Effects of handling and surgical implant on juvenile wild trout: Physiological and behavioral impact.

Fish involved in telemetry studies are usually handled, anaesthetized, and subjected to internal tag implantation, all of which have the potential to disrupt the fish's physiology, migratory patterns, food-seeking behavior, growth, and survival. As fish tagging is a widespread standard method in aquaculture, it is crucial to gain a better understanding of the short- and long-term effects on various aspects of fish welfare. The experimental outcomes of each phase of the surgical process and how it affects the fish's ability to recover during the post-tagging period, particularly in small salmonids, are poorly understood. Thus, it is essential to measure the extent of these adverse effects to precisely extrapolate differences in surgical intervention for tagged fish compared to their unmanipulated counterparts. In our study, we explored the post-tagging impacts on wild-captured brown trout (Salmo trutta) (1+) juveniles. We examined the time effect on fish recovery after each phase of the surgical procedure, which included anaesthetized fish, followed by surgery with/without internal tagging, within the first 2 and 25 h post-surgery. Common blood parameters (glucose levels and haematocrit) and endpoints related to swimming behavior, including fish movement, direction, and social distancing aspects, were studied. The findings of the study indicated no significant changes in glucose and haematocrit levels over the observational period. Fish subjected to anesthesia and tagging procedures exhibited a significant decrease in swimming activity, recovering to baseline levels 1 h after anesthesia and 24 h after surgical (tagging) procedures in recirculating aquaculture systems. Tagged fish showed less social proximity than non-tagged fish in the first 2 h post-surgery. A significant effect size was observed between nonsurgical (anesthesia) and surgical (non-tagged/tagged) groups, highlighting a more pronounced impact associated with surgical tag interventions. Our data indicate that the magnitude of behavioral response was significantly influenced by tag weight (~1.4% body/tag weight ratio in water) among manipulated fish groups. Consequently, our study reveals that wild-captured S. trutta juveniles, tagged with commercially available low-weight acoustic tags (V6, 69 kHz), experienced negative effects on swimming performance. In surgical studies, acknowledging potential influences is crucial for accurately inferring fish physiological and behavioral status. Emphasizing fish recovery potential in both short- and long-term periods is essential for quantifying tagging effects. Future research should prioritize exploring alternative tagging technologies and refining methodologies, with a particular focus on assessing telemetry's impact on socioeconomically relevant small salmonids.

Exploring the Effects of Graphene-Based Nanoparticles on Early Salmonids Cardiorespiratory Responses, Swimming and Nesting Behavior.

Graphene-based nanomaterials are exceptionally attractive for a wide range of applications, raising the likelihood of the release of graphene-containing nanoparticles into aquatic environments. The growing use of these carbon nanomaterials in different industries highlights the crucial need to investigate their environmental impact and evaluate potential risks to living organisms. The current investigation evaluated the nanotoxicity of graphene (nanoflakes) and graphene oxide (GO) nanoparticles on the cardiorespiratory responses (heart rate, gill ventilation frequency), as well as the swimming and nesting behavioral parameters of early stage larvae and juvenile salmonids. Both short-term (96 h) and long-term (23 days) exposure experiments were conducted using two common species: brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). The findings demonstrated notable alterations in fish nesting behavior, swimming performance, and cardiorespiratory functions, indicating the potential toxicity of nanoparticles. This impact was observed at both physiological and whole-organismal levels in salmonids at early stages. Future investigations should explore different types of nanocarbons and their potential enduring effects on fish population structure, considering not only individual survival but also broader aspects of development, including feeding, reproductive, and other social dynamics.

A comparative analysis of multi-biomarker responses to environmental stress: Evaluating differences in landfill leachate and pathogenic oomycete effects between wild and captive Salmo trutta.

