Master’s Degree Thesis in Ecology, 30 hp Spring 2019

BUBBLES GUIDE MIGRATING SMOLTS

AROUND HYDROPOWER PLANTS

Shona MacArthur

Supervisor: Jonatan Klaminder

Bubbles guide migrating smolts around hydropower plants Shona MacArthur Abstract The development of hydroelectric power production and the damming of water courses that ensues causes a threat to the migration patterns of Atlantic salmon (Salmo salar), by altering their habitat and compromising river connectivity. Because the species’ survival heavily depends on the completion of their migration between rivers and oceans, the design of guidance structures that promote successful passages around dams is a critical goal. Physical structures have been used to steer downwards-migrating smolts through safe fishways but they have not always proven effective, both from an ecological and economical perspective. In this study, the potential of bubbles in guiding salmon trajectories around a hydropower dam was tested as an alternative to existing guiding systems. Here the hypotheses were: i) bubbles guide downstream-migrating smolts around dams; ii) the reaction of smolts to a bubble barrier is linked to their individual boldness; iii) bubbles have varying effects on different school sizes. The first and third hypotheses were tested in the field, by recording sonar footage at the entrance of a fishway, downstream of a bubble barrier. The second hypothesis was evaluated by conducting a scototaxis experiment and an assessment of the reaction of young salmon to bubbles in the laboratory. My analysis of the fish abundance data collected in the river confirmed the first and third hypotheses. In the field, the presence of a predator (Esox lucius) was revealed to be a more influential factor than bubbles in regulating the number of smolts steered towards the fishway, indicating that predators generated a larger anxiety-like response in smolts than bubbles. In the controlled laboratory setting, there was no significant correlation between anxiety-like behaviour and reaction to bubbles and the second hypothesis did not appear to be valid. Therefore, the cause of avoidance of the barrier remains unclear, but bubble barriers seem to be an efficient and cost-effective structure for guiding downstream-migrating salmon smolts. Key Words: Atlantic salmon, migration, bubble barrier, hydropower plant, predation

Table of Contents 1 Introduction………………………………………………………………………………..…1

2 Methods……………………………………………………………………………………..…...3

2.1 Field study ………………………………………………………………………………………3

2.1.1 Data collection………………………………………………………………………….…...3

2.1.2 Data extraction………………..……………………………………………………………3

2.1.3 Statistical analysis…………………………………………………………………………4

2.2 Laboratory experiment…………………………………………………………………..4

2.2.1 Scototaxis………………………………………………………………………………..……4

2.2.2 Bubble experiment……………………………………………………………………..…4

2.2.3 Statistical analysis…………………………………………………………………………5

3 Results……………………………………………………………………………………..………6 3.1 Field experiment…………………………………………………………………………….6

3.2 Laboratory assay………………………………………………………………………….…8

4 Discussion………………………………………………………………………………………9

5 Conclusions…………………………………………………………………………………..11

Acknowledgements………………………………………………………………………11

References………………………………………………………………………………………..11

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1 Introduction The Atlantic salmon (Salmo salar) is a key species of the freshwater and marine ecosystems it inhabits, contributing to the regulation of food webs, limitation of sedimentation and nutrient cycling among others (Kulmala et al., 2012). The study by Kulmala et al., (2012) points out that, besides its ecological importance, salmon has always had a central role for human populations, providing food, recreational activities and a substantial source of revenue. Salmon is an anadromous species, whose life cycle is defined by the key events of downstream migration towards the ocean and upstream migration to spawn. The young are born in freshwater, where they spend their first years before undergoing smoltification, a series of physiological adaptations for life at sea (Ackefors et al., 1991; Karlsson and Karlström, 1994), while adults undertake an upstream migration to freshwater in order to spawn (Marschall et al., 2011). The journeys taken by salmon along rivers to and from the sea are therefore vital to the species and the maintenance of their ecological and economical importance. However, anthropogenic disturbances of freshwater systems can be detrimental to salmon populations, by altering migration routes through habitat destruction and pollution (Kulmala et al., 2012). The construction of dams, associated with the expansion of hydroelectric power production, forces migrating salmon to navigate often small bypasses in order to complete their journey, with an increased mortality caused by the risk of going through turbines or not finding a migration passage, resulting in an accelerated decline of salmon populations (Karlsson and Karlström, 1994; Rechisky et al., 2013). The physical harm that can be done to the migrating salmon by these structures is not the only possible negative impact. Indeed, when not directly lethal, the passage of dams can increase the susceptibility of smolts to predation by extending the time spent in reservoirs and delaying their journey (McCormick et al., 1998; Jepsen et al., 1998).

