Xenobiotic metabolizing enzymes in rainbow trout exposed to river water

• 1.1. Juvenile rainbow trout were exposed to river water in a flow-through system. After 15 days of exposure, hepatic biotransformation activities and related parameters were measured and compared to those of the control group organisms that were maintained in tap water under identical experimental conditions.
• 2.2. Liver somatic index (LSI), microsomal protein and cytochrome P-450 contents, benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD) and UDP glucuronyl transferase activities were not significantly affected.
• 3.3. Aminopyrine-N-demethylase (APD) activity showed a slight yet significant increase in exposed trout.

     

Xenobiotic metabolizing enzymes in rainbow trout exposed to river water.

1. Juvenile rainbow trout were exposed to river water in a flow-through system. After 15 days of exposure, hepatic biotransformation activities and related parameters were measured and compared to those of the control group organisms that were maintained in tap water under identical experimental conditions. 2. Liver somatic index (LSI), microsomal protein and cytochrome P-450 contents, benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD) and UDP glucuronyl transferase activities were not significantly affected. 3. Aminopyrine-N-demethylase (APD) activity showed a slight yet significant increase in exposed trout.     

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Microbial community structure and operational performance of a fluidized bed biofilm reactor treating oil sands process-affected water

This study evaluated the treatment of oil sands process-affected water (OSPW) using a fluidized bed biofilm reactor (FBBR) with granular activated carbon (GAC) as support media. The bioreactor was operated for 120 days at different organic and hydraulic loading rates. The combined GAC adsorption and biodegradation process removed 51% of chemical oxygen demand (COD), 56% of acid-extractable fraction (AEF) and 96% of classical naphthenic acids (NAs) under optimized operational conditions. Bioreactor treatment efficiencies were dependent on the organic loading rate (OLR), and to a lower degree, on the hydraulic loading rate (HLR). Further ultra performance liquid chromatography/high resolution mass spectroscopy (UPLC/HRMS) analysis showed that the removal of classical NAs increased as the carbon number increased. Compared with planktonic bacterial community in OSPW, more diverse microbial structures were found in biofilms colonized on the surface of GAC after 120-day treatment, with various carbon degraders namely Polaromonas jejuensis, Algoriphagus sp., Chelatococcus sp. and Methylobacterium fujisawaense in the GAC-biofilm reactor. The results of this study, therefore, showed that the GAC-biofilm seems to be a promising biological treatment method for OSPW remediation.     

Cellular responses in the skin of rainbow trout (Oncorhynchus mykiss) exposed to Rhine water

Trout, Oncorhynchus mykiss , were exposed to water from the Rhine for 24 days and their skin examined by light and electron microscopy. Relative to control fish mitotic figures were common and seen throughout the epidermis. Pavement cells in fish exposed to Rhine water contained significantly more secretory vesicles than control fish. Necrotic pavement cells were apparent after 24 h, and apoptotic cells from day 4 on. Mucous secretion was intense and the differentiation of mucous cells was stimulated. Some of these cells synthesized mucus of high electron density, probably of a serous composition. Leucocytes invaded the dermis and epidermis, and towards the end of the experiment many apoptotic and necrotic lymphocytes were found. In the dermis fibroblasts were abundant and actively producing collagen. Pigment containing cytoplasmic extensions of melanocytes penetrated into the epidermis. After 14 and 24 days of exposure many pigment cells, melanocytes, iridocytes and xanthocytes became apoptotic. Most of these changes are known from fish exposed to heavy metals, acid water or other stressful treatments, indicating that exposure to Rhine water is a stressful experience for trout.     

Differential responses to copper in rainbow trout (Oncorhynchus mykiss) acclimated to sea water and brackish water

Rainbow trout, Oncorhynchus mykiss, acclimated to 33% sea water (12 mg·ml^-1 salinity) experienced significant (10 meq·1^-1) increases in plasma [Na⁺] and [Cl^-] within 5 h of exposure to 6.3 mol copper·1^-1 indicating severe impairment of branchial ionoregulatory capacity. All plasma ion levels subsequently stabilised once the transbranchial [Na⁺] gradient was reduced to zero. The similar ionic strength of the external medium and their body fluids appeared to protect trout maintained in 33% sea water from further ionoregulatory stress and any secondary physiological disturbances during exposure to copper. Despite three- and fourfold greater transbranchial [Na⁺] and [Cl^-] gradients, trout acclimated to full-strength sea water (35 mg·ml^-1 salinity) suffered no major changes in plasma Na⁺, Cl^-, K⁺, or Ca²⁺, blood gases or haematology during 24 h exposure to 6.3 mol copper·1^-1. This reduction in toxicity in full strength sea water cannot be explained by differences in copper speciation. We suggest that during acute exposure to waterborne copper, active NaCl extrusion is unaffected due to the basolateral location of the gill Na⁺/K⁺-ATPase, but that ionoregulatory disturbances can occur due to gill permeability changes secondary to the displacement of surface-bound Ca²⁺. However, in full strength sea water the three-fold higher ambient [Ca²⁺] and [Mg²⁺] appear to be sufficient to prevent any detrimental permeability changes in the presence of 6.3 mol copper·1^-1. Plasma [NH ₊ ⁴ ] and [HCO _- ³ ] were both significantly elevated during exposure to copper, indicating that some aspects of gill ion transport (specifically the apical Na⁺/NH ₊ ⁴ and Cl^-/HCO _- ³ exchanges involved in acid/base regulation and nitrogenous waste excretion) are vulnerable to inhibition in the presence of waterborne copper.Abbreviations C _aO₂ arterial oxygen content - Hb haemoglobin - Hct haematocrit - MABP mean arterial blood pressure - MCHC mean cell haemoglobin content - MO₂ rate of oxygen consumption - P _a CO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - S salinity - SW sea water - T _Amm total ammonia (=NH₃+NH ₊ ⁴ ) - T _{CO 2} total carbon dioxide - TEP transepithelial potential - TOC total organic carbon - %Hb-O₂ percentage of haemoglobin saturated with oxygen     