Phenotypic plasticity is one of the major means by which organisms can manage with environmental factor changes. Captivity-related stress and artificial rearing settings have been shown to dramatically alter fish response plasticity in terms of physiology, behavior, and health, potentially reducing overall fitness and fish survival. Understanding the variations in plasticity between captive-bred (kept in a homogenous environment) and wild fish populations in response to varied environmental pressures is becoming increasingly important, particularly in risk assessment research. In this study, we investigated whether captive-bred trout (Salmo trutta) are more susceptible to stress stimuli than their wild counterparts. In both wild and captive-bred trout, we investigated a battery of biomarkers that depicts the effects at various levels of biological organization in response to landfill leachate as a chemical pollutant, and after exposure to pathogenic oomycetes Saprolegnia parasitica. According to the findings, wild trout were more susceptible to chemical stimuli based on cytogenetic damage and catalase activity changes, whereas captive-bred trout were more sensitive to biological stress as evidenced by changes in overall fish activity and increasing cytogenetic damage in gills erythrocytes. Our findings emphasize the significance of exercising caution when conducting risk assessments of environmental pollutants using captive-bred animals, especially when seeking to extrapolate hazards and better understand the consequences of environmental contamination on wild fish populations. Additional comparative studies are required to investigate the impact of environmental stressors on multi-biomarker responses in both wild and captive fish populations in order to uncover changes in the plasticity of various traits that can result in adaptation or maladaptation to environmental stimuli within these fish populations, affecting data comparability and transferability to wildlife.

The impact of various carbon nanomaterials on the morphological, behavioural, and biochemical parameters of rainbow trout in the early life stages.

With the increasing production and the number of potential applications of carbon nanomaterials, mainly from the graphene family, their release into the natural environment, especially to aquatic ecosystems, is inevitable. The aim of the study was to determine the effects of various carbon nanomaterials (graphene nanoflakes (GNF), graphene oxide (GO), reduced graphene oxide (RGO) and silicon carbide nanofibers (NFSiC) in the concentration of 4 mg L-1 on the early life stages of the rainbow trout Oncorhynchus mykiss. The survival rates of O. mykiss were not affected after 36 days of exposure to studied materials, except for RGO, which caused significant mortality of both embryos and larvae compared to the control conditions. Larvae exposed to GO and NFSiC were characterized by a smaller standard body length at hatch, whereas at the end of the experiment, the growth of fish exposed to all materials was accelerated, especially in GO and RGO treatment, in which higher body weight and length were accompanied by lower volume of the yolk sac. Neither the markers of the oxidative damage nor the antioxidant enzymes activities were significantly affected in embryos, newly hatched larvae and larvae after 26-day exposure to studied carbon nanomaterials. Also, no neurotoxic effect expressed by the activity of the whole-body acetylcholinesterase was observed. Nevertheless, the significant increase in the velocity and the overall activity of larvae exposed to GNF (not investigated after exposure to other materials) must be highlighted. The most pronounced effect of RGO might be connected with its large particle size, sharp edges, and the presence of TiO2 nanoparticles. The results indicate for the first time that various carbon nanomaterials potentially released into aquatic ecosystems may have serious developmental implications for the early life stages of salmonid fish.

Technical suitability and reliability of an in vivo and non-invasive biosensor-type glucose assessment as a potential biomarker for multiple stressors in fishes: an evaluation on Salmonids.

Regardless of the wide use of glucose measurements in stress evaluation, there are some inconsistencies in its acceptance as a stress marker. To meet the challenge and test the reliability/suitability of glucose measurement in practice, we simulated different environmental/anthropogenic exposure scenarios in this study. We aimed to provoke stress in fish followed by a 2-week stress recovery period and under the cumulative effect of leachate fish exposed to pathogenic oomycetes (Saprolegnia parasitica) to represent a possible infection in fish. We selected stream-resident and anadromous brown trout ecotypes (Salmo trutta) representing salmonids with different migratory behaviour strategies. Here, we analysed glucose content in fish-holding water, blood and gills to determine glucose suitability as a potential biomarker of fish response to environmental challenges. Additionally, swimming behavioural parameters and haematocrit were measured. The results indicated that the quantity of glucose released in the holding water of stressed fish increased considerably and remained substantially higher throughout the stress recovery period than the control level. Correspondingly, the circulating levels of glucose in blood and gills decreased over time in fish exposed to different stressors. A significant decrease in swimming activity of fish was observed during the first hours of leachate exposure and increased in fish exposed to S. parasitica compared to control. Our study is the first to ensure the validity and reliability of glucose response in evaluating physiological stress in fish under chemical and biological stimuli, indicating its sensitivity and response range of glucose measurement in fish-holding water.

Swimming behaviour in two ecologically similar three-spined (Gasterosteus aculeatus L.) and nine-spined sticklebacks (Pungitius pungitius L.): a comparative approach for modelling the toxicity of metal mixtures.

