases (Ellis, 1981).

The proportion of dead fish in the non-infested groups was lowduring the whole experiment, but some mortality occurred alreadyin the early phase of the experiment. In contrast, for the infectedgroups, mortality was generally much higher and highest in theAcid14/Rec2 and the Acid7/Rec7 groups. This indicates that therecovery periods of 2 and 7 days were too short for the fish torecover before transferring them to seawater. No significantdifferences in mortality were seen between the Control group andthe Acid2/Rec14 group indicating that a recovery for 14 days did, tocertain degree, restore the fish before exposure to salmon lice inseawater. This recovery period seemed, however, to be too short fora full recovery as seen from the somewhat higher mortality in thelatter group. The proportion of dead fish increased from 23 May (day12 p.i.) to 28 June (day 48 p.i.) in the lice infested fish. This did nothappen in the non-infested fish. It therefore seems that the mortalityobserved in the initial phase was due to the fishes previousexperience of poor water quality/not recovered and, to some extent,in combination with the stress influence of the salmon licecopepodids (Bjørn and Finstad, 1998; Finstad et al., 2000). Whenthe lice had evolved into preadult stages, causing physical damage tothe fish (see Revie et al., 2009 for descriptions and illustrations), theproportion of dead fish in the groups that had experienced poorwater quality/recovery increased dramatically. This is probably dueto the higher susceptibility and higher lice infection intensities inthese groups combined with mobile lice as a multiple stressor.

The gill NKA activity for non-infested smolts in the Control groupwas within the range shown to be normal for smolts (Nilsen et al.,2003; Handeland et al., 2003; Finstad et al., 2007) while values for theAcid groups were lower and in accordance with previous results fromFinstad et al. (2007). In the infested groups, the Acid group at 19 June

(day 39 p.i.) had significantly lower values indicating lower gillenzymatic levels compared to Control groups at the same period. Aninhibition of gill NKA activity caused by acid water and aluminium hasbeen shown in several previous studies (e.g. Staurnes et al., 1984,1993; Kroglund and Staurnes, 1999; Magee et al., 2003). Plasmachloride values in the non-lice groups were lowest in the Controlgroup after transfer to seawater and in accordance with the lowobserved mortality in this group at 23 May (day 11 p.i.). Furtherdevelopment of this parameter in all non-infested groups progressedtowards normal levels during the experiment. Plasma chloride valuesin lice infested groups revealed higher values in the Acid7/Rec7 andthe Acid14/Rec2 groups in accordance with the increased mortalitywithin these groups. For Acid, Acid2/Rec14 and Control groups (liceinfested) there were lower plasma chloride levels in these groups atthe end of the experiment which was in accordance with the lowermortality seen in these groups.

Acid rain (Hesthagen and Hansen, 1991; Kroglund et al., 2002;Hesthagen and Østborg, 2008; Hesthagen et al., 2011) and salmonlice have been identified as a major population regulation factors(Heuch et al., 2005; Revie et al., 2009). Furthermore, in large areas ofwestern Norway, these factors geographically co-occur throughspring acidification of rivers (Skjelkvåle et al., 2001) and high risks ofsalmon lice infection in fjords and coastal areas (Finstad et al., 2011).The combination of these two factors has also been seen to reducepostsmolt survival (Finstad et al., 2007). That is — the combinedeffect of Ali/low pH/salmon lice (multiple stressors) will increase themortality and physiological response more than the two factorsalone. In the present study we have addressed in more detail theeffects of episodic acidic water and recovery in freshwater (Kroglundet al., 2012) followed by salmon lice infestations in seawater. Theecological perspective of this can be directly addressed to lastdecade's change from chronic acidification over Norway and Europe(Skjelkvåle et al., 2001), to more episodic spring acidification ofrivers followed by a recovery period (Kroglund et al., 2012). In thepresent study, salmon lice-inducedmortality increased in all episodicAcid/Rec groups. However, the group given the longest recoveryperiod experienced the lowest mortality compared to the othertreatment groups. A period of recovery after Acid exposure mayrestore the fish back to normal physiological levels but in the presentexperiment a period of 14 days of recovery after a 2 day exposure toacidwaterwas too short to fully restore thefish back to normal levels.Even short-time episodic acidification followed by recovery duringspringtime and the vulnerable smoltification process, may thereforehave little and often unnoticed effects in wild salmonids. Mortalitiesin the freshwater stage are seldom observed, as the acid waterstressor may not kill the fish in freshwater but reduce the overallhealth of the fish and delay the mortality until the postsmolts meetother stressors in themarine phase such as salmon lice and other fishdiseases.

Acknowledgements

The Norwegian Fisheries and Aquaculture Industry Research Fund(FHF) and the Norwegian Research Council (NFR 172514/S40) fundedthis project. We wish to thank Knut Bergesen and the personnel atNINA's Research Station at Ims for aid during the project period. Theexperiment described has been approved by the local responsiblelaboratory animal science specialist under the surveillance of theNorwegian Animal Research Authority (NARA) and registered by theAuthority.

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