Guiding structures such as screens or walls have been installed to orient migrating fish around dams through safe bypasses in an attempt to minimise mortality (Popper and Carlson, 1998). However, especially for downstream migrating smolts, the success of fishways is dependent on the trajectory taken by salmon when approaching the dam, and costly guiding structures have had limited efficiency in steering salmon away from the main current that passes through the turbines (Arnekleiv et al., 2007). Moreover, underwater guiding systems can themselves cause physical harm to the fish (Nestler and Davidson, 1995). The success of those structures may further be compromised by accumulation of debris and drifting material, with the need for constant maintenance and cleaning adding to the initial cost of construction (Bainbridge, 1964). Thus, the improvement of guiding systems upstream of dams is necessary to ensure safe passage of smolts.

Marine predator species such as humpback whales (Megaptera novaeangliae), orcas (Orcinus orca) and bottlenose dolphins (Tursiops truncatus) are known for their use of bubbles as a visual communication signal that allows for a synchronised attack of prey (Moreno and Macgregor, 2019). In addition to this function, bubbles are used as a physical tool to herd prey fish and facilitate feeding, as they may cause confusion and schooling behaviour (Fertl and Wilson, 1997; Fertl and Würsig, 1995). This highlights the capacity of bubbles to initiate a response by some fish species, by affecting their behaviour and obstructing their movements. The use of bubbles as a guidance mechanism gained attention for management purposes, especially for their potential as a fish exclusion device (Patrick et al., 1985). The possibility of using bubble barriers to divert fish around hydropower plants has been investigated, but few studies have evaluated the efficiency of bubbles as a guiding system, especially for downstream-migrating smolts (Arnekleiv et al., 2007; Marschall et al., 2011). This highlights the need for a deeper understanding of the possibilities offered by bubbles in steering migrating salmon. When bubble barriers have proven effective, the reason for the avoidance by smolts was not clearly determined (Popper and Carlson, 1998), but because of visual blockage and fear of unknown environments, their response may be linked to anxiety (Ward et al., 2004).

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The movements and trajectory of fish are the results of their response to a multitude of factors such as sound, light, pressure, chemical cues, temperature and water speed, making guidance of smolts a challenge (Popper and Carlson, 1998; Coutant and Whitney, 2000). Atlantic salmon migration follows some diel patterns, with photoperiod playing a part, but it is water temperature that particularly affects the smolts’ development and journey (Ibbotson et al., 2006). Indeed, Ibbotson et al., (2006) observed a shift of migration from night to day when temperatures rise above a mean threshold value. However, another environmental factor plays a role in the migration movements of salmon and its influence could prevail over abiotic cues when attempting to guide smolts. Predation on young salmon, by birds and other fish, is the main cause of mortality during migration, with pike (Esox lucius), burbot (Lota lota) and pike-perch (Sander lucioperca) being the most impactful predators (Hvidsten and Mokkelgjerd, 1987; Jepsen et al., 1998). Increased pike activity has been shown to suppress migration, while avoidance of predators can be facilitated in turbid water and explains the preference of smolts for night migration when temperatures are low (Jepsen et al., 1998; Thorpe et al., 1994; Ibbotson et al., 2006). Fish predators are typically recognised by smolts through sensory cues, such as odour, to which they have an innate response allowing them to assess risk and avoid the danger (Hawkins et al., 2004; Hawkins et al., 2007). Predators have a high influence on migration patterns of Atlantic salmon and by combining a greater susceptibility to predation and an increased mortality caused by physical harm, hydroelectric power production constitutes a primary threat to migration. As a consequence, understanding fish behaviour is crucial in order to design successful bypasses, using sensory cues that affect salmon trajectories according to their natural reactions to environmental biotic and abiotic factors (Coutant and Whitney, 2000). Exploiting sensory cues through the use of light, sounds or bubbles as an alternative to guiding fish around dams gained attention in the 1980s, but has not always been successfully developed (Patrick et al., 1985). Since then, studies have looked further into the possibilities of using such cues as guidance mechanisms. The overall purpose of this study was to determine if the use of bubbles as a guiding system could successfully steer downwards migrating smolts towards a safe passage around a dam. Furthermore, the aim was to assess whether all salmon would react in the same way when put in contact with bubbles, depending on behaviour traits such as boldness. Ultimately, this would help to know if the use of a bubble wall to orient fish in a river may carry out a selection of certain characteristics, by guiding some fish towards a safe passage, while some behaviour traits may lead others to go through the turbines. As seen for prey fish herded by marine predators, bubbles and predation may cause schooling behaviour (Fertl and Würsig, 1995), so there could be discrepancies observed in the reaction to bubbles depending on schooling. The hypotheses tested were:

