Fish injuries resulting from transient operating conditions in a Brazilian hydropower plant: morphological, physiological and biochemical evaluation in Pimelodus maculatus (Siluriformes: Pimelodidae)

Abstract This study investigated injuries sustained by Pimelodus maculatus caused by transient operating conditions in a Brazilian hydroelectric power plant. Fish (n = 43) that inhabited the tailrace and appeared on the water surface after changes in the operating conditions were evaluated (IF). Additionally, fish (n = 10) captured using longline in the vicinity of the tailrace were used for comparisons (NF). Blood samples were evaluated for cortisol, glucose, and lactate concentrations. Tissue samples (gill, liver, brain, and muscle) were analyzed for oxidative stress biomarkers (TBARS, protein carbonyl content, and catalase activity) and histology (gill, liver, and spleen). The most prominent findings in IF were barotraumas with intra-abdominal hemorrhage and swim bladder rupture. Cortisol and glucose concentrations were lower for IF when compared to NF. IF also suffered from oxidative stress, as indicated by increased TBARS in the liver and brain, as well as increased protein carbonyl content in the gills and liver. The main histological alterations induced by the adverse conditions were circulatory disorders, such as telangiectasia and gill aneurysm, hepatic congested veins, and hemorrhage in the liver and spleen. The abrupt drop of pressure resulting from changes in the operating conditions of the turbines caused barotraumas and oxidative stress, which were the leading causes of fish mortality


INTRODUCTION
Fish passage through turbines from upstream to downstream is considered one of the major causes of fish mortality at hydroelectric power plants (HPPs) in the Northern Hemisphere (Cada, 2001;Brown et al., 2014).In Brazilian dams, however, fish mortality related to HPP operation is mainly associated with fish movements from downstream to upstream where fish can enter the tailrace and draft tube during transient operating conditions of the turbine, such as shutdown, startup, and synchronous condenser states (Silva et al., 2018).In all cases, the leading causes of fish mortality and injuries are strike (mechanical shock against building structures, as well as runner blades), shear stress (friction between two water bodies at different velocities), cavitation (bubble formation that causes shock waves at high pressure), and abrupt pressure changes under the runner (Beirão et al., 2016;Carvalho, Marques, 2018).
Rapid decompression in the turbines causes the fish to suffer internal and external tissue injuries, known as barotrauma.Barotrauma includes various injuries such as hemorrhage, embolism, stomach eversion, exophthalmia, swim bladder, and other organ rupture (Algera et al., 2020).
When fish undergo external adverse conditions, such as passage through turbines or rapid decompression, physiological and biochemical responses are triggered, along with compensatory or adaptive mechanisms, in an attempt to overcome the stressor.For instance, the stress hormone cortisol triggers physiologic responses like increasing metabolic parameters, such as glucose and lactate, to recover fish homeostasis (Barton, Iwama, 1991;Trushenski et al., 2010).Also, fish can activate their antioxidant defense systems by producing catalase enzymes, to overcome oxidative cell damage, such as lipid and protein peroxidation, measured as TBARS (thiobarbituric acid reactive substances), and protein carbonylation, respectively (Velisek et al., 2011).Through histology, it is also possible to verify fish health because tissue changes express stress-induced conditions (Copper et al., 2018).Thus, evaluating blood parameters, oxidative tissue stress, and histology are tools for monitoring and understanding the health of downstream fish captured after abrupt decompression exposure at HPPs, as well as turbine-passed fish.
In Brazil, catfish comprise one of the groups most frequently found downstream during mortality episodes owing to transient operating conditions of HPPs (Silva et al., 2018).Pimelodus maculatus (Lacepède, 1803) is a yellow-mandi catfish widely distributed in several South American hydrographic basins, including the Uruguay River (Zaniboni Filho, Schulz, 2003), where Machadinho HPP is located.This is the fish species most frequently identified with lethal injuries after switching the turbine operating regime at this HPP, as in other dams in Brazil (Andrade et al., 2012).
Although several studies reported fish mortality at HPPs, the physiological and biochemical responses are unclear because they commonly focus on macroscopic lesions.Therefore, this study aimed to investigate the injuries of P. maculatus exposed to abrupt decompression inside the draft tube at Machadinho HPP by evaluating morphological, physiological, and biochemical parameters.

