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 tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.

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. The largest Gene Ontology groups were signal transduction, transcription, apoptosis, cell growth, cytoskeleton, cell adhesion/mobility, cardiovascular development, xenobiotic metabolism, protein metabolism, lipid metabolism and transport, and amino acid metabolism. Together these findings suggest that retene affects multiple signaling cascades in the heart of rainbow trout embryos, and potentially disturbs processes related to cardiovascular development and function.

Identification and dose dependency of ibuprofen biliary metabolites in rainbow trout.

The biotransformation of the anti-inflammatory drug ibuprofen (IBF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to IBF via intraperitoneal (i.p.) injection, and via water at four (0.17, 1.9, 13 and 145 µg L(-1)) exposure levels for 4d. Following exposure, the bile was collected and analyzed by LC-MS/MS methods. The identification of the formed metabolites in i.p. injected fish bile was based on the exact mass determinations by a time-of-flight mass analyzer (Q-TOF-MS) and on the studies of fragments and fragmentation patterns of precursor ions by ion trap mass analyzer (IT-MS). In addition to unmetabolized IBF, several phase I and phase II metabolites were found in the bile. The main metabolites were acyl glucuronides and taurine conjugates of IBF and of hydroxylated IBFs. The bioconcentration factors (BCFbile), defined as the ratio of the sum of IBF and its metabolites in fish bile to the concentration of IBF in water, was determined following enzymatic deconjugation and was found to range from 14000 to 49000. The highest BCFbile was found at the lowest exposure concentration (0.17 µg L(-1)). The results show that rainbow trout has a high capacity for biotransformation of IBF, and the exposure of fish to sub µg L(-1) concentrations of IBF can be determined by the analyses of the biliary metabolites of the compound.

The anti-inflammatory drugs diclofenac, naproxen and ibuprofen are found in the bile of wild fish caught downstream of a wastewater treatment plant.

Pharmaceutical residues are ubiquitous in rivers, lakes, and at coastal waters affected by discharges from municipal wastewater treatment plants. In this study, the presence of 17 different pharmaceuticals and six different phase I metabolites was determined in the bile of two wild fish species, bream (Abramis brama) and roach (Rutilus rutilus). The fish were caught from a lake that receives treated municipal wastewater via a small river. Prior to analyses, the bile content was enzymatically hydrolyzed to convert the glucuronide metabolites into the original pharmaceuticals or phase I metabolites. The solid phase extracts of hydrolyzates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The anti-inflammatory drug naproxen could be detected in all the six bream and roach bile samples. Diclofenac was found in five of the bream and roach samples, while ibuprofen was detected in three bream and two roach samples. The observed bile concentrations of diclofenac, naproxen, and ibuprofen in bream ranged from 6 to 95 ng mL(-1), 6 to 32 ng mL(-1), and 16 to 34 ng mL(-1), respectively. The corresponding values in roach samples ranged from 44 to 148 ng mL(-1), 11 to 103 ng mL(-1) and 15 to 26 ng mL(-1), respectively. None of the other studied compounds could be detected. The study shows that pharmaceuticals originating from wastewater treatment plant effluents can be traced to the bile of wild bream and roach living in a lake where diclofenac, naproxen, and ibuprofen are present as pollutants.

Fast genomic biomarker responses of retene and pyrene in liver of juvenile rainbow trout, Oncorhynchus mykiss.

We studied the transcriptive effects of two PAHs, retene (RET) and pyrene (PYR), in three equimolar sublethal concentrations (0.9-10 µg/L) in the liver of juvenile rainbow trout, Oncorhynchus mykiss. After 24 h of in vivo exposure, expressions of selected genes (CYP1A, Hsp30, Hsp70, Grp78, Sep15, GP1) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). These PAHs changed the studied gene transcriptions differently, but not significantly, except for CYP1A, which was induced only by RET. RET induced CYP1A gene expression even at low, environmentally realistic concentrations in the liver of juvenile rainbow trout.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment.