1: Bubble barriers can be used to guide salmon towards fishways. This hypothesis was tested in the field. 2: The fish’s reaction to bubbles is linked to anxiety-like behaviour traits. This hypothesis was tested in the laboratory. 3: Smolts in distinct school sizes are affected differently by bubble barriers. This hypothesis was tested in the field.

To test these hypotheses, a bubble wall was installed upstream of a fish passage in the Ume river in Norrfors, northern Sweden. A sonar was placed between the bubbles and the entrance of the bypass, to assess their efficiency in steering smolts of different school sizes in the right direction. Additionally, a laboratory experiment was conducted to determine if the reaction of juvenile salmon to bubbles was linked to their boldness, by conducting a scototaxis experiment and assessing their reaction to bubble curtains.

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2 Methods 2.1 Field study 2.1.1 Data collection A 50-meter-long bubble barrier was installed near Stornorrfors hydropower plant in the Ume river, northern Sweden, by Johan Leander (unpublished) as an extension to an established physical guiding structure upstream of a fishway. In order to collect data about the passage of smolts, an ARIS sonar was installed at the entrance of the bypass (Figure 1) and videos were recorded between June 19th and July 15th 2018. The bubble wall was alternately on and off until 8.00 on June 22nd, when it was switched off for the remainder of the recording. Twenty-four-hour recording started on June 21st at 15.00, resulting in a total of 536 hours 36 minutes of footage with the bubble wall off and 14 hours 32 minutes with the bubbles on.

Figure 1. Aerial view of Ume river at the entrance of the fishway at Stornorrfors hydropower plant. The existing guiding structure is visible in red, while the location of the bubble barrier is represented in blue. The area visible in the sonar recordings is marked in green. 2.1.2 Data extraction Data was extracted from the entirety of the footage, using the ARISFish software (version 2.6.3) and noting the frames with presence of pike and of smolts. For the latter, school size was specified according to three categories: small (5 fish or fewer), medium (6 to 20 fish) and large (21 fish or more). The angle of the sonar changed at different stages and it ultimately tilted downwards on June 29th at 14.35, modifying the depth of vision in the river for the remainder of the recording. In order to account for possible differences in observations because of the depth of swimming of salmon, the angle of the camera was also specified, so it could be accounted for in analysis as an explanatory variable.

