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.

Interactions of semiconductor Cd-based quantum dots and Cd2+ with gut bacteria isolated from wild Salmo trutta fry.

Background: With the rapid development of nanotechnology, more and more nanoproducts are being released into the environment where they may both pose ecological risks and be toxic to living organisms. The ecotoxicological impact of quantum dots (QDs), a class of nanoparticles (NPs), on aquatic organisms is becoming an emerging issue, this due to their nano-specific properties, to the physico-chemical transformation in the environment and to the possible release of toxic metals from their structure such as Cd. Methods: In this work, (i) spectroscopic measurements of commercially available Cd-based QDs (CdSe/ZnS-COOH) were made at various pH values (5.0 and 7.0) to study their interactions (at a concentration of 4 nm) with various strains of Gram-positive and Gram-negative gut bacteria after short-term exposure and (ii) the antibacterial efficacy of QDs and Cd2+ (at a concentration 0.09-3.56 mM) against gut bacteria isolated from wild freshwater Salmo trutta fry was studied at different temperatures (15 °C and 25 °C) and pH values (5.0 and 7.0) by applying a well-established disc diffusion assay. Results: Twenty-six gut bacterial isolates from wild Salmo trutta fry were identified as Aeromonas spp., A. popoffii, A. salmonicida, A. sobria, Carnobacterium maltaromaticum, Buttiauxella sp., Listeria sp., Microbacterium sp., Shewanella putrefaciens and Serratia sp. Cd-based (CdSe/ZnS-COOH) QDs at a concentration of 4 nm were found to be stable in aqueous media (with pH 7.0) or starting to form aggregates (at pH 5.0), thus, apparently, did not release heavy metals (HMs) into the media over 48 h in conditions of light or dark and did not show antibacterial efficacy on the gut bacteria isolated from wild Salmo trutta fry after short-term (9 h and 48 h) incubations. Cd2+ was found to produce significant dose-dependent toxic effects on bacterial growth, and the size of the inhibition zones on some of the tested strains significantly correlated with temperature. The most sensitive and the most resistant to Cd2+ were the Gram-positive bacteria, for which the minimum inhibitory concentration (MIC) values of Cd2+ were 0.09-0.27 mM and 3.11-3.29 mM respectively and varied significantly between the tested temperatures (15 °C and 25 °C). The MIC values of Cd2+ for the Gram-negative bacteria (18 out of 22 strains) ranged from 0.44 to 0.71 mM and did not differ significantly between the tested temperatures. Among the selected Gram-positive and Gram-negative strains, those with the higher sensitivity towards Cd2+ also revealed relatively stronger signals of QDs photoluminescence (PL) when transferred after incubation into fresh medium without QDs. In addition, the formation of endogenous metalloporphyrins observed spectroscopically in some bacterial strains indicates certain differences in metabolic activity that may play a protective role against potential oxidative damage.

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.

Impact evaluation of marking Salmo trutta with Alizarin Red S produced by different manufacturers.

Fish otolith marking with the alizarin dye is a commonly used tool in sustainable fishery management. However, the reported effects of this dye on fish health are rather controversial and are possibly linked to differences in the composition of different brands of Alizarin red S (ARS). Laboratory experiments designed to elucidate effects of different concentrations of theoretically the same ARS as indicated by the CAS (Chemical Abstracts Service) number on fish at different development stages were carried out. The acute toxicity of ARS to Salmo trutta was found to be concentration- and fish developmental stage-dependant. Our study results showed that S. trutta sensitivity to ARS varies depending on its developmental stages as follows: fry (50-days after hatching) > alevins (30-days after hatching) > alevins (1-day after hatching). One of the tested ARS brands (purchased from VWR International LLC (Matsonford Road, USA)) was found to be several times more toxic to fish than another (purchased from Sigma-Aldrich (St. Louis, USA)), although according to the certificates of analysis, the tested substances were identical. Survival and growth of the S. trutta fry, which was marked with different ARS brands and stocked in the same natural stream, was investigated for two consecutive years. The results obtained indicate remarkable differences (p < 0.05) in the effects produced by the tested ARS brands, thus confirming our laboratory findings. The performed elemental analysis of the tested ARS dyes revealed significant differences in chemical impurities that these dyes contain. This study has, for the first time, expressed concern about the probable long-term impact of some ARS brands on the marked fish and their potential to bias the results of the studies dealing with ARS-marked fish.

