0000000000607083
AUTHOR
Eeva-riikka Vehniäinen
Multixenobiotic resistance efflux activity in Daphnia magna and Lumbriculus variegatus
Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8–9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without…
Endogenous AhR agonist FICZ accumulates in rainbow trout (Oncorhynchus mykiss) alevins exposed to a mixture of two PAHs, retene and fluoranthene
AbstractMultiple studies have reported synergized toxicity of PAH mixtures in developing fish larvae relative to the additive effect of the components. From a toxicological perspective, multiple mechanisms are known to contribute to synergism, such as altered toxicodynamics and kinetics, as well as increased oxidative stress. An understudied contributor to synergism is the accumulation of endogenous metabolites, for example: the aryl hydrocarbon receptor 2 (AhR2) agonist and tryptophan metabolite 6-Formylindolo(3,2-b)carbazole (FICZ). Fish larvae exposed to FICZ, alongside knock-down of cytochrome p450 (cyp1a), has been reported to induced symptoms of toxicity similar to those observed foll…
Toxicity of silver nanoparticles to Lumbriculus variegatus is a function of dissolved silver and promoted by low sediment pH
Toxicity of silver nanoparticles (AgNPs) to benthic organisms is a major concern. The use of AgNPs in industry and consumer products leads to increasing release of AgNPs into the aquatic environment-sediments being the major sink. Effects of sediment pH on the toxicity of AgNPs to benthic oligochaeta Lumbriculus variegatus were studied in a 23-d toxicity test. Artificially prepared sediments (pH 5 and 7) were spiked with varying concentrations of uncoated AgNP, polyvinylpyrrolidone (PVP)-coated AgNP, and silver nitrate (AgNO3 ) as dissolved Ag reference. Number of individuals and biomass change were used as endpoints for the toxicity. The toxic effects were related to the bioaccessible conc…
Fluence Rate or Cumulative Dose? Vulnerability of Larval Northern Pike (Esox lucius) to Ultraviolet Radiation
Newly hatched larvae of northern pike were exposed in the laboratory to four fluence rates of ultraviolet radiation (UVR; 290-400 nm) over three different time periods, resulting in total doses ranging from 3.0 +/- 0.2 to 63.0 +/- 4.4 kJ.m(-2). Mortality and behavior of the larvae were followed for 8-12 days, and growth measured at the end of the experiment. Also, the principle of reciprocity-that the UVR-induced mortality depends on the cumulative dose, independent of fluence rate-was tested. Fluence rates higher than 1480 +/- 150 mW.m(-2) caused mortality and growth retardation. The highest fluence rate (3040 +/- 210 mW.m(-2)) caused 100% mortality in 5 days. All fluence rates caused beha…
Partitioning of nanoparticle-originated dissolved silver in natural and artificial sediments
Sediments are believed to be a major sink for silver nanoparticles (AgNPs) in the aquatic environment, but there is a lack of knowledge about the environmental effects and behavior of AgNPs in sediments. The release of highly toxic Ag+ through dissolution of AgNPs is one mechanism leading to toxic effects in sediments. We applied an ultrasound-assisted sequential extraction method to evaluate the dissolution of AgNPs and to study the partitioning of dissolved Ag in sediments. Silver was spiked into artificial and 2 natural sediments (Lake Höytiäinen sediment and Lake Kuorinka sediment) as silver nitrate (AgNO3), uncoated AgNPs, or polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs). In addition, …
Retene causes multifunctional transcriptomic changes in the heart of rainbow trout (Oncorhynchus mykiss) embryos
Fish are particularly sensitive to aryl hydrocarbon receptor (AhR)-mediated developmental toxicity. The molecular mechanisms behind these adverse effects have remained largely unresolved in salmonids, and for AhR-agonistic polycyclic aromatic hydrocarbons (PAHs). This study explored the cardiac transcriptome of rainbow trout (Oncorhynchus mykiss) eleuteroembryos exposed to retene, an AhR-agonistic PAH. The embryos were exposed to retene (nominal concentration 32 μg/L) and control, their hearts were collected before, at and after the onset of the visible signs of developmental toxicity, and transcriptomic changes were studied by microarray analysis. Retene up- or down-regulated 122 genes. Th…