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2.1.3 Statistical analysis To test the first and third hypotheses, the collected data was analysed in RStudio (version 1.1.463). Analysis was done on the number of frames of presence of smolts, knowing the total number of frames recorded, resulting in proportion data. A general linear model (glm) with quasi-binomial errors was used to account for overdispersion, with the proportion data treated as the response variable. For analysis of the presence of smolts with no distinction of school size, the minimal model had sonar tilt, bubbles and pike presence as factors, with an interaction factor between bubbles and pike presence. When school sizes were distinguished, the factors for analysis of small schools were bubbles, pike presence and their interaction factor. For medium schools, the factors were sonar tilt, bubbles, pike presence and the interaction factor between bubbles and pike presence. For large schools, the factors were pike presence, bubbles, sonar tilt and the interaction factor between bubbles and sonar tilt. The functions tapply and predict were used to back-transform the output of the glm form logits into mean proportions. 2.2 Laboratory experiment This study was approved by the ethical committee of animal experiment at Umeå University (dnr: A11-13) and the theoretical online courses “Swedish legislation and ethics, animal welfare and 3R” and “Laboratory Animal Science for Researchers – Fish” were passed before the start of the study. The experiments were conducted in climate rooms set at 4°C, so the water temperature would be close to the natural river temperature. The light was set to be similar to daylight hours at that time of year, with twelve-hour days and twelve-hour nights. The study was done on forty one-year old hatchery reared Atlantic salmon that were kept in individual 10 L see-through tanks with oxygenated aged tap water in a separate room from the experiments, set to the same temperature and light conditions. 2.2.1 Scototaxis The experiment was carried out in two steps. Firstly, a behaviour study to assess boldness was performed on April 2nd 2019. A scototaxis experiment was conducted on the forty fish, using four 10 L square arenas with the bottom divided in two halves, one white and one black, enclosed in a cupboard to isolate the experiment from the surrounding environment (Maximino et al., 2010). Data was collected by recording a twenty-minute video using a camera fixed above the arenas, which allowed for the simultaneous filming of four fish. The video files were then analysed using the programme ToxTrac (version v2.83), and the relevant statistics (visibility and invisibility rates) were exported into an excel sheet (Rodriguez et al., 2018). A second analysis of the files in ToxTrac was performed on the first three minutes of the videos, allowing to focus on the initial behaviour of the fish when introduced to the tanks. Because the fish were netted into the arenas one at a time, there was an average delay of 37.8 seconds, ranging from 29 to 52 seconds (16% and 29% of the footage respectively), between the introductions of the first and fourth fish for each recording. Analysis was done in RStudio (version 1.1.463). Fish 19 was removed from the analysis because the values given in the programme did not correspond to actual visibility rates. A one-sample t-test was performed on visibility rate data for the entire twenty-minute recordings as well as for the first three minutes, to establish a 95% confidence interval for the proportion of time spent on white background for the 39 fish. 2.2.2 Bubble experiment Secondly, the fish were individually put in contact with bubble walls in a larger 125 L arena, containing oxygenated aged tap water. This experiment was done one day after the scototaxis experiment for fish 1 to 4, and the following day for fish 5 to 40. The tank contained three bubble barriers at equal distances of 22 cm, that were made by puncturing 12 mm diameter

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tubes every centimetre for the width of the arena and maintained straight along the bottom of the tank by being fixed to a metal plaque. The tubes were folded in two, creating a thicker curtain of bubbles, with a long loop allowing for symmetry along the sides of the tank, where the tubes were held straight with duct tape above the water level. This setup meant that no gaps were left where the fish could have bypassed the bubbles (Figure 2). Another tube connected to a compressed air tap was divided into three branches, enabling to switch between the bubble walls with no intermission. The fish were introduced in the tank behind a see-through hatch, allowing them to visually assess their environment. The furthest wall (A) was switched on before the fish were introduced for an acclimatisation period of two minutes. Then, the hatch was lifted, letting the fish explore their environment. If the fish had not gone to encounter the wall after 5 minutes, the middle wall (B) was switched on and the furthest turned off. If the fish had still not interacted with the wall after 10 minutes, the closest wall (C) was turned on and wall B turned off. The use of three walls allowed to move the bubbles closer to the fish in the event of freezing behaviour, in an attempt to encourage interaction with the barrier. When there was an interaction of the fish with the bubbles, whether they were crossed or not, the experiment was ended after the 5 minutes of run time given for each of the barriers. If no interaction had occurred, the experiment was ended after a total time of 15 minutes, following 5 minutes with bubble wall C switched on.

Figure 2. Arena used in the bubble experiment, seen from the side of video recording. Bubble walls A, B and C (from left to right) are shown switched on. The fish were introduced in the right part of the tank, behind a see-through hatch whose position is visible by the two black sections holding it in place. Data was recorded as video files, with a webcam filming the tank from the side. The videos were subsequently analysed to extract the relevant data, with times calculated from the opening of the hatch: time until initial reaction to the bubble wall, specifying the nature of the reaction (freezing, fleeing or inspection); time to pass the positions of the different walls, whether on or off; if it occurred, time taken to cross bubbles, specifying which wall and the time taken until crossing. 2.2.3 Statistical analysis Analysis was then done in RStudio (version 1.1.463), using data from the scototaxis and bubble wall experiments to test the second hypothesis. A Spearman’s correlation table was made

