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Owing to their widespread use, pharmaceuticals have become important pollutants of the aquatic environment. Pharmaceuticals and parasites can affect the immunity, physiology and behaviour of target organisms, both singly and through interaction. In this study, we related pharmaceutical concentration, parasite infection and condition in brown trout (Salmo trutta m. fario) collected at localities upstream and downstream of a sewage treatment plant (STP). The sites were separated by a weir preventing fish migration. Pharmaceutical number and concentrations differed significantly between sites. Antibiotics and antidepressants were found at the highest concentrations at both localities. Though fish condition and overall parasite abundance did not differ between sites, there was a significant difference in parasite community composition. A higher abundance of monogenean ectoparasites, along with a lower abundance of endoparasites, was observed at the downstream locality. The adverse effect of pharmaceutical load on ecto- and endoparasitic species on brown trout was further confirmed at the individual level using multivariate analysis. Both the number and concentration of pharmaceuticals in fish tissues reflected their high incidence in the environment, confirming the ability of trout to bioaccumulate pharmaceuticals. This could have important consequences on fish health, particularly in small streams where STP effluent represents a high proportion of stream flow.

Aquaculture is increasing at the global scale, and beneficial reuse of wastewater is becoming crucial in some regions. Here we selected a unique tertiary treatment system for study over a one-year period. This experimental ecosystem-based approach to effluent management included a treated wastewater pond (TWP), which receives 100% effluent from a wastewater treatment plant, and an aquaculture pond (AP) that receives treated water from the TWP for fish production. We examined the fate of a wide range of pharmaceutically active compounds (PhACs) in this TWP-AP system and a control pond fed by river water using traditional grab sampling and passive samplers. We then employed probabilistic approaches to examine exposure hazards. Telmisartan, carbamazepine, diclofenac and venlafaxine, exceeded ecotoxicological predicted no effect concentrations in influent wastewater to the TWP, but these water quality hazards were consistently reduced following treatment in the TWP-AP system. In addition, both grab and passive sampling approaches resulted in similar occurrence patterns of studied compounds, which highlights the potential of POCIS use for water monitoring. Based on the approach taken here, the TWP-AP system appears useful as a tertiary treatment step to reduce PhACs and decrease ecotoxicological and antibiotic resistance water quality hazards prior to beneficial reuse in aquaculture.

Monitoring the contamination level in aquatic environments and assessing the impact on aquatic life occurs throughout the world. In the present study, an approach based on a combination of biomarkers and the distribution of various industrial and municipal pollutants was used to investigate the effect of aquatic environmental contamination on fish. Monitoring was performed in ten rivers in the Czech Republic (Berounka, Dyje, Elbe, Lužnice, Odra, Ohře, Otava, Sazava, Svratka, and Vltava rivers, with one or two locations in each river) at the same sites that were regularly monitored within the Czech National Monitoring Program in 2007–2011. Health status, hepatic ethoxyresorufin-O-deethylase (EROD) activity, total cytochrome P450 content, and the plasma vitellogenin concentration were assessed in wild chub (Squalius cephalus) males caught at the monitored sites. The contamination level was the highest in the Svratka River downstream of Brno. Among all measured persistent organic pollutants (POPs), polychlorinated biphenyls and dichlorodiphenyltrichloroethane and its metabolites were the major contributors of POPs in fish muscle. Elbe, Odra, and Svratka rivers were identified as the most polluted. Fish from these locations showed reduced gonad size, increased vitellogenin concentration in male plasma, EROD, and total cytochrome P450 content. These biomarkers can be used for future environmental monitoring assessments. Overall, this study improves our understanding of the relationship between human activities and pollutant loads and further contributes to the decision to support local watershed managers to protect water quality in this region.