MATERIAL AND METHODS
Study area and fish sampling.The Machadinho HPP is located on the upper Uruguay River (27°31'31"S and 51°47'07"W).It borders Santa Catarina and Rio Grande do Sul states in southern Brazil.It has three Francis turbines, each with a 380 MW output, totaling 1,140 MW of installed capacity.From September 2019 to June 2021, the tailrace was inspected 11 times during changes in the operating regimes (Tab.1).The inspections consisted of observing fish that appeared on the water surface for approximately 20 min.When sighted floating with erratic swimming, P. maculatus individuals were sampled (n = 43) for immediate analysis.The species was also captured in the immediate vicinity of the tailrace (n = 10) using longline gear for comparative analyses.A voucher specimen was deposited in the Ichthyological Collection of the Universidade Estadual de Londrina, Brazil (MZUEL 9599).
For convenience, the term "Injured Fish (IF)" refers to fish collected on the water surface during tailrace inspections, whereas "Naïve Fish (NF)" refers to those captured using longline in the proximity of the tailrace.
Sampling and analyses.Fish were measured, weighed, and then anesthetized using 50 mg L -1 eugenol.Blood was sampled from the caudal vein for glucose (Accu Cheek Active, Roche, Germany), lactate (Accutrend Plus, Roche, Germany), and cortisol analyses.A 3.0 mL syringe with anticoagulant (EDTA, 10%) was used, and the collected blood was stored in 2 mL sterile microtubes.Cortisol determination used plasma from centrifuged blood (2,500 x g, 15 min, 4 ºC), maintained frozen in liquid nitrogen Transients in hydropower plant cause fish injuries (-180 ºC) until analyses, and measured in duplicate using ELISA kits (Arbor Assays Cortisol Immunoassay, Arbor Assays, USA).After blood sampling, fish were euthanized with an overdose of the anesthetic (100 mg L -1 eugenol) and submitted to necropsy.Necropsy consisted of external and internal macroscopic analysis.
Gill, liver, brain, and muscle samples were collected and stored in liquid nitrogen (-180 ºC) until required for biochemical analyses.Levels of the biomarkers thiobarbituric acid reactive substances (TBARS) and protein carbonyl, as well as catalase activity, were measured using the methods described by Reznick, Packer (1994), Buege, Aust (1978), and Nelson, Kiesow (1972), respectively with some modifications as detailed in Thiel et al. (2020).For IF, biochemical and hematological analysis were carried out with 30 randomly sampled fish.
Gill, liver, and spleen tissues sampled for histology were fixed in 10% buffered formalin solution for 24 h, washed in 70% alcohol, and stored in 70% alcohol until analysis.Next, tissues were dehydrated in a series of alcohol solutions (70, 80, 90, and 100%) and embedded in paraffin following a routine technique.Subsequently, 5 μm thick cross-sections were taken using a Leica RM2255 rotary microtome (Leica Microsystems, Germany).Then, each tissue was stained with hematoxylin and eosin using the automatic Leica AutoStainer XL (Leica Microsystems, Germany).Images were captured under a light microscope (Leica DM3000 LED, Leica Microsystems, Germany) with a connected camera and analyzed with Leica Application Suite 3.4.0(Leica Microsystems, Switzerland).

Statistical analyses.
Comparisons among groups were made by one-way ANOVA followed by Tukey-Kramer test after checking for homoscedasticity (Levene's test) and normality (Shapiro-Wilk test).Outliers were detected and removed from the analysis using the Tukey outlier detection test (k = 2.1).The Kruskal-Wallis test was performed when assumptions were violated.The frequencies of lesions were compared between groups using Fisher's test.All analyses were performed using Statistica Software 7.0 (Stat Soft, USA) at a 0.05 significance level.

Startup
The turbine runner goes from rest to nominal speed in seven min (slow startup scheme), the unit is synchronized to the grid, and the load is increased (load acceptance).

Synchronous condenser to generating mode
At the synchronous condenser mode, the turbine is operated in air conditions rotating at the synchronous speed driven by the generator acting as a motor.Depending on the energy demand, the turbine goes from synchronous condenser mode to generating mode.The scheme adopted by the Machadinho HPP to return to generating mode involves a sequence of shutdown and slow startup.