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between the continuous variables, using the function rcorr from the Hmisc package, in order to obtain the significance levels of the correlations. 3 Results 3.1 Field experiment The mean proportion of presence of smolts at the entrance of the fishway was 6.7% when the bubble wall was on (table 2) , which, as shown from the glm results in table 1, is significantly higher (t=-3.49; p<0.001) than the 3.3% of presence when the wall was switched off. In the presence of pike, the mean proportion of presence of smolts was 0.4%, which is significantly lower (t=-11.20; p<0.001) than the 6.8% observed in absence of a predator. The downwards tilt of the sonar also had a significant effect on the proportion of footage where smolt were seen (t= -8.21; p<0.001), with salmon visible on average 2.6% of the time after the sonar tilted down, versus 15.7% of the time before the tilt. The interaction of bubbles and pike presence is also significant on the proportion of presence of smolts at the fishway entrance (t= 4.37, p<0.001). Smolts were most present when the bubble wall was on and no predator was present (figure 3.a), while the combination of the presence of pike and having the bubble barrier switched off resulted in the lowest mean proportion of smolt presence. It is also visible from the bar plot that, whether the bubbles were on or off, the presence of pike lead to a decrease of the mean proportion of presence of smolts. Table 1. Results from the glm for presence of smolts independently of school size. ‘***’ indicates significance at p<0.001

Estimate Std. Error t value Pr (>|t|) Intercept -0.46 0.29 -1.59 0.11

Bubbles -2.40 0.29 -8.21 4.03E-16 *** Tilted sonar -1.34 0.38 -3.49 4.98E-04 *** Predator -3.42 0.31 -11.2 < 2e-16 *** Bubbles x Predator 2.60 0.59 4.37 1.31E-05 ***

Table 2. Mean proportions of presence of smolts (P) with and without distinction of school size at the entrance of the fishway depending on use of bubble wall, predator presence and sonar tilt.

Smolt presence (P) Small school presence (P)

Bubbles on Pike presence Tilted sonar Bubbles on Pike presence Tilted sonar

No 0.033 0.068 0.16 0.024 0.049 0.021

Yes 0.067 0.004 0.026 0.019 0.0013 0.024

Medium school presence (P) Large school presence (P)

Bubbles on Pike presence Tilted sonar Bubbles on Pike presence Tilted sonar

No 0.0019 0.0047 0.025 0.0063 0.012 0.093

Yes 0.025 0.001 0.0013 0.013 0.0022 0.0009

Similarly, the presence of large schools of smolts was significantly improved by the use of the bubble barrier (t=-3.156, p<0.01) (table 3), with their mean proportion of presence increasing from 0.6% to 1.3% (table 2). The presence of pike was also significant in decreasing the presence of large schools (t=-3.933, p<0.001) from an average of 0.1% to 0.02% (table 2). The

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sonar tilt meant that large schools were significantly less visible at greater depth (t=-10.193, p<0.001), and the interaction of depth of recording and the use of the bubble barrier was significant and greatly diminished the mean proportion of presence of large schools (figure 3.d). Table 3. Results from the glm for presence of large schools, with 21 smolts or more. Significance levels: ‘*’ indicates significance at 0.01<p<0.05; ‘**’ 0.001<p<0.01; ‘***’ p<0.001

Estimate Std. Error t value Pr (>|t|)

Intercept -1.16 0.37 -3.14 1.72E-03 ** Predator -1.92 0.49 -3.93 8.66e-05 *** Bubbles -2.16 0.68 -3.16 1.62E-3 ** Tilted sonar -5.22 0.51 -10.19 < 2e-16 *** Bubbles x Tilted sonar 3.00 1.48 2.03 0.042 *