Photoinduced lethal and sublethal toxicity of retene, a polycyclic aromatic hydrocarbon derived from resin acid, to coregonid larvae
A comparative investigation on the acute phototoxicity of retene to vendace (Coregonus albula) and whitefish (C. lavaretus), both having pelagial larvae in spring, was conducted. To test the concept of early warning of sublethal biomarkers in relation to lethality to posthatch stages, we examined the effects of ultraviolet-B (UV-B) and retene on the levels of cytochrome P4501A (CYP1A) and heat shock protein 70 (HSP70) by exposing the animals to elevated levels of these factors for 48 and 72 h, respectively. Whereas UV-B and retene on their own were not lethal, simultaneous retene and UV-B exposure caused very high mortality to both species. The median lethal concentration (LC50; i.e., the c…
Toxicity Testing of Silver Nanoparticles in Artificial and Natural Sediments Using the Benthic Organism Lumbriculus variegatus
The increased use of silver nanoparticles (AgNP) in industrial and consumer products worldwide has resulted in their release to aquatic environments. Previous studies have mainly focused on the effects of AgNP on pelagic species, whereas few studies have assessed the risks to benthic invertebrates despite the fact that the sediments act as a large potential sink for NPs. In this study, the toxicity of sediment-associated AgNP was evaluated using the standard sediment toxicity test for chemicals provided by the Organization of Economic Cooperation and Development. The freshwater benthic oligochaete worm Lumbriculus variegatus was exposed to sediment-associated AgNP in artificial and natural …
Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, Part 2 – Proteomics and metabolomics
Polycyclic aromatic hydrocarbons (PAHs) are global contaminants of concern. Despite several decades of research, their mechanisms of toxicity are not very well understood. Early life stages of fish are particularly sensitive with the developing cardiac tissue being a main target of PAHs toxicity. The mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons (PAHs) retene, pyrene and phenanthrene were explored in rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to sublethal doses of each individual PAH causing no detectable morphometric alterations. Changes in the cardiac proteome and metabolome were assessed after 7 o…
The effects of treated effluents on the intensity of papillomatosis and HSP70 expression in roach.
Epidermal papillomatosis in fish has been proposed as an indicator of environmental stress but experimental evidence of connection between contaminants and papillomatosis in fish is scarce. We studied changes in the intensity of epidermal papillomatosis and the expression of heat shock protein 70 (HSP70) in roach, Rutilus rutilus, exposed to treated pulp mill and municipal effluents. In male roach, the increase in papillomatosis intensity was higher in fish exposed to 15% than in fish exposed to 1.5% concentration of municipal effluent. No differences were observed in papillomatosis development in females, or in HSP70 expression. In all the experiments conducted, the increasing effect of ef…
How to preserve and handle fish liver samples to conserve RNA integrity
As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at − 20 °C for 6 months. Freeze-thaw cycles of …
Toxicity of silver nanoparticles to Lumbriculus variegatus is a function of dissolved silver and promoted by low sediment pH
Toxicity of silver nanoparticles (AgNPs) to benthic organisms is a major concern. The use of AgNPs in industry and consumer products leads to increasing release of AgNPs into the aquatic environment—sediments being the major sink. Effects of sediment pH on the toxicity of AgNPs to benthic oligochaeta Lumbriculus variegatus were studied in a 23‐d toxicity test. Artificially prepared sediments (pH 5 and 7) were spiked with varying concentrations of uncoated AgNP, polyvinylpyrrolidone (PVP)‐coated AgNP, and silver nitrate (AgNO3) as dissolved Ag reference. Number of individuals and biomass change were used as endpoints for the toxicity. The toxic effects were related to the bioaccessible conce…
Different sensitivities of whitefish (Coregonus lavaretus) and northern pike (Esox lucius) eleutheroembryos to photoinduced toxicity of polycyclic aromatic hydrocarbons.