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Vertical distribution of pharmaceuticals in lake sediments-citalopram as potential chemomarker.

The use of pharmaceuticals has increased enormously over the last few decades and serious concerns about their environmental fate and effects have arisen. Thus far, there is little knowledge about the historical pollution of the environment by pharmaceuticals. In the present study, sediment columns from three lake sites adjacent to wastewater treatment plants were collected, further divided in 2- or 2.5-cm slices, and analyzed for 15 pharmaceuticals by liquid chromatography-tandem mass spectrometry. In addition, sediment columns were historically interpreted by analyzing fecal sterols (coprostanol and cholesterol) as well as organic material and organic carbon. Several pharmaceuticals were detected in sediments, the most abundant being citalopram, bisoprolol, and propranolol. At site A, pharmaceuticals prevailed only in the uppermost 15 cm, whereas at site B they existed in the whole sediment column (0-30 cm). Pharmaceuticals were not found in site C sediments. Based on the sterol analyses, municipal wastewater contamination at sites A, B, and C was found in the uppermost 15, 30, and 20 cm, respectively. For the first time, contamination of sediments by pharmaceuticals was demonstrated below the subsurface (up to a depth of 30 cm). When considering the consumption and the observed concentration profiles of pharmaceuticals, a clear increasing trend of citalopram toward the surface was evident at site 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 fluorescence: opaqueness of the sample, and enzymes (DT-diaphorase and plausibly NADPH-CYP450 oxidoreductase) that reduce resorufin to a non-fluorescent form.

Agricultural potential of anaerobically digested industrial orange waste with and without aerobic post-treatment.

The potential of anaerobically digested orange waste with (AAD) and without (AD) aerobic post-treatment for use in agriculture was evaluated through chemical analyses, short-term phytotoxicity and long-term plant assays. Chemical analyses showed that AD contained ammonia and organic acids, and aerobic post-treatment did not significantly remove these phytotoxins. The N:P2O5:K2O ratio in AD was 1:0.26:0.96 and aerobic post-treatment did not change the composition in AAD except for K2O (1:0.26:1.24). Heavy metal contents in AD and AAD were more or less the same and were below the upper limit recommended for non-sewage sludge application on agricultural soils. Short-term phytotoxicity tests showed that seed germination and root elongation of Chinese cabbage and ryegrass were severely inhibited at digestate concentrations of 60-100%. Germination index values were well below the score of 50% required to indicate the phytotoxic-free nature of compost. Long-term plant assays showed that AD and AAD, when supplemented with a base fertilizer, resulted in higher plant growth, and fresh weight and dry matter production than AD without base fertilizer. The results thus indicate that aerobic post-treatment did not have any significant beneficial effect on reducing phytotoxicity, and AD could be used as such on agricultural soils, especially with high P.

Phytotoxicity of low-weight carboxylic acids.

Presence of low-weight carboxylic acids (LWCAs) can be the reason for phytotoxicity of green manures, treated bio-waste or digestates from biogas production applied to soils. As the phytotoxic concentrations of LWCA are poorly known, this work presents data on six acids (C(1)C(6): formic, acetic, propionic, butyric, valeric, and caproic). Phytotoxicity was measured in acute (72 or 120 h) and subchronic (21 d) assays for seed germination, seedling elongation, and plant growth for garden cress Lepidium sativum and ryegrass Lolium multiflorum. The dose-response relationship was modeled using Weibull model. Results showed a trend that toxicity of LWCA increases with the length of the carbon chain, formic acid (C(1)) being the least and caproic acid (C(6)) the most toxic. EC50 values in the acute seed germination of cress ranged between 1.9 and 4.2mM and for ryegrass between 1.8 and 6.4mM. In subchronic assays EC50 values for germination were in a range from 11 to 46mMkg(-1)dm for cress, and from 18 to 127 mM kg(-1) dm for ryegrass. EC50 values for early seedling growth of cress based on acute assays ranged from 0.7 to 2.3mM and that of ryegrass from 1.2 to 1.8mM. Range of EC50 values for shoot biomass of cress was between 8 and 40 mM kg(-1) dm and of ryegrass between 12 and 93 mM kg(-1) dm.