Sewage treatment plants (STPs) are one of the major source of pharmaceuticals and personal care products in the aquatic environment. Generally, the effects of individual chemicals on fish are studied under laboratory conditions, which leads to results that are potentially not realistic regarding the effects of these chemicals under environmental conditions. Therefore, in this study, common carps were held in exposed pond that receive water from STP effluents for 360 days under natural conditions. Elimination of xenobiotics starts in the fish intestine, in which the microbial community strongly influences its function. Moreover, the fish intestine functions as crucial organ for absorbing lipids and fatty acids (FA), with consequent transport to the liver where their metabolism occurs. The liver is the primary organ performing xenobiotic metabolism in fish, and therefore, the presence of pollutants may interact with the metabolism of FA. The catalytic activity of CYP1A and CYP3A-like enzymes, their gene expression, FA composition and intestinal microbiome consortia were measured. The catalytic activity of enzymes and their gene and protein expression, were induced in hepatic and intestinal tissues of fish from the exposed pond. Also, fish from the exposed pond had different compositions of FA than those from the control pond: concentration of 18:1 n-9 and 18:2 n-6 were significantly elevated and the longer chain n-3 FA 20:5 n-3, 22:5 n-3 and 22:6 n-3 were significantly lowered. There were clear differences among microbiome consortia in fish intestines across control and exposed groups. Microbiome taxa measured in exposed fish were also associated with those found in STP activated sludge. This study reveals that treated STP water, which is assumed to be clean, affected measured biomarkers in common carp.

Concerns about the effect of sewage treatment plant (STP) effluent on the health of freshwater ecosystems have increased. In this study, a unique approach was designed to show the effect of an STP effluent-dominated stream on native wild brown trout (Salmo trutta L.) exposed under fully natural conditions. Zivny stream is located in South Bohemia, Czech Republic. The downstream site of Zivny stream is an STP-affected site, which receives 25% of its water from Prachatice STP effluent. Upstream, however, is a minimally polluted water site and it is considered to be the control site. Native fish were collected from the upstream site, tagged, and distributed to both upstream and downstream sites. After 30, 90, and 180 days, fish were recaptured from both sites to determine whether the downstream site of the Zivny stream is associated with the effects of environmental pollution. Several biomarkers indicating the oxidative stress and antioxidant enzyme activities, cytochrome P450 activity, xenoestrogenic effects, bacterial composition, and lipid composition were investigated. Additionally, polar chemical contaminants (pharmaceuticals and personal care products (PPCPs)) were quantified using polar organic chemical integrative samplers (POCIS). Fifty-three PPCPs were detected in the downstream site; 36 of those were constantly present during the 180-day investigation period. Elevated hepatic 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD) (after 90 days) and blood plasma vitellogenin concentrations in males were detected in fish downstream of the STP effluent during all sampling events. An increase in the fishes' total fat content was also observed, but with low levels of ω-3 fatty acid in muscle tissue. Two bacterial taxa related to activated sludge were found in the intestines of fish from downstream. Our results show that Prachatice STP is a major source of PPCPs in the Zivny stream, which has biological consequences on fish physiology.

The fate of fourteen target perfluoroalkyl substances (PFASs) are described within a small stream affected by a sewage treatment plant (STP) effluent. Concentrations of target PFASs in samples of water, benthic macroinvertebrates and brown trout (Salmo trutta) are presented. Two hundred brown trout individuals originating from clean sites within the same stream were tagged and stocked into an experimental site affected by the STP's effluent. As a passive sampling approach, polar organic chemical integrative samplers (POCIS) were deployed in the water to reveal the water-macroinvertebrates-fish biotransformation processes of PFASs. Bioconcentration/bioaccumulation of target compounds was monitored one, three, and six months after stocking. Twelve of the fourteen target PFASs were found in concentration above the LOQ in at least one of the studied matrices. The compound pattern varied significantly between both the studied species and water samples. Concerning the accumulation of PFASs in fish, the highest concentrations were found in the liver of individuals sampled after three months of exposure. These concentrations rapidly decreased after six months although the water concentrations were slightly increasing during experiment.

Intensive aquaculture is an important and fast-growing food production industry generating significant amounts of nutrient-rich sludge, which represents a potential environmental threat. Vermicomposting aquacultural sludge has been suggested, but remained poorly understood – only survival and growth of initial earthworm stocks have been assessed so far. The present study provides a comprehensive evaluation of the production system, examining vermicomposting of three types of sludge each at four inclusion levels and the possibility of further utilising end-products (vermicomposts and earthworms). Through an 18-week experiment, high survival of initial earthworm stocks, exceeding 90% among treatments up to week 6, was documented. Higher inclusion levels and sludge types richer in nutrients positively influenced individual weight of initial stocks and their reproduction indices (cocoon and juvenile production). The most progressive treatments sustained >300 juveniles in experimental incubators containing 200 g dw of initial substrates. Original sludge and final vermicomposts were found suitable for use in agriculture, complying with limits for heavy metals given in the most usually applied regulations. In relation to the heavy metals, earthworms were found to be a generally safe feed for fish. Only arsenic concentrations may occasionally exceed given limits. Still, observed concentrations are considered safe, presuming arsenic presence primarily in organic forms having largely reduced toxicity. Vermicomposting is recommended as a clean and sustainable technology transforming aquaculture sludge into highly valuable vermicompost and earthworm biomass.