Small and medium schools of smolts were present on average 1.9% and 2.5% of the time respectively when the bubble barrier was switched on (table 2). As opposed to smolts regardless of school size and large schools, there is no significant difference for the presence of between 6 and 20 salmon due to the bubble wall (small schools: t=-1.354, p>0.05; medium schools: t=0.716, p>0.05) (tables 4, 5). Pike presence significantly decreased the mean presence of schools of twenty fish or fewer (small schools: t=-7.822, p<0.001; medium schools: t=-4.127, p<0.001) by a factor of 38.8 for small schools and a factor of 4.7 for medium schools. The interaction of pike presence and use of the bubble wall was also significant in determining the presence of small schools (t=2.656, p<0.01), and the bar plot (figure 3.b) shows that small schools were seen the least when the barrier was off and pike was present. Contrary to smolts regardless of school size and medium schools (figure 3.c), small schools were most present when predators were absent and the bubble wall was off. Table 4. Results from the glm for presence of small schools of 5 smolts or fewer. Significance levels: ‘**’indicates significance at 0.001<p<0.01; ‘***’ p<0.001

Estimate Std. Error t value Pr (>|t|) Intercept -2.95 0.093 -31.68 < 2e-16 *** Bubbles -0.64 0.47 -1.35 0.17 Predator -3.98 0.51 -7.82 8.44e-15 *** Bubbles x Predator 3.00 1.13 2.66 7.97E-3 **

Table 5. Results from the glm for presence of medium schools, with 6 to 20 smolts. Significance levels: ‘*’ indicates significance at 0.01<p<0.05; ‘**’ 0.001<p<0.01; ‘***’ p<0.001

Estimate Std. Error t value Pr (>|t|) Intercept -3.32 0.46 -7.20 8.36e-13 *** Tilted sonar -2.73 0.47 -5.84 6.03e-09 *** Bubbles 0.37 0.52 0.72 0.47

Predator -2.26 0.55 -4.13 3.84e-05 *** Bubbles x Predator 1.75 0.75 2.34 0.019 *

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Figure 3. Mean proportion of presence of smolt at the entrance of the fishway, depending on the interaction of pike presence and use of bubble wall for smolts independently of school size (a), small schools (b) and medium schools (c). For large schools (d), the significant interaction is between use of bubbles and sonar tilt. From the bar plots in figure 3 and the results of glm analysis (tables 1, 3, 4, 5), it is clear that the combined effect of presence of pike and the bubble wall being switched off importantly decreased the presence of smolts at the entrance of the fishway, except for large schools of over 20 fish, for which the interaction was not significant. The bubble wall alone was significant in increasing smolt presence at the entrance of the fishway for large schools and for smolt regardless of school size, which is consistent with the first hypothesis tested. However, these results show that the presence of a predator is an important factor in determining smolt presence. In the case of smolts regardless of school size (figure 3.a), the presence of smolts was lower in case of higher predation risk, even when the bubble wall was on, than in the absence of predator and with the barrier switched off. Therefore, these results validate the first hypothesis that bubbles are successful in guiding smolts in the right direction, as well as the third hypothesis that school size affects the response of smolts to bubbles. However, predation may have the potential to overrule that effect. 3.2 Laboratory assay Analysis of the visibility rate data of the 20-minute tracking showed that, with 95% confidence, the fish spent on average less than half of their time in the arena on the white background (95% CI =0.385 - 0.467); hence, there was a significant preference for the black surface. During the first three minutes, the mean proportion of time spent by salmon on the white half of the tank was less than a third (95% CI= 0.18 - 0.32); hence, the preference for the black surface was

No pike Pike

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initially stronger. These results show that smolts favour a half of the arena over the other and therefore display a scototaxis behaviour depending on their boldness. The Spearman’s correlation coefficients and their significance levels obtained from the analysis of the scototaxis experiment and the assessment of the reaction of salmon to bubbles do not show any significant correlations (table 6). The reaction of the fish to bubble barriers is not linked to their scototaxis visibility rate and therefore to their boldness. As a consequence, the second hypothesis cannot be supported by these results. Table 6. Results from the correlation between visibility rate measured in the scototaxis experiment and the variables measures in the bubbles experiment. The numbers in italics are the corresponding significance levels for the Spearmans’s correlation coefficients given between variables.