Toxicity of waste gasification bottom ash leachate.

Toxicity of waste gasification bottom ash leachate from landfill lysimeters (112 m(3)) was studied over three years. The leachate of grate incineration bottom ash from a parallel setup was used as reference material. Three aquatic organisms (bioluminescent bacteria, green algae and water flea) were used to study acute toxicity. In addition, an ethoxyresorufin-O-deethylase (EROD) assay was performed with mouse hepatoma cells to indicate the presence of organic contaminants. Concentrations of 14 elements and 15 PAH compounds were determined to characterise leachate. Gasification ash leachate had a high pH (9.2-12.4) and assays with and without pH adjustment to neutral were used. Gasification ash leachate was acutely toxic (EC(50) 0.09-62 vol-%) in all assays except in the algae assay with pH adjustment. The gasification ash toxicity lasted the entire study period and was at maximum after two years of disposal both in water flea (EC(50) 0.09 vol-%) and in algae assays (EC(50) 7.5 vol-%). The grate ash leachate showed decreasing toxicity during the first two years of disposal in water flea and algae assays, which then tapered off. Both in the grate ash and in the gasification ash leachates EROD-activity increased during the first two years of disposal and then tapered off, the highest inductions were observed with the gasification ash leachate. The higher toxicity of the gasification ash leachate was probably related to direct and indirect effects of high pH and to lower levels of TOC and DOC compared to the grate ash leachate. The grate ash leachate toxicity was similar to that previously reported in literature, therefore, confirming that used setup was both comparable and reliable.

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 caused plausible p53 activation in the brain and epidermis of some individuals. Also at a fluence rate occurring in nature, the DNA damage in the brain and eyes of pike and changes in protein expression were followed by severe behavioral disorders, suggesting that neural molecular changes were associated with functional consequences.

Pharmaceuticals in settleable particulate material in urban and non-urban waters.

Wastewater treatment plants (WWTP) are important sources of settleable particulate material (SPM), heading to sediments with natural suspended solids. To date, there is little information about the fate of pharmaceuticals in sediment systems. In this study, the objective was to determine if pharmaceuticals are detected in SPM at locations near WWTPs or even in rural areas, thus being susceptible for sedimentation. SPM samples were collected from 10 sites in Finland, grouped as reference, rural and wastewater effluent sites. SPM collectors were placed about 35 cm above bottom for about 2 months during summer. After extraction, a set of 17 pharmaceuticals was analyzed. Several pharmaceuticals were detected in SPM accumulated at sites next to WWTPs. The concentration of citalopram was notably high (300-1350 ng g⁻¹ dw). Also bisoprolol and ciprofloxacin were detected at high concentrations (6-325 and 9-390 ng g⁻¹ dw, respectively). In contrast, none of the pharmaceuticals were detected from reference sites and only two were found from a single rural site. There is no previous information about the presence of pharmaceuticals in SPM. The results showed that pharmaceuticals are sorbed to particles in WWTP and nearby, eventually ending up in sediments. These results also indicate that pharmaceuticals are not markedly contaminating sediments of rural areas in Finland.

Uptake from water, biotransformation, and biliary excretion of pharmaceuticals by rainbow trout.