Although pharmaceuticals are frequently studied contaminants, their fate in the environment is still not completely clear. During a one year study, a complex approach including water, sediment and fish sampling was used to describe the behaviour of pharmaceuticals and their metabolites (PTMs) in the environment. Eighteen pharmaceuticals and seven of their metabolites were determined in a pond used for the tertiary treatment of wastewater effluent. A liquid chromatography-tandem mass spectrometry method was applied to determine the PTMs concentrations in all matrices. Seasonal variations in concentrations were evaluated. The partitioning of contaminants between pond compartments was estimated by means of solid water distribution coefficients (Kd) and bioaccumulation factors (BAF) for the livers of fish. Kd values were almost stable throughout the year, which may be a sign of the continuous transport of PTMs between water and sediment under the experimental conditions. Almost all of the studied compounds, with exception of sertraline (BAF of 6200), were found to not be bioaccumulative in fish livers. The pond removal efficiency was calculated for all PTMs, and favourable conditions for natural pharmaceutical removal were proposed. Further aspects regarding fish pharmaceutical exposure need to be studied.

Reservoirs are known to alter temperature and flow regimes, shift nutrient cycles, reduce downstream species diversity and enable a predominantly upstream spread of non-native species. However, information about the seasonal dynamics of the spread of non-natives from a reservoir to its tributaries and the further consequences regarding the spatial distribution of native species is rare. We observed the occurrence of fish in the Vltava River and its tributaries (Elbe catchment area, central Europe) upstream of the Lipno Reservoir for five consecutive years. We radio-tagged two non-native and four native species. To detect assemblage spatial variability, we sampled sites in the study area by electrofishing twice per year (spring and autumn). We expected seasonal trends in non-native species appearance in upstream reservoir tributaries and, conversely, low motivation of native fishes to descend to the reservoir. By analysing nearly 3000 individuals of 21 species from the longitudinal profile of the study area, we observed an effect of reservoir distance on the native species ratio in the upper Vltava catchment area, i.e., an increase in distance increased the native species proportion, and the opposite was observed for non-native species. Analyses of 3798 tracking positions of 193 tagged individuals showed massive spring dispersal of non-native species from the reservoir to the main tributary, the Vltava River, and their return to the reservoir for wintering. Their upstream movement positively correlated with an increase in flow rate. Native Salmo trutta showed a specific shift from the Vltava River to smaller streams during the summer, when the presence of non-native species in the Vltava River was most significant. These findings indicate that non-native species repeatedly spread from the reservoir to the upstream river stretch and its tributaries and potentially compete with native species for resources.

With increasing demand for aquaculture products, water reuse is likely to increase for aquaculture operations around the world. Herein, wastewater stabilization ponds (WSP) represents low cost and sustainable treatment technologies to reduce nutrients and various contaminants of emerging concern from effluent. In the present study, we examined bioaccumulation of selected pharmaceuticals from several therapeutic classes by two important fish species in aquaculture with different feeding preferences (Cyprinus carpio and Sander lucioperca) and their common prey to test whether species specific accumulation occurs. Forty and nineteen from 66 selected pharmaceuticals and their metabolites were positively found in water and sediment samples, respectively from the representative WSP. After a six-month study, which corresponds to aquaculture operations, fourteen pharmaceuticals and their metabolites were detected (at a frequency of higher than 50% of samples) in at least one fish tissue collected from the WSP. We observed striking differences for species and organ specific BAFs among study compounds. Though muscle tissues consistently accumulated lower levels of the target analytes, several substances were elevated in brain, liver and kidney tissues (e.g., sertraline) of both species. Low residual concentrations of these target analytes in aquaculture products (fish fillets) suggest WSPs are promising to support the water-food nexus in aquaculture.