Visibility rate (20min); p value Start visibility rate (3min); p value

Time until reaction (s) 0.22 0.18 -0.20 0.21

Time to pass position C (s) -0.05 0.78 -0.19 0.27

Time to pass position B (s) 0.13 0.49 -0.22 0.25

Time to pass position A (s) -0.13 0.61 -0.19 0.44

Time to cross bubbles (s) -0.13 0.56 0.07 0.76

4 Discussion The use of the bubble wall in the river resulted in a higher presence of salmon at the entrance of the fishway, indicating that bubbles can function as a guiding system for migrating smolts, in support with the first hypothesis. Notably, the function of the bubbles remained significant despite the fact that results were influenced by the sonar tilting, resulting in a reduced proportion of presence of smolts, especially for medium and large schools. This is coherent with the fact that smolts tend to swim close to the surface (Arnekleiv et al., 2007; Coutant and Whitney, 2000), meaning that the tilt diminished the proportion of footage where they were visible. The effects of the bubble wall were still noticeable but would perhaps have been more accurate if the field of vision had stayed shallower for the entire experiment. Moreover, the method used to analyse smolt presence does not give an estimate of the number of smolts using the fishway, but of the amount of time when smolts were visible near the entrance throughout the experiment. A higher proportion of presence may signify a greater number of smolts, but it could also be that the same smolts spent more time in the zone leading to the fishway. In either case, the results still manifest the efficiency of the bubble barrier in modifying smolt trajectory, but they may not reflect the amount of salmon that use the fishway. A method that allows to count smolts going through the fishway could be a better assessment of the efficiency of the bubble wall. The recording in the river spanned twenty-seven days, but the data collected with use of the bubble barrier was concentrated during the first three days. Catch data of smolts collected by Johan Leander in previous years (unpublished) suggests that the number of migrating smolts decreases towards the end of June. If the days of use of the bubble barrier coincided with a peak in migration, the disparity in data recording may induce some differences in smolt presence due to the temporality of migration, and this may not reflect the efficiency of the bubble wall. In order to obtain more accurate results, it would be interesting to use the bubble barrier during a longer period of time, to account for possible fluctuations in smolt abundance in the river. The third hypothesis was supported by the results showing differences in the reaction to bubbles depending on school size, especially as the presence of fish in groups of 6 to 20 was not significantly affected by the bubbles alone. More generally, in order to optimise and expand the use of bubbles as a guiding mechanism, it is important to understand the reason of the salmon’s avoidance of bubbles and the behaviour traits

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responsible for the fish’s reaction, as investigated in the test of the second hypothesis (Popper and Carlson, 1998; Coutant and Whitney, 2000). Salmon were shown to display a scototaxis behaviour, by choosing to spend most of their time on the black background of the arena. Indeed, this suggests an anxiolytic-like behaviour, as the test is designed to measure (Maximino et al., 2010; McCallum et al., 2019). Thus, my test accurately captured some boldness traits for the studied fish. During the first three minutes of the experiment, this behaviour was even more pronounced, meaning that, on average, the fish tended to stay on the black background when first introduced to a new environment before starting to explore it. However, the slight delay between the introductions of the four fish caused by the design of the experiment meant that a proportion of the time accounted for as spent on the black background was actually time when the fish were not yet introduced to the arena. When focusing on the first three minutes of footage, this delay represents a more considerable proportion of the time of the experiment than when analysing the whole twenty-minute footage, and the lower initial visibility rate found may be due to this bias. Overall, no link was found between the scototaxis behaviour of the salmon and their reactions to bubble curtains in the laboratory. This suggests that the boldness of smolts does not influence the efficiency of a bubble wall in steering them away from the turbine intake. However, the salmon used in this experiment were one-year old and had not undergone smoltification and it is plausible that behaviour changes brought about by smolting could affect the responses of salmon to exterior stimuli (McCormick et al., 1998; Popper and Carlson, 1998). In that case, the absence of a link between boldness and reaction to bubbles found in the laboratory assay may not be representative of the behaviour of migrating smolts. An additional difference between the field and laboratory assays is the risk of predation. Because the interaction of predation and the use of the bubble barrier had a significant effect in the field, it is reasonable to assume that the absence of a predator in the laboratory experiment may influence the response of smolts to bubble barriers. This raises the question of whether the success of the bubble wall in the field is due to other behaviour traits than boldness, and what cues eventually cause the smolts to modify their trajectory. Salmon tend to fear unknown environments, as they may be a source of danger (Maximino et al., 2010). Thus, a possible explanation for avoidance is that bubbles provide a visual blockage of what lies behind them, causing the smolts to alter their trajectory to stay in visible surroundings. Moreover, visual stimuli have been shown to affect salmon movement, with the use of strobe lights proving effective in causing avoidance responses by smolts (Coutant and Whitney, 2000). Other sensory cues could induce a change of trajectory, such as water displacement, which may originate from the bubbles and be perceived by smolts through their lateral line system, as well as low frequency sounds (Knudsen et al., 1992; Popper and Carlson, 1998). Finally, environmental cues play a role in determining salmon trajectory, including current speed and flow, which are altered in the vicinity of hydroelectric dams (Popper and Carlson, 1998). In addition to these factors, the heavy predation to which smolts are subjected is key in deciding their trajectory around an obstacle, as was seen in the field study. Indeed, the presence of pike at the entrance of the fishway had a greater effect on the presence of smolts in that area than the use of the bubble barrier. The susceptibility of smolts to predation is heightened in anthropogenically disturbed environments and is also particularly high during migration towards the sea, which could greatly affect the efficiency of guiding systems if not taken into account (McCormick et al., 1998; Koed et al., 2006). The risk of predation may cause smolts to adopt a schooling behaviour (Fertl and Wilson, 1997), which may explain the effect of pike on the presence of schools of all sizes, as well as the effect of the interaction between predation and the use of the bubble barrier on small and medium schools. Furthermore, it is possible that bubbles also affect the movements of pike, as various fish species have been shown to react to bubble barriers (Fertl and Wilson, 1997). This could lead to a greater presence of predators in the section leading to the fishway and cause avoidance of the area by smolts despite the use of guiding systems. The timing of migration plays an important role in the effect