Sticklebacks (Gasterosteiformes) are increasingly used in ecological and evolutionary research and have become well established as role model species for biologists. However, ecotoxicology studies concerning behavioural effects in sticklebacks regarding stress responses, mainly induced by chemical mixtures, have hardly been addressed. For this purpose, we investigated the swimming behaviour (including mortality rate based on 96-h LC50 values) of two ecologically similar three-spined (Gasterosteus aculeatus) and nine-spined sticklebacks (Pungitius pungitius) to short-term (up to 24 h) metal mixture (MIX) exposure. We evaluated the relevance and efficacy of behavioural responses of test species in the early toxicity assessment of chemical mixtures. Fish exposed to six (Zn, Pb, Cd, Cu, Ni, and Cr) metals in the mixture were either singled out by the Water Framework Directive as priority or as relevant substances in surface water, which was prepared according to the environmental quality standards (EQSs) of these metals set for inland waters in the European Union (EU) (Directive 2013/39/EU). The performed behavioural analysis showed the main effect on the interaction between time, species, and treatment variables. Although both species exposed to MIX revealed a decreasing tendency in swimming activity, these species' responsiveness to MIX was somewhat different. Substantial changes in the activity of G. aculeatus were established after a 3-h exposure to MIX solutions, which was 1.43-fold lower, while in the case of P. pungitius, 1.96-fold higher than established 96-h LC50 values for each species. This study demonstrated species-specific differences in response sensitivity to metal-based water pollution, indicating behavioural insensitivity of P. pungitius as model species for aquatic biomonitoring and environmental risk assessments.

Biomarker responses in perch (Perca fluviatilis) under multiple stress: Parasite co-infection and multicomponent metal mixture exposure.

Parasitic infections may cause damage to the host immune system (i.e. fish), thereby endangering its health and weakening its responses to other types of stressors. Therefore, exposure to different kinds of natural or anthropogenic stressors can lead to unexpected toxicity outcomes in aquatic organisms. This study examined the haematological, genotoxic and cytotoxic effects of the co-infection with the protozoan parasite (Trichodina sp.) and the pathogenic oomycete (Saprolegnia parasitica) in Perca fluviatilis alone and in combination with chemical stress (environmentally-relevant aqueous concentrations of metal mixtures). Haematological analyses such as red cell and white cell indices revealed that chemical and biological stressors, used singly and in combination, exerted adverse effects on fish health. Changes in haematological indices induced by exposure to each of the above-mentioned stressors separately and by combined exposure to all of them suggested the multiple stress-induced inflammation process in the exposed fish. The cytogenetic damage inflicted by the S. parasitica and Trichodina sp. co-infection and multiple stress was revealed in fish erythrocytes. This information is expected to contribute to the elucidation of how multiple stressors impact on responses of haematic indices, geno- and cytotoxicity endpoints in P. fluviatilis. Assessment of the risk associated with multiple stressors is expected to prove valuable for the effective aquatic environment management (Løkke et al., 2013 and references therein).

Biological effects of multimetal (Ni, Cd, Pb, Cu, Cr, Zn) mixture in rainbow trout Oncorhynchus mykiss: Laboratory exposure and recovery study.

The present study tested the biological consequences of exposure to a multimetal mixture as a multiple chemical stressor on Oncorhynchus mykiss at molecular, cellular, physiological and whole-organism levels and on biomarker responses of this fish during the depuration period. To represent environmentally relevant multiple chemical stressors, in our study, we used the mixture of Zn, Cu, Ni, Cr, Pb and Cd at the concentrations corresponding to Maximum-Permissible-Concentrations (MPCs) acceptable for the EU inland waters. This study was undertaken with a view to elucidate if changes in the MPC of the test mixture components (Ni, Pb, Cd) could cause significantly different biomarker responses in O. mykiss from those previously determined in the carnivorous and omnivorous fishes exposed to the mixture of the same metals but at different MPCs of Ni, Pb and Cd. This study has revealed that exposure to mixtures of metals at MPC produces genotoxic effects in fish blood erythrocytes and a lethargic effect on O. mykiss behaviour, and, also, significantly increases the levels of Cd, Cr and Ni accumulated in the gills tissue. O. mykiss successfully depurated Cr and Ni in less than 28 days, however, the level of Cd decreased by only approximately 40% over the same period. A significant capacity of O. mykiss to restore its DNA integrity (Comet assay) after exposure to metal mixtures was revealed. However, the 28-day recovery period proved to be insufficiently long for erythrocytes with nuclear abnormalities to recover to the unexposed level. In conclusion, changes in the MPCs of Ni, Pb and Cd in the test mixture produce biological effects similar to those previously determined in S. salar, R. rutilus and P. fluviatilis exposed to the mixture of the same metals but at lower MPCs of Ni and Pb and at higher MPC of Cd.