The aim of this study was to characterize biomarker responses, haematological profiles, structural changes and uptake in juvenile rainbow trout exposed to clotrimazole (CLO) at three concentrations (0.01 - [lowest environmentally relevant concentration], 1.0 [highest environmentally relevant concentration] and 10 μg L(-1)) in a semi-static system over a period of 42 days. Antioxidant defence enzymes, which responded to CLO exposure, changed the oxidative stress status of cells, but no differences were observed in lipid peroxidation. Clotrimazole triggered a biphasic response of CYP3A-like activity in liver microsomes, which may indicate a detoxification process in the liver. Histopathological alterations were most pronounced in kidneys and testes in the group exposed to 10 μg L(-1). Structural changes in the kidney included tubulonephrosis and hyaline droplet degeneration in the tubular epithelial cells. The relative proportions of germ cells in testes were changed: The number of spermatozoa was reduced, and the spermatogonia and spermatocytes were increased. The highest CLO concentration was detected in fish liver (3710 ng per gram wet tissue) and kidney (4280 ng per gram wet tissue). Depuration half-life was estimated to be 72, 159, and 682 h in liver, muscle, and kidney, respectively. Taken together, these results provide valuable toxicological data on the effects of CLO on aquatic non-target organisms, which could be useful for further understanding of the potential risks in the real aquatic environment.

Stream ecosystems face ever-increasing pressures by the presence of emergent contaminants, such as, personal care products. N, N-diethyl-3-methylbenzamide (DEET) is a synthetic insect repellent that is being found in surface waters environments in concentrations up to 33.4 μg/L. Information concerning DEET's toxicity in the aquatic environment is still limited and focused only on its acute effects on model species. Our main objective was to assess the effects of DEET exposure to a caddisfly non-target species using sub-lethal endpoints. For that, we chose Sericostoma vittatum, an important shredder in Portuguese freshwaters that has been already used in different ecotoxicological assays. Besides acute tests, S. vittatum were exposed during 6 days to a gradient of DEET concentrations (8, 18 and 40.5 mg/L) to assess effects on feeding behaviour and biochemical responses, such as, lipid peroxidation levels (LPO), catalase and acetylcholinesterase (AChE) activities, and also assess effects on energy reserves and consumption. Acute tests revealed a 48 h-LC50 of 80.12 mg/L and DEET exposure caused feeding inhibition with a LOEC of 36.80 mg/L. Concerning the biochemical responses, DEET caused no effects in LPO nor on catalase activity. A non-significant decrease in AChE activity was observed. Regarding energetic reserves, exposure to DEET caused a significant reduction in S. vittatum carbohydrates levels. These results add important information for the risk assessment of insect repellents in the aquatic environment and suggest that reported environmental concentrations of DEET are not toxic to non-target freshwater insects.

The concentrations of seven perfluoroalkyl substances (PFASs) were investigated in 36 European chub (Squalius cephalus) individuals from six localities in the Czech Republic. Chub muscle and liver tissue were analysed at all sampling sites. In addition, analyses of 16 target PFASs were performed in Polar Organic Chemical Integrative Samplers (POCISs) deployed in the water at the same sampling sites. We evaluated the possibility of using passive samplers as a standardized method for monitoring PFAS contamination in aquatic environments and the mutual relationships between determined concentrations. Only perfluorooctane sulphonate was above the LOQ in fish muscle samples and 52% of the analysed fish individuals exceeded the Environmental Quality Standard for water biota. Fish muscle concentration is also particularly important for risk assessment of fish consumers. The comparison of fish tissue results with published data showed the similarity of the Czech results with those found in Germany and France. However, fish liver analysis and the passive sampling approach resulted in different fish exposure scenarios. The total concentration of PFASs in fish liver tissue was strongly correlated with POCIS data, but pollutant patterns differed between these two matrices. The differences could be attributed to the metabolic activity of the living organism. In addition to providing a different view regarding the real PFAS cocktail to which the fish are exposed, POCISs fulfil the Three Rs strategy (replacement, reduction, and refinement) in animal testing.

Methamphetamine, mainly consumed as an illicit drug, is a potent addictive psychostimulant that has been detected in surface water at concentrations ranging from nanograms to micrograms per litre, especially in Middle and East Europe. The aim of this study was to expose brown trout (Salmo trutta fario) to environmental (1 μg L−1) and higher (50 μg L−1) concentrations of methamphetamine for 35 days with a four-day depuration phase to assess the possible negative effects on fish health. Degenerative liver and heart alterations, similar to those described in mammals, were observed at both concentrations, although at different intensities. Apoptotic changes in hepatocytes, revealed by activated caspase-3, were found in exposed fish. The parent compound and a metabolite (amphetamine) were detected in fish tissues in both concentration groups, in the order of kidney > liver > brain > muscle > plasma. Bioconcentration factors ranged from 0.13 to 80. A therapeutic plasma concentration was reached for both compounds in the high-concentration treatment. This study indicates that chronic environmental concentrations of methamphetamine can lead to health issues in aquatic organisms.