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of predation on smolts. Firstly, vulnerability to predation has been shown to decrease with prey size, meaning that older, larger smolts may be less affected by predation (Lundvall et al., 1999; Kekäläinen et al., 2008). Secondly, the effect of predation by pike depends on the life cycle of the latter, as they have been shown not to prey on smolts during their spawning period (Jepsen et al., 2000). The synchronicity of smolt migration and pike spawning may therefore also affect the success of fishways and guiding structures. Smolts have shown habituation when repeatedly exposed to stimuli, so it could be hypothesised that multiple interactions with bubbles could alter the smolts’ behaviour towards them, making the effectiveness of bubble barriers uncertain on the long term (Knudsen et al., 1992; Folkedal et al., 2010). In case of a river with multiple dam passages, smolts suffer an increased mortality due to accumulated stress, which could be amplified by a decreased efficiency of the guiding structures as the smolts approach the sea (Elder et al., 2016; Marschall et al., 2011). Generalising the use of sensory cues to steer smolts around dams necessitates a better understanding of the reason for their success, and their design should take into account the responses of fish to those stimuli. It appears that combining several sensory cues, such as sounds, vibrations and visual stimuli might be the best option in order to trigger avoidance from smolts, by designing guiding structures that use, for instance, both bubbles and light and take into account environmental factors such as the direction of the current (Bainbridge, 1964; Sager et al., 1987; Popper and Carlson, 1998). 5 Conclusions Overall in this study, the bubble wall has proven effective in the field in increasing the presence of smolts going towards the fishway and could therefore be a realistic solution in preserving the migration routes of Atlantic salmon. However, a multitude of factors can affect the trajectory of smolts, as well as their schooling behaviour, especially the presence of a predator. The laboratory assay conducted did not produce evidence that avoidance of bubbles was linked to the boldness of salmon, so the behaviour traits influencing individual reaction to bubbles remain unclear. It is therefore necessary to carry out further research into the cues that smolts react to in order to design successful bypasses that can ensure migration and the preservation of Atlantic salmon populations. Acknowledgements I would like to thank my supervisor, Jonatan Klaminder, for giving me the possibility to conduct this project and, along with Micael Jonsson, Johan Leander and Johan Fahlman, for providing help and feedback throughout the whole process of my thesis work. I would also like to thank Johan Leander for allowing me to use the data he collected and helping me set up the laboratory experiments, and Johan Fahlman for guiding me through the ToxTrac software. Finally, I would like to thank my classmates, Rick Heeres for giving me advice on how to approach thesis work, and Arno Veenstra for giving me feedback, help in the laboratory and following my work during its entire progress. References Ackefors, H., Johansson, N., Wahlberg, B. 1991. The Swedish compensatory programme for

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