Acclimation effect on fish behavioural characteristics: determination of appropriate acclimation period for different species.

In the present study, the authors investigated the effect of acclimation duration (up to 4 h) on behavioural characteristics of taxonomically and functionally different fish species, i.e., the migratory rheophilic salmonids rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), and the non-migratory eurytopic European perch (Perca fluviatilis) and three-spined stickleback (Gasterosteus aculeatus). Specifically, the authors explored fish behavioural patterns based on specific endpoints (average, maximum and angular velocity) during the acclimation period, and determined the acclimation period suitable for the tested fish species. The performed behavioural data analysis showed that the minimum time needed to adjust fish activity to a more stable (baseline) level should be at least 2 h for O. mykiss and S. salar and 1 h for G. aculeatus. Nonetheless, P. fluviatilis behaviour did not show significant changes during the 4 h acclimation. The results of this study revealed that the effect of the acclimation duration on such rheophilic species as O. mykiss and S. salar was greater than that on the eurytopic species P. fluviatilis and G. aculeatus, indicating that acclimation period is important in managing fish stress before behavioural observations. For all species, the highest variability was found in the endpoint of maximum velocity, and the lowest in that of angular velocity. This study showed that before starting actual toxicity testing experiments, it is important to determine an appropriate, species-specific acclimation period.

Juvenile fish responses to sublethal leachate concentrations: comparison of sensitivity of different behavioral endpoints.

The aim of the present study was to investigate changes in the locomotor activity of rainbow trout (Oncorhynchus mykiss) juveniles under the impact (2 h) of landfill leachate (as a multicomponent mixture) based on different endpoints such as average, maximum and angular velocities, movement duration, body mobility, and blood glucose level. Fish were exposed to five different sublethal leachate concentrations (0, 0.0625, 0.125, 0.25, and 0.5%). The locomotor activity of the leachate-exposed fish significantly decreased at 0.25 and 0.5% concentrations. Significant changes in fish behavior in response to sublethal leachate concentrations were determined during the first minutes of exposure. Angular velocity proved to be the most sensitive of all the endpoints tested. A positive correlation was observed among behavioral responses, but no correlation was established between the blood glucose level and behavioral endpoints. The blood glucose endpoint was found to be insensitive, and we suggest that it should be used only in combination with other endpoints to complement toxicity data. To enhance the understanding of rainbow trout behavioral characteristics in relation to time, and relations among behavioral endpoints of the fish under short-term exposure to a multicomponent mixture, in the current study, we investigated dynamics of the selected behavioral endpoints over time, relations among these endpoints and compared behavioral response rapidness and efficacy.

Rapidness- and Sensitivity-Based Comparison of Behavioral and Respiratory Responses of European Perch and Rainbow Trout to Metal Mixture Exposure.

The aim of the study was to investigate acute (96-h LC50) toxicity of the complex metal (Zn, Pb, Cd, Cu, Ni and Cr) mixture (MIX) to European perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss), and to examine differences between locomotor and respiratory endpoints based on rapidness and sensitivity of fish responses to short-term (2 h) metal mixture exposure. MIX was prepared taking into consideration the maximum-permissible-concentrations (MPC) set for these metals in EU inland waters (Directive 2008/105/EC). The studied behavioral and respiratory responses of O. mykiss, in contrast to those of P. fluviatilis, were found to be significantly affected by the relationship between exposure duration and treatment. In O. mykiss, gill ventilation frequency (GVF) was found to be the most rapid and sensitive endpoint of all the investigated ones. However, the investigated behavioral and respiratory endpoints of P. fluviatilis showed its behavioral insensitivity to MIX exposure.

Genotoxic and cytotoxic effects of 50 Hz 1 mT electromagnetic field on larval rainbow trout (Oncorhynchus mykiss), Baltic clam (Limecola balthica) and common ragworm (Hediste diversicolor).