An urgent need exists to assess the exposure of fish to pharmaceuticals. The aim of the present study was to assess the uptake and metabolism of waterborne pharmaceuticals in rainbow trout (Oncorhynchus mykiss). A further objective was to determine the possibility of monitoring exposure to low levels of pharmaceuticals by bile assays. Rainbow trout were exposed for 10 d under flow-through conditions to mixtures of five pharmaceuticals (diclofenac, naproxen, ibuprofen, bisoprolol, and carbamazepine) at high and low concentrations. The low concentration was used to mimic the conditions prevailing in the vicinity of the discharge points of wastewater treatment plants. The uptake and the bioconcentration were determined by blood plasma and bile analyses. The average bioconcentration factor in plasma ranged from below 0.1 for bisoprolol to 4.9 for diclofenac, the values being approximately similar at low and high ambient concentrations. The biotransformation of diclofenac, naproxen, and ibuprofen was considered efficient, because several metabolites could be detected in concentrations clearly exceeding those of the unmetabolized compounds. The glucuronides were the dominant metabolites for all three pharmaceuticals. The total bioconcentration in the bile was two to four orders of magnitude higher than in the plasma. The results of this work show that the exposure of fish to pharmaceuticals in environmentally relevant concentrations may be monitored by blood plasma and bile analyses, the latter allowing detection at markedly lower ambient concentration.

Detection of naproxen and its metabolites in fish bile following intraperitoneal and aqueous exposure.

PURPOSE: The anti-inflammatory drug naproxen (NPX) has been found as a micropollutant in river water downstream the discharge points of wastewater treatment plants (WWTP). In this study, rainbow trout (Oncorhynchus mykiss) was exposed to NXP and the uptake and metabolism of the drug was studied. METHODS: Following exposure through intraperitoneal injection (i.p., 0.5 mg NPX/100 g fish biomass) and through water (1.6 µg L(-1)), the bile was collected and analyzed with various LC-MS/MS methods. The identification of the formed metabolites in i.p. injected fish was based on the exact mass determinations by a time-of-flight mass analyzer (Q-TOF-MS) and on the studies of fragments and fragmentation patterns of precursor ions by an ion trap mass analyzer (IT-MS). RESULTS: No matter the exposure route, the main metabolites were found to be acyl glucuronides of NPX and of 6-O-desmethylnaproxen. Also, unmetabolized NPX was detected in the bile. The total bioconcentration factors (BCF(total-bile)) of NPX and the metabolites in the bile of fish exposed through water ranged from 500 to 2,300. CONCLUSION: The findings suggest that fish living downstream WWTPs may take up NPX and metabolize the compound. Consequently, NPX and its metabolites in bile can be used to monitor the exposure of fish to NPX.

Microbial transformation of pharmaceuticals naproxen, bisoprolol, and diclofenac in aerobic and anaerobic environments.

Although biotransformation is generally considered to be the main process by which to remove pharmaceuticals, both in sewage treatment plants and in aquatic environments, quantitative information on specific compounds is scarce. In this study, the transformations of diclofenac (DCF), naproxen (NPX), and bisoprolol (BSP) were studied under aerobic and anaerobic conditions using inocula taken from activated and digested sludge processes, respectively. Whereas concentration decays were monitored by LC-tandem mass spectrometry, oxygen consumption and methane production were used for the evaluation of the performance of overall conditions. DCF was recalcitrant against both aerobic and anaerobic biotransformation. More than one third of the BSP disappeared under aerobic conditions, whereas only 14% was anaerobically biotransformed in 161 days. Under aerobic conditions, complete removal of NPX was evident within 14 days, but anaerobic transformation was also efficient. Formation of 6-O-desmethylnaproxen, a previously reported aerobic metabolite, was also detected under anaerobic conditions and persisted for 161 days.

Metabolites of the aquatic pollutant diclofenac in fish bile.

The uptake and metabolism of anti-inflammatory drug diclofenac (DCF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to DCF intraperitoneally, and via water at concentration of 1.7 µg L(-1). The bile was collected and the formed metabolites were identified. The identification was based on the exact mass determinations by a time-of-flight mass analyzer and on the studies of fragments and fragmentation patterns of precursor ions by an ion trap mass analyzer. The main metabolites found were acyl glucuronides of hydroxylated DCFs. In addition, one ether glucuronide of hydroxylated DCF was found. Also, unmetabolized DCF was detected in the bile. The total bioconcentration factors (BCF(total-bile) for DCF and its metabolites) in rainbow trout bile, varied between individuals and was roughly estimated to range from 320 to 950. These findings suggest that fish living downstream the wastewater treatment plants (WWTPs) and which are chronically exposed to the drug may accumulate the drug and its metabolites in the bile.