Biochemical responses in gills of rainbow trout exposed to propiconazole

The aim of this study is to investigate the toxic effect of PCZ, a triazole fungicide commonly present in surface and ground water, on the ROS defense system and Na₊-K₊-ATPase in gills of rainbow trout exposed to sublethal concentrations (0.2, 50 and 500 μg L⁻¹) for 7, 20 and 30 days. After prolonged exposure of PCZ at higher test concentrations (50 and 500 μg L⁻¹), oxidative stress was apparent as reflected by the significant higher ROS levels in fish gill, as well as the significant inhibition of SOD and CAT activities. In addition, Na₊-K₊-ATPase activities were significantly lower than those of the control with increasing PCZ concentration and prolonged exposure period. The results of this study indicate that chronic exposure to PCZ has altered multiple physiological indices in fish gill; however, before these parameters are used as unique biomarkers for monitoring residual pharmaceuticals in aquatic environments, more detailed laboratory experiments need to be performed.     

Impact of ozonation pre-treatment of oil sands process-affected water on the operational performance of a GAC-fluidized bed biofilm reactor

Treatment of oil sands process-affected water (OSPW) using biodegradation has the potential to be an environmentally sound approach for tailings water reclamation. This process is both economical and efficient, however, the recalcitrance of some OSPW constituents, such as naphthenic acids (NAs), require the pre-treatment of raw OSPW to improve its biodegradability. This study evaluated the treatment of OSPW using ozonation followed by fluidized bed biofilm reactor (FBBR) using granular activated carbon (GAC). Different organic and hydraulic loading rates were applied to investigate the performance of the bioreactor over 120 days. It was shown that ozonation improved the adsorption capacity of GAC for OSPW and improved biodegradation by reducing NAs cyclicity. Bioreactor treatment efficiencies were dependent on the organic loading rate (OLR), and to a lesser degree, the hydraulic loading rate (HLR). The combined ozonation, GAC adsorption, and biodegradation process removed 62 % of chemical oxygen demand (COD), 88 % of acid-extractable fraction (AEF) and 99.9 % of NAs under optimized operational conditions. Compared with a planktonic bacterial community in raw and ozonated OSPW, more diverse microbial communities were found in biofilms colonized on the surface of GAC after 120 days, with various carbon degraders found in the bioreactor including Burkholderia multivorans, Polaromonas jejuensis and Roseomonas sp.     

Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization

Naphthenic acids are a complex family of naturally occurring cyclic and acyclic carboxylic acids that are present in the acidic fraction of petroleum. Naphthenic acids are acutely toxic to aquatic organisms. Previous studies showed that wetland sediments exposed to oil sands process water containing naphthenic acids had higher rates of naphthenic acid degradation in vitro compared with unexposed wetlands. In this study we compare the microbial community structures in sediments from wetlands exposed to different amounts of oil sands process water using BIOLOG, phospholipid fatty acid analysis and denaturing gradient gel electrophoresis of total bacterial DNA. Community profiles were compared using cluster analysis. BIOLOG profiles were primarily influenced by seasonal trends rather than naphthenic acids content. In contrast, phospholipid fatty acid analysis comparisons clustered communities that had higher levels of residual oil, although this association was not strong. In contrast, cluster diagrams produced from the denaturing gradient gel electrophoresis data clearly separated bacterial communities according to naphthenic acids concentrations, indicating that naphthenic acids content was a major influence on the composition of the bacterial community. In addition, denaturing gradient gel electrophoresis profiles indicated that naphthenic acids-exposed bacterial communities were homogeneous on a scale of meters, whereas unexposed (off-site) wetlands were less homogeneous.     