Shutdown
The turbine runner comes to stop after working as synchronous condenser, by opening the deaeration valve to expel air from the runner to the atmosphere and break the runner by friction with water under the runner. 5/17

DISCUSSION
Hydrostatic pressure variation on the turbine runner during transient operating conditions causes fish to decompress and, consequently, suffer from barotrauma (Beirão et al., 2016).This is the same kind of pressure drop experienced by turbine-passed fish during steady state operating conditions, typical of partial load or full load operation at hydropower plants worldwide (Cada et al., 2006;Brown et al., 2014).Pimelodus maculatus collected in the tailrace after changing the operating condition were found with various barotraumas, such as swim bladder rupture, hemorrhage, embolism, stomach eversion, and exophthalmia.Bone fractures as result of mechanical shocks were also observed, as described for other species (Brown et al., 2009;Algera et al., 2020).
The swim bladder controls fish buoyancy in the water column, acting as a gas reservoir, and its retention capacity depends on fish morphology (Stephenson et al., 2010;Pflugrath et al., 2012).Pimelodus maculatus is a benthic and physoclistous species that exchanges gas through the circulatory system.It often maintains negative buoyancy by retaining a little air in the swim bladder (Silva et al., 2018).Therefore, when this fish undergoes rapid decompression, the dissolved gases boil (Henry's Law) and expand (Boyle's Law), causing the swim bladder to overinflate and burst (Brown et al., 2009), a condition that affected 90% of the IF evaluated in this study.Hemorrhage is a frequent finding that results from gas embolism, i.e., bubble formation in blood vessels.It increases blood volume and reduces blood density, causing organ rupture and blood loss, leading to death (Brown et al., 2012(Brown et al., , 2014)).More than 70% of IF presented widespread bleeding, which extended to the peritoneal cavity, liver, spleen, kidney, gills, eyes, skin, and fins.
Adaptive stress responses, classified as primary and secondary, follow a pattern in most fish, and several of these responses can be analyzed through blood (Wandelaar-Bonga, 1997;George et al., 2013).Among the primary responses, cortisol is the most important corticosteroid hormone produced during stressor events.The hypothalamicpituitary-interrenal axis controls the release of cortisol that acts mainly on the gills, intestine, and liver, regulating metabolic energy and hydromineral balance (Barton, Iwama, 1991).Secondary responses are triggered by primary responses and are mainly metabolic in origin.The release of glucose from the liver by glycogenolysis, known as hyperglycemia, is a metabolic response mediated by catecholamines, acting as a substrate for energy production.Also, in anaerobic situations, lactate is released as an energy source for cellular metabolism (Olsen et al., 1995;Polakof et al., 2012).
The reference values of blood stress parameters are unknown for fish exposed to pressure drops.However, it is possible to infer those values from fish farming studies.Barcellos et al. (2003), for example, have found increased cortisol (seven times) and glycose (three times) levels in the juvenile silver catfish Rhamdia quelen (Quoy & Gaimard, 1834) after harvesting.In juvenile cobia Rachycentron canadum (Linnaeus, 1766) exposed to air, cortisol, glucose, and lactate have increased six, three, and eight times, respectively (Trushenski et al., 2010).
The cortisol and glucose concentrations in fish under stress rise to establish homeostasis (Iversen et al., 2009).Accordingly, we were expecting higher cortisol and glucose levels in IF compared to those in NF owing to the severity and irreversibility of the trauma herein described.Notably, even fishing is a stressor able to drive both stress indicators away from the baseline in NF.However, systemic response did not occur in IF, possibly because of hemorrhage.Typically, cortisol travels through the bloodstream signaling the onset of a stressful situation (Wandelaar-Bonga, 1997).However, the severe bleeding found in most IF (70%) blocked the activation of physiological responses that would otherwise have increased cortisol and glucose levels.Therefore, in the present study, neither cortisol nor glucose was suitable as a stress marker in the assessment of fish exposed to pressure drop.
To the best of our knowledge, the present study is the first to perform biochemical analyses of oxidative stress parameters on fish affected by HPP operation, a common approach in ecotoxicological studies (Parvez, Raisuddin, 2005;Menezes et al., 2011;Toni et al., 2011).Oxidative stress is the imbalance between the generation of oxidant compounds and the action of antioxidant defense systems.A deficiency in the antioxidant system or insufficient ability to repair oxidative damage can increase reactive oxygen species and cause cell death (Barbosa et al., 2010;Halliwell, Gutteridge, 2015).