The photoinduced toxicity of two polycyclic aromatic hydrocarbons (PAHs), retene (RET) and pyrene (PYR), to the eleutheroembryos of whitefish and northern pike was studied. Fish were exposed to three concentrations of RET and PYR, and irradiated with ultraviolet radiation (UVR) or visible light for 3 h on two consecutive days. UVR covered the absorption maxima of RET and PYR at UVB and UVA, the daily UVR doses were 30 and 28 kJ m−2, respectively. After 72 h, mortality and behavioral abnormalities were observed. Survivors were analyzed for the proteins of CYP1A, CYP3A, Hsp70, and the activity of glutathione reductase (GR). While neither PAHs nor UVR on their own were lethal, simultaneous exp…
Boreal fishes and ultraviolet radiation : actions of UVR at molecular and individual levels
Eeva-Riikka Vehniäinen on väitöskirjatyössään tutkinut UV-säteilyn vaikutuksia siian, hauen ja muikun poikasissa. Ihmisen aiheuttama otsonikerroksen ohentuminen on lisännyt maan pinnalle saapuvan ultravioletti-B-säteilyn (UVB) määrää viime vuosikymmenten aikana, ja lisääntymisen odotetaan jatkuvan vielä noin 2050-luvulle saakka. UVB-säteily on haitallista eliöille, koska se aiheuttaa vaurioita solun keskeisissä rakenteissa: DNA:ssa, proteiineissa ja lipideissä.Vehniäinen arvioi lisääntyvän UVB-säteilyn aiheuttamaa riskiä taloudellisesti ja ekologisesti tärkeille makeanveden kalalajeillemme, siialle, muikulle ja hauelle, niiden herkimmässä elämänvaiheessa välittömästi kuoriutumisen jälkeen. …
Exposure to retene, fluoranthene, and their binary mixture causes distinct transcriptomic and apical outcomes in rainbow trout (Oncorhynchus mykiss) yolk sac alevins
Polycyclic aromatic hydrocarbons (PAHs) are widely spread environmental contaminants which affect developing organisms. It is known that improper activation of the aryl hydrocarbon receptor (AhR) by some PAHs contributes to toxicity, while other PAHs can disrupt cellular membrane function. The exact downstream mechanisms of AhR activation remain unresolved, especially with regard to cardiotoxicity. By exposing newly hatched rainbow trout alevins (Oncorhynchus mykiss) semi-statically to retene (32 µg l−1; AhR agonist), fluoranthene (50 µg l−1; weak AhR agonist and CYP1a inhibitor) and their binary mixture for 1, 3, 7 and 14 days, we aimed to uncover novel mechanisms of cardiotoxicity using a…
More accuracy to the EROD measurements—The resorufin fluorescence differs between species and individuals
Ethoxyresorufin-O-deethylase (EROD) activity is a biomarker of exposure to planar aromatic hydrocarbons, and it is often measured from the S9 fraction. The effect of the liver S9 fraction of seven boreal freshwater fish species on the fluorescence of resorufin was studied. The S9 fractions diminished resorufin fluorescence by 40–80%, and there were large differences between species. Thus, using a resorufin standard curve without the S9 fraction leads to a large underestimation of the EROD activity. Therefore a microwell plate EROD method was developed that takes into account the effect of each sample on resorufin fluorescence. At least two mechanisms were involved in the decrease of the flu…
Tolerance of whitefish (Coregonus lavaretus) early life stages to manganese sulfate is affected by the parents
European whitefish (Coregonus lavaretus) embryos and larvae were exposed to 6 different manganese sulfate (MnSO4) concentrations from fertilization to the 3-d-old larvae. The fertilization success, offspring survival, larval growth, yolk consumption, embryonic and larval Mn tissue concentrations and transcript levels of detoxification-related genes were measured in the long-term incubation. Full factorial breeding design (4 females x 2 males) enabled examining the significance of both female and male effects, and female-male interactions in conjunction with the MnSO4 exposure on the observed endpoints. The MnSO4 exposure reduced the survival of the whitefish early life stages. Also the offs…
Changes in Cardiac Proteome and Metabolome Following Exposure to the Pahs Retene and Fluoranthene and Their Mixture in Developing Rainbow Trout Alevins