Environmental monitoring and surveillance studies of pharmaceuticals routinely examine occurrence of substances without current information on human consumption patterns. We selected 10 streams with diverse annual flows and differentially influenced by population densities to examine surface water occurrence and fish accumulation of select psychoactive medicines, for which consumption is increasing in the Czech Republic. We then tested whether passive sampling can provide a useful surrogate for exposure to these substances through grab sampling, body burdens of young of year fish, and tissue specific accumulation of these psychoactive contaminants. We identified a statistically significant (p 0.05) relationship between ambient grab samples and passive samplers in these streams when psychoactive contaminants were commonly quantitated by targeted liquid chromatography with tandem mass spectrometry, though we did not observe relationships between passive samplers and tissue specific pharmaceutical accumulation. We further observed smaller lotic systems with elevated contamination when municipal effluent discharges from more highly populated cities contributed a greater extent of instream flows. These findings identify the importance of understanding age and species specific differences in fish uptake, internal disposition, metabolism and elimination of psychoactive drugs across surface water quality gradients.

Aquatic organisms can be affected not only via polluted water but also via their food. In the present study, we examined bioaccumulation of seventy pharmaceuticals in two benthic organisms, Hydropsyche sp. and Erpobdella octoculata in a small stream affected by the effluent from a sewage treatment plant (STP) in Prachatice (South Bohemia region, Czech Republic). Furthermore, water samples from similar locations were analyzed for all seventy pharmaceuticals. In water samples from a control locality situated upstream of the STP, ten of the seventy pharmaceuticals were found with average total concentrations of 200 ng L−1. In water samples collected at STP-affected sites (downstream the STP′s effluent), twenty-nine, twenty-seven and twenty-nine pharmaceuticals were determined at average total concentrations of 2000, 2100 and 1700 ng L−1, respectively. Six of the seventy pharmaceuticals (azithromycin, citalopram, clarithromycin, clotrimazole, sertraline, and verapamil) were found in Hydropsyche. Four pharmaceuticals (clotrimazole, diclofenac, sertraline, and valsartan) were detected in Erpobdella. Using evaluation criterion bioconcentration factor (BCF) is higher than 2000 we can assign azithromycin and sertraline as bioaccumulative pharmaceuticals. Even pharmaceuticals present at low levels in water were found in benthic organisms at relatively high concentrations (up to 85 ng g−1 w.w. for azithromycin). Consequently, the uptake of pharmaceuticals via the food web could be an important exposure pathway for the wild fish population.

Seasonal changes in the concentration of 21 pharmaceuticals in a wastewater treatment plant (WWTP) in Ceske Budějovice were investigated over 12 months. The target compounds were 10 antibiotics, 4 antidepressants, 3 psychiatric drugs, 2 antihistamines and 2 lipid regulators. 272 Wastewater samples (136 influents and 136 effluents) were collected from March 2011 to February 2012 and analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry. All studied pharmaceuticals were frequently detected in both the influent and the effluent wastewater samples, except for meclozine, which was only found in the influent. The mean concentration of pharmaceuticals varied from 0.006 μg L −1 to 1.48 μg L −1 in the influent and from 0.003 μg L −1 to 0.93 μg L −1 in the effluent. The concentration of most pharmaceuticals was higher during winter.

Tissue-specific bioconcentration of selected antidepressants was studied in rainbow trout (Oncorhynchus mykiss) exposed to undiluted effluent from a Swedish municipal sewage treatment plant for 13 days. Citalopram, sertraline and venlafaxine were found in the brains and livers of most fish, but not in blood plasma or muscle. Venlafaxine was the only drug found in plasma (3/20 fish). Fluoxetine was not detected in any fish tissue, in accordance with a low concentration in the effluent and a comparably high limit of quantification in tissues. Concentrations of citalopram, sertraline and venlafaxine in fish brain were up to 1/12, 1/8 and 1/26, respectively, of the lowest concentrations found in the brains of mammals treated with therapeutic doses. Thus, given co-exposure to several antidepressants and an assumed similar potency in fish, the margin of safety for target-related effects in fish residing in effluent-dominated streams is relatively low. Furthermore, the non-detectable levels of these drugs in blood plasma suggest that analyses of concentrations in target tissues (brain) would be more informative in field studies and other studies with environmentally realistic exposure concentrations.