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.

Toxicological Potential of Cadmium Impact on Rainbow Trout (Oncorhynchus mykiss) in Early Development.

Cadmium (Cd) is a toxic element widely distributed in the aquatic environment and producing a wide variety of harmful effects. In this study, the acute toxicity (96 h LC50) of Cd to rainbow trout Oncorhynchus mykiss embryos and larvae was determined. The obtained results showed that hatched larvae were the most sensitive to Cd exposure. After 4 days of exposure, embryos were found to have accumulated greater concentrations of Cd than larvae. Exposure to Cd at sublethal concentrations produced deleterious, exposure duration-related effects on biological parameters (mortality, heart rate and gill ventilation frequency) of larvae. Cd induced a significant elevation of all the studied geno- and cytotoxicity endpoints in larval erythroblasts.

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.

Interaction of carboxylated CdSe/ZnS quantum dots with fish embryos: Towards understanding of nanoparticles toxicity.

Due to colloidal instability even with protective coatings, nanoparticles tend to aggregate in complex environments and possibly interact with biota. In this study, visualization of quantum dots (QDs) interaction with rainbow trout (Oncorhynchus mykiss) embryos was performed. Studies on zebrafish (Danio rerio) and pearl gourami (Trichogaster leerii) embryos have shown that QDs interact with embryos in a general manner and their affects are independent on the type of the embryo. It was demonstrated that carboxylated CdSe/ZnS QDs (4 nM) were aggregating in accumulation media and formed agglomerates on the surface of fish embryos under 1-12 days incubation in deep-well water. Detailed analysis of QDs distribution on fish embryos surface and investigation of the penetration of QDs through embryo's membrane showed that the chorion protects embryos from the penetration through the chorion and the accumulation of nanoparticles inside the embryos. Confocal microscopy and spectroscopy studies on rainbow trout embryos demonstrated that QDs cause chorion damage, due to QDs aggregation on the surface of chorion, even the formation of the agglomerates at the outer part of the embryos and/or with the mucus were detected. Aggregation of QDs and formation of agglomerates on the outer part of the embryo's membrane caused the intervention of the aggregates to the chorion and even partially destroyed the embryo's chorion. The incorporation of QDs in chorion was confirmed by two methods: in living embryos from a 3D reconstruction view, and in slices of embryos from a histology view. The damage of chorion integrity might have adverse effects on embryonic development. Moreover, for the first time the toxic effect of QDs was separated from the heavy metal toxicity, which is most commonly discussed in the literature to the toxicity of the QDs.

Embryotoxicity of Quantum Dots in Rainbow Trout Oncorhynchus mykiss During the Hatching Period.

Due to the active development and application of nanotechnology, nanoparticles have emerged as a new class of environmental pollutants. The aim of the study was to investigate quantum dots (QDs) access routes and distribution in embryos and larvae of rainbow trout Oncorhynchus mykiss and to determine the toxicity of QDs to rainbow trout larvae depending on the duration of exposure. CdSe/ZnS-COOH QDs at sublethal concentration was used during the toxicity test (1, 4 and 14 days). The results showed that QDs could get from the solutions into the larvae after hatching. QDs induced a significant increase in mortality, gill ventilation frequency and behavioral responses and a decrease in relative body mass in larvae at the end of the test. Larvae exposed to QDs were found to possess developmental malformations (blood clots). It was found that biological responses of larvae significantly depended on the duration of exposure to QDs.

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.

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.

