Methodologies for investigating the potential for subtle damage to fish

The concerns regarding the potential for subtle or delayed effects on smolts from turbine passage highlight a need for methodologies which can detect such subtle or chronic effects, and more information on their significance for onward survival.

Most studies on downstream fish passage through conventional hydropower schemes have been designed to estimate direct mortality (e.g. Bell & Kynard, 1985; Mathur et al., 1995; Normandeau Associates, 2009). However, there may be a spectrum of severity of injuries which could disadvantage fish and decrease the likelihood of survival. To assess such sub-lethal effects, some studies have captured fish at the outflow of turbines for visual inspection for signs of external injury (e.g. Bracken & Lucas, 2013; Kibel et al., 2009). This may be extended to gross pathological examination and histopathology of potentially affected tissues or organs.

Damage is most frequently categorized as several types (for example descaling, laceration, beheading, haemorrhage, haematoma, eye damage, split fins, or spinal fracture, although this is mostly limited to the assessment of mortalities and not sensitive examination of subtle effects to live fish (see reviews by Monten (1985) and Larinier & Travade (2002)). These categories lack information on the severity of injury, which becomes important when considering novel technologies which involve more slowly moving parts. In their studies, Kibel et al. (2009) used a scoring system to rate the severity of injury, with the following categories:

1 - Death or serious injury likely to cause death within 24 hours. Deep wounding exposing internal organs;

2 - Moderate damage, including abrasions to skin. Fin damage and significant scale loss above 15%;

3 - Very little damage. Limited if any fin damage. Between 1% and 15% scale loss;

4- No damage.

Some studies have kept turbine passed fish under observation following turbine passage either to detect delayed mortality or to assess behaviour for subtle effects which may affect survival. Cada et al. (2003), for example, examined startle response following turbine passage, and Bracken & Lucas (2013) assayed symetrical swimming motion in turbine-passed lampreys.

Serum biochemistry may be a useful tool for identifying subtle effects which are not readily apparent on visual examination. There is an extensive literature on the use of endocrine measures of stress in fish. These are reviewed in relation to potential turbine passage applications by Hasler et al. (2009). The difficulty with this approach for assessing stress to fish from hydropower turbine passage is the need for strict time control, as the response of some of these hormones (cortisol, for example) is rapid. Capture methods, and any pre- or post- trial handling of fish may confound the hormone response, and control groups (for example, sampled from a bypass channel) may be similarly stressed to turbine- passed fish. Mauls & Mesa (1994) measured cortisol in fish which were electric-

fished and immediately sacrificed after passage through a large-scale hydropower system, but found that there was no significant effect when compared to fish which had passed through a bypass system. The relative effects of passage experience and sampling technique were not separated in that case.

A more promising avenue for identifying tissue damage is the measurement of intracellular enzymes in the blood serum. When cells are damaged or die, these enzymes are released into the blood. Measuring the levels of these enzymes in collected serum samples can allow inferences about the magnitude and type of tissue damage (Hasler et al., 2009). This type of clinical pathology is routine for domesticated animals, and is becoming an established tool in aquaculture. The enzymes Creatine Kinase (CK), Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and Alanine Aminotransferase (ALT) in particular, but others also, have received research attention (e.g. Rodger et al., 1991; Yousaf & Powell, 2012). For the most part, these attentions have focussed on the response of these enzymes after a disease or chemical challenge, or with differing dietary constituents for the purposes of aquaculture management, but some studies have examined their usefulness for detecting mechanical trauma to fish.

Congleton & Wagner (2004) measured serum constituents of naturally migrating wild and stocked Chinook salmon smolts, and considered ALT, AST and LDH to be general indicators of tissue damage, and CK to indicate damage to the muscle or heart, where it is most concentrated. In a study on damage caused by handling methods for farmed channel catfish, Grizzle et al. (1992) found the highest plasma activities of AST and LDH in the group with the highest incidence of external injuries from handling methods. Dobšíková et al. (2006) and Dobšíková & Svobodova (2009) found AST, CK and LDH levels in common carp (Cyprinus carpio L.) to be significantly influenced by handling and transportation. The use of such assays are also supported by successful application in measuring angling stress (Butcher et al., 2011; Cooke et al, 2013; Killen et al., 2003; Morrissey et al., 2005; Rapp et al., 2012; Wells et al., 1986), and the effects of pollution (e.g. Escher et al., 1999).