UV filters belong to a group of compounds that are used by humans and are present in municipal waste-waters, effluents from sewage treatment plants and surface waters. Current information regarding UV filters and their effects on fish is limited. In this study, the occurrence of three commonly used UV filters - 2-phenylbenzimidazole-5-sulfonic acid (PBSA), 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) and 5-benzoyl-4-hydroxy-2-methoxy-benzenesulfonic acid (benzophenone-4, BP-4) - in South Bohemia (Czech Republic) surface waters is presented. PBSA concentrations (up to 13μgL(-1)) were significantly greater than BP-3 or BP-4 concentrations (up to 620 and 390ngL(-1), respectively). On the basis of these results, PBSA was selected for use in a toxicity test utilizing the common model organism rainbow trout (Oncorhynchus mykiss). Fish were exposed to three concentrations of PBSA (1, 10 and 1000µgL(-1)) for 21 and 42 days. The PBSA concentrations in the fish plasma, liver and kidneys were elevated after 21 and 42 days of exposure. PBSA increased activity of certain P450 cytochromes. Exposure to PBSA also changed various biochemical parameters and enzyme activities in the fish plasma. However, no pathological changes were obvious in the liver or gonads.

Endocrine-disruptive potential and concentrations of polar organic contaminants were measured in seven headwaters flowing through relatively unpolluted areas of the Czech Republic. Towns with Wastewater Treatment Plant (WWTP) discharges were the first known sources of anthropogenic pollution in the areas. River water was sampled several kilometers upstream (US) and several tens of meters downstream (DS) of the WWTP discharges, by use of Pesticide and Pharmaceutical Polar Organic Integrative Samplers (POCIS-Pest, POCIS-Pharm). Extracts of passive samplers were tested by use of a battery of in vitro bioassays to determine overall non-specific cytotoxicity, endocrine-disruptive (ED) potential and dioxin-like toxicity. The extracts were also used for quantification of polar organics. There was little toxicity to cells caused by most extracts of POCIS. Estrogenicity was detected in all types of samples even though US locations are considered to be background. At US locations, concentrations of estrogen equivalents (EEq) ranged from less than the detection limits (LOD) to 0.5 ng EEq/POCIS. Downstream concentrations of EEqs ranged from less than LOD to 4.8 ng EEq/POCIS. Concentrations of EEq in POCIS extracts from all DS locations were 1 to 14 times greater than those at US locations. Concentrations of EEq measured in extracts of POCIS-Pest and POCIS-Pharm were in a good agreement. Neither antiestrogenic nor anti/androgenic activities were detected. Concentrations of 2,3,7,8-TCDD equivalents (TEqbio) were detected in both types of POCIS at concentrations ranging from less than the LOD to 0.39 ng TEqbio/POCIS. Nearly all extracts of POCIS-Pharm contained greater concentrations of TEqbio activity than extracts of POCIS-Pest. Concentrations of pesticides and pharmaceuticals in extracts of POCIS were generally small at all sampling sites, but levels of some pharmaceuticals were significantly greater in both types of POCIS from DS locations. Chemical analyses along with the results of bioassays documented impacts of small towns with WWTPs on headwaters. Keywords: Androgen, Dioxin-like activity, Estrogen, In vitro assay, POCIS, Waste Water Treatment Plant

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout ( Oncorhynchus mykiss ). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 μg l −1 ) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Blood is an indicator of physiological condition of an animal. Therefore, the chronic effects of propiconazole, a triazole fungicide present in aquatic environment, on hematology of rainbow trout were investigated in this study. Fish were exposed at various concentrations of PCZ (0.2, 50 and 500 μg L(-1)) for 7, 20 and 30 d. Multiple biomarkers were measured, including hematological indices (hemoglobin concentration, red blood cells count, hematocrit, leukocyte count, mean erythrocyte hemoglobin, mean erythrocyte volume and mean color concentration) and plasma biochemical parameters (ammonia, glucose, total proteins, creatine kinase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase). Through principal component analysis and integrated biomarker response assessment, influence extent induced by PCZ-stress of each test group was distinguished. Additional, all parameters measured in this study displayed different dependent patterns to PCZ concentrations and exposure time by two-way ANOVA. The results of this study indicate that chronic exposure of PCZ has altered multiple physiological indices in fish hematology and CK activity may be an early biomarker of PCZ toxicity; however, before these parameters are used as special biomarkers for monitoring residual PCZ in aquatic environment, more detailed experiments in laboratory need to be performed in the future.