Oxidative stress responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to pro-oxidants and a complex environmental sample

A wide range of pollutants in the aquatic environment have the capacity to induce toxic effects expressed as cellular oxidative stress. In the current study, the potential of an in vitro toxicity testing system was therefore investigated using rainbow trout (Oncorhynchus mykiss) hepatocytes to assess different endpoints of oxidative stress. The pro-oxidants CuSO₄ and paraquat were used as models for comparison to a complex environmental sample. Results following 6, 24, 48 and 96 h exposure to different concentrations of these substances show cellular effects on intracellular ROS formation, glutathione levels and redox status, expression of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, γ-glutamyl-cysteine synthetase (GCS) and thioredoxin, as well as cytotoxicity parameters. The most consistent effects (maximum values within brackets), observed in dose and time parameters for both model compounds and environmental sample, were the depletion of total glutathione (9.4% of control), induced levels of oxidized glutathione (695% of control), and gene expression regulation depicted relative to the control gene beta-actin of GCS mRNA (239% of control) and catalase (29% of control). In conclusion, the responses on several antioxidant defence system parameters demonstrated the validity of the in vitro toxicity testing system. Not only could multiple effects be detected at sub-lethal exposure concentrations, but these effects also gave valuable insight to the toxic mechanisms at the molecular level.     

Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes

The mechanism underlying copper hepatotoxicity was investigated in primary cultures of rainbow trout hepatocytes maintained in Leibovitz-15 media. CuSO4 treatment (0, 25, 50, 100 and 200 microM) resulted in a dose-dependent elevation in heat shock protein 70 (hsp70) expression at 24 and 48 h post-exposure. There was no effect of copper (200 microM CuSO4) on hepatotoxicity at 24 h, whereas longer exposures (48 h) resulted in increased lactate dehydrogenase (LDH) leakage and apoptosis, demonstrated by fluorescence nuclear staining and DNA fragmentation. Vitamin C (1 mM), a free radical scavenger, inhibited this copper-induced apoptosis implying a role for reactive oxygen species in copper toxicity. However, no parallel inhibition of either LDH leakage or hsp70 protein expression was observed with vitamin C suggesting that at least two independent mechanisms are involved in the cellular response to copper. Also, copper exposed (24 h) cells were unable to mount an hsp70 response to a standardized heat shock (+15 degrees C for 1 h), even in the presence of vitamin C. Together, these results suggest that hepatotoxicity of copper includes impairment of hsp70 response to subsequent stressors and/or signals, which is crucial for protecting cells from proteotoxicity.     

Morphological organ alterations and infectious diseases in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss exposed to polluted river water

Poor water quality is discussed as a major factor causing a decline of brown trout populations in Swiss rivers. For our study we have chosen a river in the Swiss midlands, where the brown trout population has decreased dramatically during the last 10 yr and where feral fish have shown distinctive pathological alterations. The objective of our study was to investigate whether river water may be responsible for impaired fish health leading to an increased mortality in the river. In an active monitoring program, groups of brown and rainbow trout were exposed to polluted river water for 24 mo. Fish held in tap water served as a reference. Mortality, macroscopic and histopathologic changes, and infectious agents were investigated. Compared with the reference group, high mortality rates and severe pathological alterations of the inner organs were observed in fish held in river water. Especially gills, liver and kidney of these fish showed significantly higher changes than fish from tap water. These changes were dominated by degenerative and inflammatory reactions. Additionally, several infectious agents were diagnosed in fish exposed to river water. The most important findings were furunculosis and proliferative kidney disease. Brown trout seemed to be more sensitive than rainbow trout to environmental stress and infectious agents.     

Treatment of raw and ozonated oil sands process-affected water under decoupled denitrifying anoxic and nitrifying aerobic conditions: a comparative study

Batch experiments were performed to evaluate biodegradation of raw and ozonated oil sands process-affected water (OSPW) under denitrifying anoxic and nitrifying aerobic conditions for 33 days. The results showed both the anoxic and aerobic conditions are effective in degrading OSPW classical and oxidized naphthenic acids (NAs) with the aerobic conditions demonstrating higher removal efficiency. The reactors under nitrifying aerobic condition reduced the total classical NAs of raw OSPW by 69.1 %, with better efficiency for species of higher hydrophobicity. Compared with conventional aerobic reactor, nitrifying aerobic condition substantially shortened the NA degradation half-life to 16 days. The mild-dose ozonation remarkably accelerated the subsequent aerobic biodegradation of classical NAs within the first 14 days, especially for those with long carbon chains. Moreover, the ozone pretreatment enhanced the biological removal of OSPW classical NAs by leaving a considerably lower final residual concentration of 10.4 mg/L under anoxic conditions, and 5.7 mg/L under aerobic conditions. The combination of ozonation and nitrifying aerobic biodegradation removed total classical NAs by 76.5 % and total oxy-NAs (O3–O6) by 23.6 %. 454 Pyrosequencing revealed that microbial species capable of degrading recalcitrant hydrocarbons were dominant in all reactors. The most abundant genus in the raw and ozonated anoxic reactors was Thauera (~56 % in the raw OSPW anoxic reactor, and ~65 % in the ozonated OSPW anoxic reactor); whereas Rhodanobacter (~40 %) and Pseudomonas (~40 %) dominated the raw and ozonated aerobic reactors, respectively. Therefore, the combination of mild-dose ozone pretreatment and subsequent biological process could be a competent choice for OSPW treatment.