Do historical sediments of pulp and paper industry contribute to the exposure of fish caged in receiving waters?

Field experiments were conducted in early summer 2004 by caging brown trout (Salmo trutta m. lacustris) in Southern Lake Saimaa (SE Finland) for 10 days. Cages were positioned into two depths at four distances (5 to 17 km) from the pulp and paper mill; one set with direct contact to the sediment and other locating at the three meters' depth from the water surface. Resin acids were measured from sediment, water, and fish bile. Possible toxicities were investigated by measuring liver EROD-activity, HSP70-protein, and gene expression profiles by microarray. The concentration of resin acids in the sediment showed clear gradient along the distance from the mill, and their concentrations in water ranged approximately from one to 30 microg L(-1). In the trout bile, concentrations were higher near the surface than on the bottom, indicating the role of current releases as sources of resin acids. When compared to trout held upstream from the effluent source, no differences were found in liver EROD nor HSP70 in bottom cages. However, near surface, a slight but statistically significant increase in EROD and HSP70 was found. Altogether 145 genes were found to be differentially regulated at downstream sites when compared 10 km upstream from the mill. The numbers of genes at each site varied from 5 to 45 without consistent differences between bottom and surface cages. Overall, neither expression profiles nor composition of the transcription signatures made possible to conclude an overt toxicity in exposed brown trout.

Uptake of organic xenobiotics by benthic invertebrates from sediment contaminated by the pulp and paper industry.

Uptake of pulp and paper mill-derived pollutants by benthic invertebrates from sediment in Southern Lake Saimaa, eastern Finland, was studied. Two groups of benthic invertebrates (Diptera and Oligochaeta) were analyzed for their concentrations of resin acids (RAs), chlorophenolics (CPs) and beta-sitosterol. The samples were collected 1 and 3km downstream from the mill. In laboratory experiments Chironomus plumosus (a dipteran) and Lumbriculus variegatus (oligochaete) were exposed for 14d to sediments collected from the same locations. The concentrations of RAs, CPs and beta-sitosterol were higher in the areas downstream from the mill than those in the upstream reference area in both the feral and laboratory-exposed animals. Examination of the possible conjugation of contaminants revealed hydrolyzable fractions of RAs in Diptera, C. plumosus and L. variegatus. The results indicate both the bioavailability uptake of contaminants and uptake by benthic fauna when exposed to pulp and paper mill-contaminated sediment.

Exposure assessment of fishes to a modern pulp and paper mill effluents after a black liquor spill.

Conjugated resin acids (RAs) in fish bile are considered a sensitive chemical indicator of exposure to pulp and paper mill effluent, and were used in this study to monitor the post-spill situation of a lake area (Southern Lake Saimaa) contaminated by black liquor discharged from a mill in June 2003. From the exposure perspective of populations of wild roach and perch, which were studied for their bile RAs at four time periods (July 2003, September 2003, May 2004, July 2004), the exceptional event passed in 2 months or less. Perch had lower concentration of RAs in bile than roach in all sampling areas and all times. Besides the current emissions present in the water column, part of the exposure status of the roach population to RAs seemed to derive from historically contaminated sediments. In order to test this hypothesis, a laboratory experiment with perch and roach, along with three teleosts (rainbow trout, brown trout and whitefish), was conducted. The species were simultaneously exposed for 7 days to RAs (23 microg/l). We calculated a perch/roach-ratio to investigate the difference in origin of exposure between perch and roach populations in the wild and in the laboratory. One year after the spill, the perch/roach-ratio of bile RAs was 0.25 (CV 25%) at 1 km from the mill. This is in contrast to that found under the sole waterborne conditions (0.44; CV 24%), supporting the idea that sediments serve as an additional source of RAs in roach. Additionally, bioconcentration factor log BCF(bile(RA)) was calculated to assess hepatobiliary performance and the capacity to excrete RAs in fish.