Recently, residual fungicides are generally recognized as relevant sources of aquatic environmental pollutants. However, the toxicological effects of these contaminants have not been adequately researched. In this study, the chronic effect of PCZ, a triazole-containing fungicide commonly present in aquatic environment, on GSH-related antioxidant system and oxidative stress indices of rainbow trout (Oncorhynchus mykiss) were investigated. Fish were exposed at sub-lethal concentrations of PCZ (0.2, 50 and 500 microg/L) for 7, 20 and 30 days. GSH levels and GSH-related enzyme activities, including GPx, GR and GST, were quantified in three tissues-liver, gill and muscle. The levels of LPO and CP were also measured as makers of oxidative damage. In addition, the correlations of the measured parameters in various tissues were evaluated by using PCA. The results of this study indicate that chronic exposure of PCZ has resulted in different responses in various tissues and the gill was the most sensitive tissue; however, before these parameters are used as potential biomarkers for monitoring residual fungicides in aquatic environment, more detailed experiments in laboratory need to be performed in the future.

The effects of CBZ (a human pharmaceutical commonly present in aquatic environment) on the quality parameters and oxidative stress of common carp spermatozoa were investigated in vitro. Fish spermatozoa were incubated with different concentration of CBZ (0.2, 2.0 and 20 mgL(-1)) for 2h. Results revealed that the percentage of spermatozoa motile and velocity were decreased significantly at higher concentration of CBZ (2.0 and 20 mgL(-1)) and a dose-dependent reduction was observed. But the viability of fish spermatozoa was not affected significantly in all CBZ treatment groups. After 2h exposure of CBZ at higher test concentration (2.0 or 20 mgL(-1)), oxidative stress was apparent as reflected by the significant higher LPO and CP levels in fish spermatozoa, as well as the significant inhibition of antioxidant enzymes activities including SOD, GR and GPx. In short, CBZ can induce ROS stress in fish spermatozoa, which could impair the sperm quality and antioxidant defense system. Our results suggested that the use of fish spermatozoa in vitro assays may provide a novel and efficiently means for monitoring residual pharmaceutical in aquatic environment.

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 microg l(-1)) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na(+)-K(+)-ATPase). Exposure to 500 microg l(-1) PCZ led to significantly inhibited (p

We investigated the effect of long-term exposure to CBZ on the antioxidant system in brain tissue of rainbow trout. Fish were exposed to sublethal concentrations of CBZ (1.0 microg/L, 0.2mg/L or 2.0mg/L) for 7, 21, and 42 days. Oxidative stress indices (LPO and CP) and activities of antioxidant enzymes (SOD, CAT, GPx and GR) in fish brain were measured. In addition, non-enzymatic antioxidant (GSH) was determined after 42 days exposure. Carbamazepine exposure at 0.2mg/L led to significant increases (p0.05) of LPO and CP after 42 days and, at 2.0mg/L, after 21 days. Activities of the antioxidant enzymes SOD, CAT, and GPx in CBZ-treated groups slightly increased during the first period (7 days). However, activities of all measured antioxidant enzymes were significantly inhibited (p0.05) at 0.2mg/L exposure after 42 days and after 21 days at 2.0mg/L. After 42 days, the content of GSH in fish brain was significantly lower (p0.05) in groups exposed to CBZ at 0.2mg/L and 2.0mg/L than in other groups. Prolonged exposure to CBZ resulted in excess reactive oxygen species formation, finally resulting in oxidative damage to lipids and proteins and inhibited antioxidant capacities in fish brain. In short, a low level of oxidative stress could induce the adaptive responses of antioxidant enzymes, but long-term exposure to CBZ could lead to serious oxidative damage in fish brain.

Alkylphenols in muscle of fish from rivers in the Czech Republic were analysed in this study.