Lipid peroxidation, measured as TBARS has been reported as the one of the main factors responsible for the loss of cellular functions under oxidative stress conditions (Almroth et al., 2005;Barata et al., 2005;Menezes et al., 2011).Lipid peroxidation acts as a mechanism of cell injury caused by oxygen free radicals in biological membranes.These reactions produce lipid hydroperoxides which break down the double bond Transients in hydropower plant cause fish injuries of unsaturated fatty acids and damage the cell membrane (Barata et al., 2005).The formation of lipid hydroperoxides can, in turn, further oxidize proteins, causing an excessive formation of carbonylated proteins, a biomarker for protein oxidation assessment (Lackner, 1998;Almroth et al., 2005).
TBARS levels have been reported to increase in the gills, liver, and muscle of lambari Astyanax jacuhiensis (Cope, 1894) (= A. lacustris (Lütken, 1875)) in the presence of five pesticides in the water (Loro et al., 2015).Also, the increased levels of TBARS in the gill, liver, and kidney previously demonstrated that Prochilodus lineatus (Valenciennes, 1837) is living under stressful field conditions in the Salado River basin (Santa Fé, Argentina) (Cazenave et al., 2009).Similarly, increased TBARS levels in the liver (328%) and brain (52%) of IF in the present study showed that changes in the operating conditions of HPP turbines also caused cellular damage to P. maculatus tissues as a direct result of stress and trauma.
IF also showed increased protein carbonyl levels in the gills (190%) and liver (32%).Similarly, increased protein carbonyl content was reported in the gill (200%), liver (111%), and kidney (190%) of Channa punctata (Bloch, 1793) exposed to the pesticide deltamethrin (Parvez, Raisuddin, 2005), as well as the liver of silver catfish exposed to herbicides (Ferreira et al., 2010;Menezes et al., 2011).Unexpectedly, IF muscle showed a reduction of protein carbonyl content (44%).In this case, exhaustion caused by the attempt to escape from the longline could explain the higher oxidative stress in NF muscle compared to that in IF.Protein carbonylation is induced by reactive oxygen species (ROS), which are produced in low amount during muscle contractility (Dalle-Donne et al., 2003).Under strenuous activity, however, ROS accumulate in the muscle, increasing oxidative stress (Duberstein et al., 2009).
Organisms have developed antioxidant defense systems to minimize oxidative damage to cellular components, as determined through the activity of antioxidant enzymes, such as catalase (Barata et al., 2005;Monteiro et al., 2006).Catalase is an enzyme that belongs to the first line of antioxidant defense and converts hydrogen peroxide into water and oxygen (Halliwell, Gutteridge, 2015).Catalase and other antioxidant enzymes can increase, decrease, or remain unchanged, depending on the intensity and duration of the stressor event, or even vary according to fish species (Ballesteros et al., 2009).
Catalase activity was reported to decreased in the liver of R. quelen infected with Aeromonas hydrofila.This was attributed to the low capacity of this species to neutralize the hydrogen peroxide produced by high TBARS, contributing, in turn, to the oxidative liver damage (Baldissera et al., 2017).A similar trend in these biomarker levels was observed in the brain of IF, where high TBARS levels occurred, along with a decrease in catalase activity, indicating oxidative brain damage.
The brain has very low antioxidant defenses compared to other tissues, e.g., the liver (Halliewell, Gutteridge, 2015).In addition, the brain is characterized by high oxygen consumption and lipid-rich content; therefore, it is highly susceptible to oxidative stress.In IF, decreased catalase activity and increased TBARS in the brain indicate the induction of oxidative damage and further indicate that such damage may be related to the severity of the lesions.It is postulated that catalase levels are low in all brain regions compared to the liver.Owing to the intrinsic characteristics of the central nervous system and its metabolism, several antioxidant defense systems are, however, present in the brain, making it more susceptible to damage caused by oxidant species ( Two fish species, Oreochromis niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822), reported to be chronically exposed to metal concentrations in water and sediments, showed marked increases in catalase activity and TBARS levels in their liver and gills (Moussa et al., 2022).Since these species have antioxidant defense capacity against different stressors, such findings suggest these organs are good indicators of pollution.In the present study, our analyses showed an increase in TBARS and protein carbonyl content in the liver, but no change in catalase activity.Perhaps the time required for an increase in catalase expression in this tissue was not sufficient to generate antioxidant responses to trauma.Thus, our data provide new information on the use of oxidative markers in IF, mainly catalase activity, TBARS levels and protein carbonyl content in the different organs analyzed.
For P. maculatus, no changes in catalase activity were observed in the liver and gills, even with an increase in TBARS levels and protein carbonyl in the liver.The lack of antioxidant enzyme response contributes to more substantial lipid and protein oxidation and, consequently, more severe oxidative damage (Toni et al., 2011). Toni et al. (2011) also did not observe changes in catalytic activity of in Cyprinus carpio (Linnaeus, 1758) exposed to tebuconazol, even with the increased levels of TBARS and protein carbonyl content.