A difficulty with using these biochemical approaches is that there is currently a lack of information in the literature base on normal reference ranges, and expected effect size. Sandnes et al. (1988), and very recently Braceland et al. (2016) published normal ranges for these enzymes in adult farmed Atlantic salmon, but other studies have found values outwith these ranges in control groups for adults (Vangen & Hemre 2003; Hemre et al., 2007), pre-smolts (Petri et al., 2006), and Atlantic salmon smolts (Hevrøy et al., 2011). A review of studies which used these enzymes in Atlantic salmon alone reveals very variable ranges of mean activities and standard deviations in control groups (Table 3.1). This lack of consistent ranges in the literature suggests that activities of these enzymes in the blood may vary widely between species, stages and individuals; with condition and environmental conditions. Therefore any attempt to identify subtle damage by using these measures should incorporate appropriate controls for comparison with the challenged group.

Table 3.1. The range of mean enzyme activities, and standard deviations within and between published studies for control groups, from studies measuring the activities of AST, ALT, CK and LDH in Atlantic salmon. For studies where no mean concentrations were given but range was present, mean is taken as the mean of the range. The unit (U) of enzymatic activity is the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under standard conditions (NC-IUB, 1978).

Enzyme Range of mean activities (U/l), between groups and studies Range of within-study standard deviations Standard deviations of the means between- studies References

AST 37-616 9-121 211.1 Hemre, et al., 2007; Hevrøy et al., 2011; Petri et al., 2006; Sandnes et al., 1988; Vangen & Hemre, 2003; Wagner & Congleton, 2004 ALT 4-50 1-9.2 14.87 Hemre, et al., 2007;

Hevrøy et al., 2011; Petri et al., 2006; Sandnes, et al., 1988; Vangen & Hemre, 2003; Wagner & Congleton, 2004 CK 1582-10297 425-6277 4408.5 Rodger et al., 1991;

Wagner & Congleton, 2004; Yousaf & Powell, 2012 LDH 235-1757 53-781 686.6 Hemre et al., 2007;

Wagner & Congleton, 2004; Yousaf & Powell, 2012

A recent and promising assay with high specificity for skeletal muscle damage measures enolase 3 enzyme (Braceland et al., 2014). Enolase 3 is defined classically as a glycolytic enzyme catalysing the conversion of 2- phosphoglycerate to phosphoenolpyruvate in the ninth and penultimate step of glycolysis (Panchioli,2001). Three isoforms of enolase, which have differing distributions in body tissues, have been identified in mammals (Tracy & Hedges, 2000). Braceland et al. (2014) showed a significant relationship between white muscle pathology and serum enolase 3 (hereafter referred to simply as enolase, since no other form is discussed further in this thesis) content through the use of histopathology and serum protein analysis in adult Atlantic salmon challenged with pancreatic disease. The output of the assay is a semi quantitative measure of enolase activity in the serum, and appears to lack the problem of high variability in apparently healthy fish that the other analytes (AST, ALT, CK and LDH) exhibit. Whilst the enolase assay has demonstrated effectiveness for identifying chronic, disease induced damage to skeletal muscle, the response of this enzyme to acute trauma is untested.

3.2 Methods

There are already many ASHT schemes operational, hence, in order to attain results with the greatest applied value, controlled field tests were carried out at a full-scale, commercially operating ASHT. These tests involved the assessment of turbine exposed and control groups of Atlantic salmon smolts. Hatchery origin Atlantic salmon smolts were used in order to attain predictably sufficient sample sizes during the planned period for the experiments. Two approaches were used to assess damage:

Firstly, visual inspection of fish and post-hoc analysis of photographs were used to identify and measure external signs of damage.

Secondly, to attempt to detect subtle damage which may not be visually apparent, levels of activity of the enzymes AST, CK, LDH and enolase were measured and compared between turbine-passed and control groups

of fish. This is a novel application of serum chemistry techniques for the assessment of mechanical trauma from hydropower turbine passage.