The acute and behavioral effects of a copper-nickel mixture on roach Rutilus rutilus.

Semi-static acute toxicity tests were conducted on adult roach, Rutilus rutilus, to estimate its sensitivity toward an equitoxic binary mixture (EBM) of copper and nickel. The sum of their individual LC50 values was considered to equal 100 %. The main endpoints of the study were mortality and behavioral responses: detection, locomotor activity, coughing rate and pectoral-fin activity. The 96-h LC50 of EBM was 14.4 (10.1 %-20.5 %), indicating a synergism of individual metals. The most meaningful behavioral results were obtained after 10-min, 1-h and 24-h exposures, and the most sensitive and informative behavioral response was found to be coughing rate. This bioassay response may be used successfully to evaluate wastewaters containing heavy metals for their toxicity toward fish.

Toxic effects of crude oil combined with oil cleaner simple green on yolk-sac larvae and adult rainbow trout Oncorhynchus mykiss.

UNLABELLED: BACKGROUND, GOAL AND SCOPE: Cleaner CRYSTAL Simple Green (SG) was used for the cleanup of the oil spill in the Baltic Sea near Lithuania in 2001. No scientific data are available on the effects and consequences of its application for local aquatic life. The aim of this study was to determine and compare sublethal effects of a) solution SG; b) crude oil alone; c) SG in combination with oil on rainbow trout Oncorhynchus mykiss at different stages of its development in laboratory conditions. METHODS: Laboratory studies were performed on adult rainbow trout (4-day duration) and on yolk-sac larvae (25-day duration) evaluating their biological parameters. Concentrations of water-soluble and thin-dispersed fractions of petroleum hydrocarbons were measured using gas chromatography. RESULTS AND DISCUSSION: SG solution (0.5 mg/l) did not affect the survival of larvae and adult fish, and no significant changes were determined in respiratory parameters of the exposed lar vae and adult fish. The most expressed alterations were found in morphological parameters (a decrease in the average body mass) of larvae and in haematological indices (a decrease in the leukocyte count) of adult fish at the end of the tests. Crude oil (1610 mg/l) did not affect the survival of adult fish during the 4-day exposure. An increase in larvae mortality rate (approximately 36%) was recorded at the end of the tests. A significant decrease in the average body mass and heart rate of larvae as well as in gill ventilation frequency of larvae and adult fish were determined. SG combined with oil induced an increase in larval mortality approximately 46% of individuals died at the end of the tests. No mortality was recorded in adult fish. The average body mass and heart rate of larvae were significantly decreased. Marked changes were also found in respiratory parameters (gill ventilation frequency of larvae and adult fish significantly decreased, while 'coughing' rate increased). A 1-day, 2-day exposure of fish to SG combined with oil induced a significant decrease in the leukocyte count of adult fish, which was also determined at the end of the tests. The augmentation of adverse impact could be explained by the data obtained from our studies. When SG was added into dilution water with crude oil the concentration of petroleum hydrocarbons in the mixture increased 3 approximately 4.5 times after 24 h and 96 h, respectively. CONCLUSIONS: The comparative study of the effects of crude oil alone, SG and SG combined with oil showed that their toxic effects on fish differed. Oil combined with SG was found to be more toxic to fish (larvae and adults) than SG alone and oil alone. Fish at early stages of development (yolk-sac larvae) were more sensitive to the effects of the compounds studied than adults. RECOMMENDATIONS AND OUTLOOK: To diminish the negative impact of oil spill cleanup using chemicals on aquatic ecosystems, it is recommended to carry out more comprehensive studies of their effects and after-effects in laboratory conditions using a wide scale of local aquatic organisms. The selected species of the most sensitive aquatic organisms should include those which are unable to escape the impact of combined action of oil and cleaners. Special attention should be directed to the research of effects of these pollutants on studied organisms at their most sensitive stages of life (reproduction, hatching, early stages of development), as after-effects of exposure to pollutants may be observed in future generations.

