Toxicity of different-sized cobalt ferrite (CoFe2O4) nanoparticles to Oncorhynchus mykiss at early development stages.

As innovative and versatile agents with potential applications in a wide range of fields including medicine, electronics, wastewater treatment, cosmetics, and energy storage devices, magnetic nanoparticles (NPs) are significant attention. However, our knowledge of the harmful effects of different-sized NPs, particularly of their effects on aquatic animals, is limited. In this study, we evaluated the impact of different-sized (sub-2, 5, and 15 nm) cobalt ferrite (CoFe2O4) NPs on the biological parameters of rainbow trout (Oncorhynchus mykiss) embryos and larvae. The NPs were characterized using techniques such as high-resolution transmission electron microscopy (HRTEM) for imaging, X-ray diffraction (XRD) for crystallographic analysis, and energy-dispersive X-ray spectroscopy (EDX) for elemental analysis, and were tested for impact through a series of toxicity, genotoxicity, and biochemical assays at a concentration of 100 mg/L. The obtained results showed that toxicity of CoFe2O4 NPs depended on the size of NPs and the developmental stage of the fish. Our results, which revealed significant changes in biological parameters of O. mykiss under exposure to CoFe2O4 NPs, imply that these NPs may be not environmentally safe. The hierarchical cluster analysis showed that embryos of the control group were clearly separated from those exposed to NPs of various sizes. However, in the exposed larvae, the effects of control and the smallest-sized NPs (sub-2 nm) differed from those induced by larger NPs (5 nm and 15 nm). Additional research is necessary to comprehend the mechanisms underlying the observed variations, which would be advantageous for both managing the risk of NPs to humans and advancing the field of aquatic nanotoxicology.

Diversity of fungus-like stramenopilous organisms (Oomycota) in Lithuanian freshwater aquaculture: Morphological and molecular analysis, risk to fish health.

The present work is the first comprehensive study of fungus-like stramenopilous organisms (Oomycota) diversity in Lithuanian fish farms aimed at proper identification of saprolegniasis pathogens, which is important for water quality control, monitoring infection levels and choosing more effective treatments for this disease in aquaculture. Pathogenic to fish, Saprolegnia and other potentially pathogenic water moulds were isolated from adult fish, their eggs, fry and from water samples. All detected isolates were examined morphologically and confirmed by sequence-based molecular methods. A total of eight species belonging to the genera Saprolegnia, Achlya, Newbya and Pythium were identified. Four species (S. parasitica, S. ferax, S. australis and S. diclina) were found to be the main causative agents of saprolegniasis in Lithuania. S. parasitica and S. ferax dominated both in hatcheries and open fishponds, accounting for 66.2% of all isolates. S. parasitica was isolated from all farmed salmonid fish species as well as from the skin of Cyprinus carpio, Carassius carassius and Perca fluviatilis. S. australis was isolated from water and once from the skin of Oncorhynchus mykiss, and S. diclina was detected only once on the skin of Salmo salar fish. In addition, Achlya ambisexualis, Saprolegnia anisospora and Newbia oligocantha isolated during this study are noted as a possible source of saprolegniasis. The results of this study are relevant for assessing the risk of potential outbreaks of saprolegniasis or other saprolegnia-like infection in Lithuanian freshwater aquaculture.

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 co-treatment with graphene oxide and metal mixture on Salmo trutta at early development stages: The sorption capacity and potential toxicity.

Graphene oxide (GO) are novel nanomaterials with a wide range of applications due to their high absorption capacity. This study was undertaken with a view to assess the bioaccumulation and acute toxicity of GO used in combination with the heavy metal mixture (Cr, Cu, Ni and Zn) to fish embryos and larvae. For this purpose, Salmo trutta embryos and larvae were subjected to the 4-day long treatment with three different concentrations of GO, the metal mixture, which was prepared of four metals at the concentrations corresponding to the maximum-permissible-concentrations for EU inland waters (Cr-0.01, Cu-0.01, Ni-0.034, and Zn-0.1 mg/L), and with GO in combination with MIX (GO+MIX). When used in combination with the metal mixture, GO exhibited a high metal sorption capacity. The obtained confocal fluorescence microscopy results showed that GO located in the embryo chorion causing its damage; in larvae, however, GO were found only in the gill region. Results of these experiments confirmed the hypothesis that GO affects the accumulation of metals and mitigates their toxic effects on organism. In embryos, the acute toxicity of exposure to GO and co-exposure to MIX+GO was found to manifest itself through the decreased heart rate (HR) and malondialdehyde (MDA) level and through the increased metallothionein (MT) concentration. Meanwhile, in larvae, GO and MIX+GO were found to induce genotoxicity effects. However, changes in HR, MDA, MT, gill ventilation frequency, yolk sack absorption and cytotoxicity compared with those of the control group were not recorded in larvae. The obtained results confirmed our hypothesis: the combined effect of MIX and GO was less toxic to larvae (especially survival) than individual effects of MIX components. However, our results emphasize that fish exposure to GO alone and in combination with heavy metal contaminants (MIX+GO) even at environmentally relevant concentrations causes health risks that cannot be ignored.