Exposure to polycyclic aromatic hydrocarbons (PAHs) is known to affect developing organisms. Utilization of different omics-based technologies and approaches could therefore provide a base for the discovery of novel mechanisms of PAH induced development of toxicity. To this aim, we investigated how exposure towards two PAHs with different toxicity mechanisms: retene (an aryl hydrocarbon receptor 2 (Ahr2) agonist), and fluoranthene (a weak Ahr2 agonist and cytochrome P450 inhibitor (Cyp1a)), either alone or as a mixture, affected the cardiac proteome and metabolome in newly hatched rainbow trout alevins (Oncorhynchus mykiss). In total, we identified 65 and 82 differently expressed proteins (…
Alterations in Daphnia magna exposed to enniatin B and beauvericin provide additional value as environmental indicators
Mycotoxins beauvericin (BEA) and enniatin B (ENN B) affect negatively several systems and demand more studies as the mechanisms are still unclear. The simultaneous presence of contaminants in the environment manifests consequences of exposure for both animals and flora. Daphnia magna is considered an ideal invertebrate to detect effects of toxic compounds and environmental alterations. In this study, the potential toxicity and the basic mechanism of BEA and ENN B individually and combined were studied in D. magna. Acute and delayed toxicity were evaluated, and transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), reproduction, and oxidative stress (vtg-S…
UV-B exposure causes DNA damage and changes in protein expression in northern pike (Esox lucius) posthatched embryos.
The ongoing anthropogenically caused ozone depletion and climate change has increased the amount of biologically harmful UV-B radiation, which is detrimental to fish in embryonal stages. The effects of UV-B radiation on the levels and locations of DNA damage manifested as cyclobutane pyrimidine dimers (CPDs), heat shock protein 70 (HSP70) and p53 protein in newly hatched embryos of pike were examined. Pike larvae were exposed in the laboratory to current and enhanced doses of UV-B radiation. UV-B exposure caused the formation of CPDs in a fluence rate-dependent manner, and the CPDs were found deeper in the tissues with increasing fluence rates. UV-B radiation induced HSP70 in epidermis, and…
Do laboratory exposures represent field exposures? Effects of sediments contaminated by wood industry on yolk-sac fry of rainbow trout (Oncorhynchus mykiss)
Purpose. Risk assessment of contaminated sediments is routinely based on laboratory exposures. The purpose of this work was to study if sediments contaminated by the chemical wood industry cause developmental defects in fish fry and how well a laboratory exposure correlates with a field exposure. Materials and methods. Newly hatched yolk-sac fry of rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory and in situ. In the laboratory, the fish were placed in contact with either clean or contaminated sediment in aquaria. In the field, half of the fish were placed in contact with the lake sediment and the other half were similarly caged 2 m above it, to discern the effects of the s…
Partitioning of nanoparticle-originated dissolved silver in natural and artificial sediments
Sediments are believed to be a major sink for silver nanoparticles (AgNPs) in the aquatic environment, but there is a lack of knowledge about the environmental effects and behavior of AgNPs in sediments. The release of highly toxic Ag+ through dissolution of AgNPs is one mechanism leading to toxic effects in sediments. We applied an ultrasound-assisted sequential extraction method to evaluate the dissolution of AgNPs and to study the partitioning of dissolved Ag in sediments. Silver was spiked into artificial and 2 natural sediments (Lake Hoytiainen sediment and Lake Kuorinka sediment) as silver nitrate (AgNO3 ), uncoated AgNPs, or polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs). In addition,…
Multixenobiotic resistance efflux activity in Daphnia magna and Lumbriculus variegatus