The aim of this research was to assess genotoxicity and cytotoxicity responses in aquatic animals exposed to 50 Hz 1 m T electromagnetic field (EMF). Rainbow trout (Oncorhynchus mykiss) at early stages of development were exposed to EMF for 40 days, whereas marine benthic invertebrates - the common ragworm Hediste diversicolor and the Baltic clam Limecola balthica - for 12 days. To define genotoxicity and cytotoxicity responses in selected animals, assays of nuclear abnormalities in peripheral blood erythrocytes of O. mykiss, coelomocytes of H. diversicolor and gill cells of L. balthica were performed. Induction of formation of micronuclei (MN), nuclear buds (NB), nuclear buds on filament cells (NBf) and cells with blebbed nuclei (BL) were assessed as genotoxicity endpoints, and 8-shaped nuclei, fragmented (Fr), apoptotic (Ap) and binucleated (BN) cells as cytotoxicity endpoints. Exposure to EMF affected all studied species but with varying degrees. The strongest responses to EMF treatment were elicited in L. balthica, in which six out of the total eight analyzed geno- and cytotoxicity endpoints were significantly elevated. Significantly induced frequencies of MN were detected in O. mykiss and H. diversicolor cells, NBf and BL only in gill cells of L. balthica, and NB in analyzed tissues of all the test species. As cytotoxicity endpoints, a significant elevation in frequencies of cells with 8-shaped nuclei was found in O. mykiss and L. balthica, while Ap and BN was observed only in L. balthica. EMF exposure did not induce any significant cytotoxic activity in H. diversicolor coelomocytes. The present study is the first to reveal the genotoxic and cytotoxic activity of 1 m T EMF in aquatic animals, and, consequently, the first one to report the adverse effect of this factor on common marine invertebrates and early life stages of fish.

Responses of biomarkers in Atlantic salmon (Salmo salar) following exposure to environmentally relevant concentrations of complex metal mixture (Zn, Cu, Ni, Cr, Pb, Cd). Part II.

The aim of this research was to assess interactions between metals at low exposure concentrations (Maximum-Permissible-Concentrations accepted for the inland waters in EU) and to assess possible influence of background exposure (10-times reduced concentration of a single metal) on toxicological significance of selected biomarkers in Salmo salar after treatment with metal mixture (Zn - 0.1, Cu - 0.01, Ni - 0.01, Cr - 0.01, Pb - 0.005 and Cd - 0.005 mg/L). The tissue-specific bioaccumulation, genotoxicity and cytotoxicity responses (erythrocytic nuclear abnormalities assay) in peripheral blood, kidneys, gills and liver erythrocytes of fish to metal mixtures were assessed after 14 days treatment. Treatment with primary mixture (MIX) or two variants of this mixture (Cr↓ (10 times reduced Cr6+ concentration) and Cu↓ (10 times reduced Cu2+ concentration)) induced the strongest responses in genotoxicity and cytotoxicity endpoints. Exposure to these mixtures highly affected Zn, Cu and Cd bioaccumulation in liver tissue. The highest amount of Ni accumulated was measured after Cd↓ treatment in all tissues. Treatments with reduced concentration of non-essential metal resulted in an increased accumulation of Pb, Ni, or Cd; treatments with reduced concentration of essential metal resulted in a reduced accumulation of certain metals (especially Cd and Pb) in tissues compared between treatments. Glucose content in blood and behavioural endpoints were evaluated after short-term exposure to metal mixtures (MIX, Cr↓, Cu↓). Significant increase in blood glucose concentration was measured after all treatments. These metal mixtures elicit significant behavioural alterations in fish. Consequently, this research revealed a significant influence of background exposure considering mixture toxicity.

Rapid Detection of Sublethal Toxicity Using Locomotor Activity of Rainbow Trout Juveniles.

This study examined changes in locomotor activity of the rainbow trout (Oncorhynchus mykiss) juveniles exposed to sublethal concentrations of hexavalent chromium (Cr6+) (as a single pollutant) and landfill leachate (as an complex mixture of mainly organic compounds, including trace amounts of metals). Fish were first examined for baseline behavior patterns for 3 h to determine the appropriate duration of acclimation and the control level. The average velocity of fish was found to be the most informative among other endpoints throughout the 3 h long acclimation. Under the effect of both test substances, fish locomotor activity significantly increased after 5 min reaching maximum values after 10 min of exposure. The juveniles exposed to leachate were more responsive than those exposed to Cr6+. However, in this study we did not succeed in identifying pollutants from the elicited fish behavioral response patterns.