Toxic effects of orimulsion on rainbow trout Oncorhynchus mykiss.

GOAL, SCOPE AND BACKGROUND: Orimulsion (stable emulsion of natural bitumen and water) is a new imported industrial fuel in Lithuania. No data on its toxicity to fish is freely available. The aim of this study was to investigate sensitivity of rainbow trout (Oncorhynchus mykiss) to acute and chronic toxicity of orimulsion and to estimate the Maximum Acceptable Toxicant Concentration (MATC) of orimulsion to fish. METHODS: Laboratory tests were conducted on rainbow trout in all stages of development (embryos, larvae, adults). Acute toxicity (96-hour duration) and long-term (28 or 60-day duration) tests evaluating the wide range spectrum of biological indices were performed under semi-static conditions. RESULTS AND DISCUSSION: Median lethal concentration (96-hour LC50) values and their 95% confidence intervals derived from the tests were: 0.1 (0.09-0.12) to embryos, 0.06 (0.05-0.07) to larvae and 2.22 (2.02-2.43) to adult fish, and 28-day LC50 to adult fish was found to be 0.26 (0.21-0.32) g/l of total orimulsion respectively. The acute toxicity of orimulsion to rainbow trout can be characterised by a narrow zone of toxic effect and a sharp boundary between lethal and sublethal concentrations. The lowest 'safe' or 'no-effect' concentration values of total orimulsion obtained in long-term tests were equal to 0.09 g/l to adult fish, 0.019 g/l to embryos, and 0.0017 g/l to larvae. Proposed value of 'application factor' for orimulsion was found to be equal to 0.03. Since orimulsion has the property to disperse in all water volume, its toxic effect on fish can be characterised by the combined effects of dispersion and water-soluble-fraction. CONCLUSIONS: Maximum Acceptable Toxicant Concentration (MATC) of 0.0017 g/l of total orimulsion to fish was derived from long-term tests based on the most sensitive parameter of rainbow trout larvae (relative mass increase at the end of the test). According to substance toxicity classification accepted for Lithuanian inland waters, orimulsion can be referred to substances of 'moderate' toxicity to fish. RECOMMENDATIONS AND OUTLOOK: For prediction and evaluation of toxic impact of orimulsion accident spills on fish, some recommendations should be given. Since orimulsion has the property to disperse in all water volume during short time periods, the amounts of both spilled orimulsion and polluted water should be ascertained. Once both parameters are known, the real concentration of orimulsion in the water body must be determined. Then this concentration must be compared with 'safe' concentration to fish. By use of 'application factor' 0.03, approximate MATC for other fish species can be estimated when only acute toxicity data (96-hour LC50 value) is available.

Effect of a heavy metal model mixture on biological parameters of rainbow trout Oncorhynchus mykiss.

The effects of a model mixture (HMMM) of seven heavy metals (Cu, Zn, Ni, Cr, Pb, Cd, Mn) on the rainbow trout Oncorhynchus mykiss at all stages of development (embryos, larvae, adults) were investigated based on the annual average concentrations of these metals in cooling waste waters discharging from Ignalina Nuclear Power Plant (Lithuania) into the Druksiai lake. According to mortality parameters, the most sensitive to HMMM were larvae, although no significant differences between the sensitivity of embryos and adult fish to HMMM were found. Maximal toxic effect of HMMM was observed during the hatching period. Long-term exposure to sublethal concentrations of HMMM affected embryo development, growth of larvae, their cardio-respiratory and behavioural responses, induced significant changes in morphological, morpho-physiological, physiological and haematological parameters of adult fish. Respiratory responses and growth parameters of fish were found to be the most sensitive to low concentrations of HMMM. Adult fish were capable of detecting and avoiding low, sublethal concentrations of HMMM. Heavy metals in a mixture at low concentrations were more toxic than single ones. According to the background of the damages induced by HMMM, after-effects in a fish organism, as well as in a whole population, can be predicted.