Multixenobiotic resistance is a phenomenon in which ATP-binding cassette (ABC) family proteins transfer harmful compounds out of cells. Daphnia magna and Lumbriculus variegatus are model species in aquatic ecotoxicology, but the presence and activity of ABC proteins have not been well described in these species. The aim of this work was to study the presence, activity, and inhibition of ABC transport proteins in D. magna and L. variegatus. The presence of abcb1 and abcc transcripts in 8-9-day-old D. magna was investigated by qRT-PCR. The activity of MXR in D. magna and L. variegatus was explored by influx of the fluorescent ABC protein substrates rhodamine B and calcein-AM, with and without…
Fluence rate or cumulative dose? : Vulnerability of larval northern pike (Esox lucius) to ultraviolet radiation
Newly hatched larvae of northern pike were exposed in the laboratory to four fluence rates of ultraviolet radiation (UVR; 290–400 nm) over three different time periods, resulting in total doses ranging from 3.0 ± 0.2 to 63.0 ± 4.4 kJ·m−2. Mortality and behavior of the larvae were followed for 8–12 days, and growth measured at the end of the experiment. Also, the principle of reciprocity—that the UVR-induced mortality depends on the cumulative dose, independent of fluence rate—was tested. Fluence rates higher than 1480 ± 150 mW·m−2 caused mortality and growth retardation. The highest fluence rate (3040 ± 210 mW·m−2) caused 100% mortality in 5 days. All fluence rates caused behavioral disorde…
Polycyclic Aromatic Hydrocarbons Phenanthrene and Retene Modify the Action Potential via Multiple Ion Currents in Rainbow Trout Oncorhynchus mykiss Cardiac Myocytes.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in aqueous environments. They affect cardiovascular development and function in fishes. The 3-ring PAH phenanthrene has recently been shown to impair cardiac excitation-contraction coupling by inhibiting Ca2+ and K+ currents in marine warm-water scombrid fishes. To see if similar events take place in a boreal freshwater fish, we studied whether the PAHs phenanthrene and retene (an alkylated phenanthrene) modify the action potential (AP) via effects on Na+ (INa ), Ca2+ (ICaL ), or K+ (IKr , IK1 ) currents in the ventricular myocytes of the rainbow trout (Oncorhynchus mykiss) heart. Electrophysiological characteristics of myo…
Responses to ultraviolet radiation in larval pike, Esox lucius, of two origins and ages
Ultraviolet radiation (UVR)-induced mortality and behavioural disorder were studied in larval northern pike of two origins and ages. Newly hatched larvae of two differently coloured populations and six-day-old larvae of one population were exposed to four fluence rates of UVR, resulting in total doses from 11.5 to 63 kJ m–2, and monitored for mortality and behaviour. The rate of mortality and the severity of behavioural disorder differed by origin and age of the animals, but the effect was fluence-rate dependent. Total melanin concentration of newly hatched larvae was measured to assess if sensitivity to UVR correlated with pigmentation, but no differences in melanin concentration between l…
Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 1 – Transcriptomics
Polycyclic aromatic hydrocarbons (PAHs) are contaminants of concern that impact every sphere of the environment. Despite several decades of research, their mechanisms of toxicity are still poorly understood. This study explores the mechanisms of cardiotoxicity of the three widespread model PAHs retene, pyrene and phenanthrene in the rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to each individual compound at sublethal doses causing no significant increase in the prevalence of deformities. Changes in the cardiac transcriptome were assessed after 1, 3, 7 and 14 days of exposure using custom Salmo salar microarrays. The highest number of differentiall…
Interacting effects of simulated eutrophication, temperature increase, and microplastic exposure on Daphnia
The effects of multiple stressors are difficult to separate in field studies, and their interactions may be hard to predict if studied in isolation. We studied the effects of decreasing food quality (increase in cyanobacteria from 5 to 95% simulating eutrophication), temperature increase (by 3 °C), and microplastic exposure (1% of the diet) on survival, size, reproduction, and fatty acid composition of the model freshwater cladoceran Daphnia magna. We found that food quality was the major driver of Daphnia responses. When the amount of cyanobacteria increased from 5 to 95% of the diet, there was a drastic decrease in Daphnia survival (from 81 ± 15% to 24 ± 21%), juvenile size (from 1.8 ± 0.…