Histology is a widely used tool to determine fish health and physical condition because tissue changes express conditions induced by stress, pathogens, inadequate nutrition, improper water quality, or injuries suffered (Copper et al., 2018).However, this approach is rarely applied in investigating the possible causes of fish mortality at HPPs (Brown et al., 2009).Our evaluation of IF histology revealed several lesions related to circulatory disorders, such as aneurysm and telangiectasia in the gills, congested veins in the liver, and hemorrhage in the liver and spleen.
Aneurysms and telangiectasia in the gill are related to physical or chemical damage, usually linked to trauma, intense handling, or chemical contaminants (Flores-Lopes, Thomaz, 2011).These circulatory injuries are associated with an increase in blood flow increase owing the dilatation of blood capillaries, starting with blood congesting the extremity of the secondary lamellae (telangiectasia) and then developing into an aneurysm, congesting all lamellae (Strzyżewska-Worotyńska et al., 2017).In most cases, these are irreversible lesions, and when many lamellae are affected, the respiratory capacity of fish is compromised (Roberts, 2002).Among the hemorrhagic findings from the necropsy of the injured fish, both aneurysm and telangiectasia resulted from abrupt hydrostatic pressure variation.
Congested veins are indicative of blockages in blood passage from the hepatic artery and spleen to the central vein, requiring greater effort from the heart to pump blood (Kostić et al., 2017).However, because of abdominal and local bleeding in organs like the liver and spleen, even greater pumping could not supply the circulatory loss in severe hemorrhage.
The liver and spleen of NF and IF presented melanomacrophage centers, which are typically located in the hematopoietic tissues of these organs, as well as kidneys (Wolke, 1992).Therefore, it is implausible that they were associated with the injuries caused by changes in the operating conditions.The melanomacrophage centers are associated with inflammatory processes, metabolizing toxic compounds by phagocytosis, and iron cycling; accordingly, they have been considered suitable water quality biomarkers of Transients in hydropower plant cause fish injuries environmental pollution (Agius, Roberts, 2003;Sayed, Younes, 2017).Some studies have related the higher incidence of melanomacrophage centers in P. maculatus liver to water contaminants (Brito et al., 2012;Paulino et al., 2014).
Mononuclear inflammatory infiltrates, which include first-line defense cells such as macrophages, lymphocytes, and plasmocytes (Manrique et al., 2017), were observed in the spleen of most fish evaluated, indicating that changes in the operating conditions did not trigger the inflammatory process.Araújo et al. (2019) have also found mononuclear inflammatory infiltrates in the liver of P. maculatus collected in the Funil's HPP (Rio de Janeiro, Brazil), suggesting that these infiltrates likely result from sublethal concentrations of pollutants.
Morphological changes were also found in IF gills, such as interlamellar hyperplasia and detachment of the lamellar epithelium.Excessive cell proliferation, such as that which occurs in hyperplasia, is an adaptive response that increases the distance between the external environment and the blood, reducing the contact surface area with water.As this process intensifies, the diffusion of respiratory gases reduces causing hypoxia and death (Mallatt, 1985;Sepici-Dinçel et al., 2009).The detachment of the lamellar epithelium can lead to osmoregulatory disorders, also caused by the difficulty in carrying out gas exchange (Pereira et al., 2014).
Pimelodus maculatus inhabiting the tailrace when exposed to the transient operating conditions showed barotrauma as the main cause of mortality, with swim bladder rupture and hemorrhage being the most recurrent traumas.Diffuse hemorrhage was the most critical effect because it caused circulatory disorders that promoted gill lesions, such as aneurysms.Furthermore, liver and spleen lesions, such as congested veins and blood circulation blockage, inhibited the activation of hormonal cascade essential for fish recovery and oxygen transport.In addition, lipid and protein oxidative damage to all analyzed tissues probably prevented the synthesis of antioxidant enzymes that would have otherwise helped in fish recovery, thus causing irreversible cell damage.

TABLE 1 |
Transient operating conditions of turbines at the Machadinho hydroelectric power plant (HPP) where catfish sustained injuries evaluated in this study.

TABLE 2 |
Types of injuries and their frequency of occurrence (%) as determined following necropsy of

TABLE 3 |
Hematological responses of Pimelodus maculatus captured using longline in the vicinity of the

TABLE 4 |
Biochemical responses in different tissues (gill, liver, brain, and muscle) of Pimelodus maculatus captured using longline in the vicinity of the tailrace (NF) or collected in the tailrace during transient operating conditions (IF) of the Machadinho hydroelectric power plant (HPP).Data are reported as mean ± standard deviation.Different letters indicate significant differences (P < 0.05) among groups for each parameter and tissue.TBARS = thiobarbituric acid reactive substances.a (n = 10) 147.3 ± 107.8 a (n = 10) 30.4 ± 19.2 a (n = 10) 3.7 ± 2.9 a (n = 8) IF 475.1 ± 331.