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.

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.

Response patterns of biomarkers in omnivorous and carnivorous fish species exposed to multicomponent metal (Cd, Cr, Cu, Ni, Pb and Zn) mixture. Part III.

Toxicity to fish of multicomponent metal mixtures at maximum-permissible-concentrations (MPC: Cd-0.005, Cr-0.01, Cu-0.01, Ni-0.01, Pb-0.005 and Zn-0.1 mg/L) set for EU inland waters was evaluated using the whole-mixture approach. An extended follow-up study on the biological effects of multicomponent metal mixtures on three ecologically different fish species, i.e. Perca fluviatilis, Rutilus rutilus, and Salmo salar is reported. The aim of this study was to assess response patterns of biomarkers (erythrocytic nuclear abnormalities (ENAs), metal accumulation and metallothioneins) in tissues of fish species after 14-day treatment with multicomponent metal mixtures at MPC and metal mixtures with one of its components at reduced MPC (↓). After treatments with Cu↓ and Cr↓, the lowest amount of Ni was found in all tissues (except the liver) of all fish species tested. After Zn↓ and Pb↓ treatments, the amount of Ni in muscle of all the tested fish species significantly decreased. The highest amounts of Cr in gills and Pb in muscle were detected in all fish species after treatments with Ni↓ and Cd↓ mixtures, respectively. R. rutilus accumulated significantly larger amounts of metals than P. fluviatilis and S. salar. The data obtained show that tissues of the omnivorous R. rutilus exposed to metal mixtures accumulated higher amounts of Cr, Cu, Ni and Zn, while tissues of carnivorous S. salar and P. fluviatilis higher amounts of Cd and Pb. The analysis of ENAs revealed concentration-dependent responses, indicating Cu↓ and Cr↓ treatments as causes of higher geno- and cytotoxicity levels.

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.

Genotoxicity and cytotoxicity response to environmentally relevant complex metal mixture (Zn, Cu, Ni, Cr, Pb, Cd) accumulated in Atlantic salmon (Salmo salar). Part I: importance of exposure time and tissue dependence.

Health impact of metal mixture at environment realistic concentrations are difficult to predict especially for long-term effects where cause-and-effect relationships may not be directly obvious. This study was aimed to evaluate metal mixture (Zn-0.1, Cu-0.01, Ni-0.01, Cr-0.01, Pb-0.005 and Cd-0.005 mg/L, respectively for 1, 2, 4, 7, 14 and 28 days at concentrations accepted for the inland waters in EU) genotoxicity (micronuclei, nuclear buds, nuclear buds on filament), cytotoxicity (8-shaped nuclei, fragmented-apoptotic erythrocytes), bioaccumulation, steady-state and the reference level of geno-cytotoxicity in hatchery-reared Atlantic salmon tissues. Metals accumulated mostly in gills and kidneys, to the lesser extent in the muscle. Uptake of metals from an entire mixture in the fish for 14 days is sufficient to reach steady-state Cr, Pb concentrations in all tissues; Zn, Cu-in kidneys and muscle, Ni-in liver, kidneys, muscle and Cd-in muscle. Treatment with metal mixture significantly increased summed genotoxicity levels at 7 days of exposure in peripheral blood and liver erythrocytes, at 14 days of exposure in gills and kidney erythrocytes. Significant elevation of cytotoxicity was detected after 2 and 14 days of exposure in gills erythrocytes and after 28 days-in peripheral blood erythrocytes. The amount of Cu, Cr, Pb and Cd accumulated in tissues was dependent upon duration of exposure; nuclear buds, 8-shaped nuclei frequencies also were dependent upon duration of exposure. This study indicates that metals at low levels when existing in mixture causes significant geno-cytotoxicity responses and metals bioaccumulation in salmon.

Accumulation of heavy metals in different body tissues of Atlantic salmon, Salmo salar L., exposed to a model mixture (Cu, Zn, Ni, Cr, Pb, Cd) and singly to nickel, chromium, and lead.

One-year-old Atlantic salmon smolts were exposed for 2 weeks either to six priority heavy metal mixture or to Ni, Pb, and Cr singly at a concentration corresponding to Lithuanian inland water standards: Cu - 0.01, Zn - 0.1, Ni - 0.01, Cr - 0.01, Cd - 0.005 and Pb - 0.005 mg/L, respectively under semi-static conditions. The presence of metal mixture in the water only partly (by 50 %) affected the accumulation of single metals in body tissues (muscle, gills, liver and kidneys) probably due to the synergistic interactions among metals. Although metal concentrations increased in most cases, only Pb exceeded recommended level for human consumption (0.2 Pb mg/L) by 1.1-fold to 2.1-fold.