Toxicity of biomining effluents to Daphnia magna: Acute toxicity and transcriptomic biomarkers
Increasing metal consumption is driving the introduction of new techniques such as biomining to exploit low grade ores. The biomining impacts notably aquatic ecosystems, yet, the applicability of ecotoxicological tests to study the complex mixture effects of mining waters is insufficiently understood. The aim of the present work was to test if transcriptomic biomarkers are suitable and sensitive for the ecotoxicity assessment of biomining affected waters. The study site had been affected by a multimetal biomine, and the studied water samples formed a concentration gradient of contamination downstream from the biomining site. Cadmium and nickel were used as positive controls in the toxicity …
Tolerance of Whitefish (Coregonus Lavaretus) Early Life Stages to Manganese Sulfate is Affected by the Parents
European whitefish (Coregonus lavaretus) embryos and larvae were exposed to 6 different manganese sulfate (MnSO4) concentrations from fertilization to the 3-d-old larvae. The fertilization success, offspring survival, larval growth, yolk consumption, embryonic and larval Mn tissue concentrations, and transcript levels of detoxification-related genes were measured in the long-term incubation. A full factorial breeding design (4 females × 2 males) allowed examination of the significance of both female and male effects, as well as female–male interactions in conjunction with the MnSO4 exposure in terms of the observed endpoints. The MnSO4 exposure reduced the survival of the whitefish early li…
Toxicity of biomining effluents to Daphnia magna : Acute toxicity and transcriptomic biomarkers
Increasing metal consumption is driving the introduction of new techniques such as biomining to exploit low grade ores. The biomining impacts notably aquatic ecosystems, yet, the applicability of ecotoxicological tests to study the complex mixture effects of mining waters is insufficiently understood. The aim of the present work was to test if transcriptomic biomarkers are suitable and sensitive for the ecotoxicity assessment of biomining affected waters. The study site had been affected by a multimetal biomine, and the studied water samples formed a concentration gradient of contamination downstream from the biomining site. Cadmium and nickel were used as positive controls in the toxicity …
Recovery of Historically Contaminated Watercourse Polluted by the Chemical Wood Industry: EROD Activity in Fish as Biomarker
Despite outstanding process alterations over decades, pulp- and paper-mill-contaminated sediments and continuing exposure by the effluents may still have effects on biota. In this study, ecotoxicological impacts in the boreal watercourse were analyzed by measuring ethoxyresorufin-O-deethylase (EROD) induction from wild fish populations and from experimentally exposed fish. In order to assess the role of sediment-borne chemicals, juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to the surface sediments of Lake Vatianjarvi and Southern Lake Saimaa, both watercourses impacted by the chemical wood industry for approximately a century. Hepatic EROD activity was also me…
Salmo trutta is more sensitive than Oncorhynchus mykiss to early-life stage exposure to retene
Salmonids are known to be among the most sensitive fish to dioxin-like compounds (DLCs), but very little is known about the sensitivity of the brown trout (Salmo trutta), which has declined and is endangered in several countries of Europe and Western Asia. We investigated the sensitivity of brown trout larvae to a widespread dioxin-like PAH, retene (3.2 to 320 μg.L−1), compared to the larvae of a salmonid commonly used in toxicology studies, the rainbow trout (Oncorhynchus mykiss). Mortality, growth, cyp1a induction and the occurrence of deformities were measured after 15 days of exposure. Brown trout larvae showed a significantly higher mortality at 320 μg.L−1 compared to rainbow trout lar…