Molecular formula assignment of dissolved organic matter by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry using two non-targeted data processing approaches: A case study from recirculating aquaculture systems.

BACKGROUND: The accumulation of dissolved organic matter (DOM) poses an issue in the management of the water quality from recirculating aquaculture systems (RAS), but its characterization is often not detailed enough to understand the DOM transformations in RAS. In this study, we investigated the application of two distinct non-targeted data processing approaches using ultra-performance liquid chromatography (UPLC) with quadrupole time-of-flight mass spectrometry (QTOF-MS) and two software with different algorithmic designs: PetroOrg and Progenesis QI to accurately characterize the molecular composition of DOM in RAS by UPLC-QTOF-MS. RESULTS: The UPLC-QTOF-MS resolution in combination with PetroOrg and Progenesis QI software successfully assigned 912 and 106 unique elemental compositions, respectively, including compounds containing carbon, hydrogen, and oxygen (CHO) and nitrogen-containing CHO compounds (CHON), in the DOM samples from RAS. The results of these two distinct data processing approaches were consistent with the list of DOM formulas from RAS identified by higher resolution mass spectrometry techniques confirming their reliability. PetroOrg approach revealed only compositional information in the DOM samples from RAS, while Progenesis QI in addition to identifying new elemental compositions, increased their chemical space by giving information about their polarity and their possible key structures. DOM samples from RAS were found to be rich in unsaturated CHO compounds, with tentatively key structures of terpenoids with medium polarity indicating natural origins in their composition. The analysis also revealed probable structures of sucrose fatty acid esters and polyethylene glycol, indicating anthropogenic sources. SIGNIFICANCE AND NOVELTY: The combination of these two non-targeted data processing approaches significantly improves the characterization of the complex mixture of DOM from RAS by UPLC-QTOF-MS reporting for the first time accurate DOM results in terms of its composition, while proposing its key structures. The presented methods can also be used to analyze different DOM samples with other HRMS techniques and software.

Impact of ozone treatment on dissolved organic matter in land-based recirculating aquaculture systems studied by Fourier transform ion cyclotron resonance mass spectrometry.

In land-based recirculating aquaculture systems (RAS), the accumulation of dissolved organic matter (DOM) can have detrimental effects on water quality impacting the system performance, microbial community, and consequently fish health and welfare. Ozone is used in the RAS water treatment process to improve water quality and remove DOM. However, little is known about the molecular composition of DOM in RAS and its transformation when exposed to ozone. In this study, we performed a detailed molecular characterization of DOM in RAS and explored its transformation induced by ozonation of RAS waters. Ultra-high resolution (UHR) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the DOM matrix of RAS waters (pump-sump and tanks) and to evaluate its transformation by ozonation. The analysis of DOM extracted from makeup water and feed samples allowed for the determination of DOM sources in RAS prior to ozonation. The CHO and unsaturated group of compounds were the most abundant class found in water samples. On the contrary, the DOM from feed samples was unique and consisted mainly of CHO, CHON and unsaturated group of compounds. After the ozonation of RAS waters, humic-like and unsaturated compounds [positive oxygen subtracted double bond equivalent per carbon (DBE-O)/C)] were decomposed, particularly the CHO-DOM that contained fewer -CH2- features. Fulvic-like compounds and several hundred saturated compounds [negative (DBE-O)/C)] were formed post ozonation, particularly the CHON and CHONS group of compounds that were associated with fish diets, makeup waters and transformation products from the ozonation of the RAS waters. This study showed that the high accuracy of the ultra-high resolution FTICR MS can be applied to characterize and monitor the changes of DOM at a molecular level in RAS waters. To our knowledge, this is the first study where FTICR MS was incorporated for the characterization of DOM and its sources in RAS.

Characterizing changes of dissolved organic matter composition with the use of distinct feeds in recirculating aquaculture systems via high-resolution mass spectrometry.

Recirculating aquaculture systems (RAS) are a new alternative to traditional aquaculture approaches, allowing full control over the fish production conditions, while reducing the water demand. The reduction of water exchange leads to an accumulation of dissolved organic matter (DOM) that can have potential effects on water quality, fish welfare and system performance. Despite the growing awareness of DOM in aquaculture, scarce scientific information exists for understanding the composition and transformation of DOM in RAS. In this study, a non-targeted approach using ultra-performance liquid chromatography coupled to a hybrid quadrupole-time of flight mass spectrometer (UPLC-QTOF-MS) was used to characterize compositional changes of low molecular weight (LMW) DOM in RAS, when operated under two different feed types. A total of 1823 chemicals were identified and the majority of those contained a CHON chemical group in their structure. Changes in the composition of LMW-DOM in RAS waters were observed when the standard feed was switched to RAS feed. The DOM with the use of standard feed, consisted mainly of lignin/CRAM-like, CHO and CHOS chemical groups, while the DOM that used RAS feed, was mainly composed by unsaturated hydrocarbon, CHNO and CHNOS chemical groups. The Bray-Curtis dissimilarity cluster demonstrated differences in the composition of DOM from RAS and was associated to the type of feed used. When the RAS feed was used, the Kendrick mass defect plots of -CH2- homologous units in the pump-sump (after the water treatment) showed a high removal capacity for CHNO, CHNOS and halogenated chemicals with high Kendrick mass defect, KMD > 0.7. To our knowledge, this is the first report of LMW-DOM characterization of RAS by high-resolution mass spectrometry (HRMS).

A salty start: Brackish water start-up as a microbial management strategy for nitrifying bioreactors with variable salinity.

Nitrifying biofilms developed in brackish water are reported to be more robust to salinity changes than freshwater biofilms. This makes them a promising strategy for water treatment systems with variable salinity, such as recirculating aquaculture systems for Atlantic salmon. However, little is known about the time required for nitrification start-up in brackish water or the microbial community dynamics. To investigate the development of nitrifying biofilms at intermediate salinity, we compared the startup of moving bed biofilm reactors with virgin carriers in brackish- (12‰ salinity) and freshwater. After 60 days, the brackish water biofilm had half the nitrification capacity of the freshwater biofilm, with a less diverse microbial community, lower proportion of nitrifiers, and a significantly different nitrifying community composition. Nitrosomonas and Nitrosospira-like bacteria were the main ammonia oxidizers in the brackish water biofilms, whereas Nitrosomonas was dominant in freshwater biofilms. Nitrotoga was the dominant nitrite oxidizer in both treatments. Despite the lower nitrification capacity in the brackish water treatment, the low ammonia and nitrite concentration with rapidly increasing nitrate concentration indicated that complete nitrification was established in both reactors within 60 days. The results suggest that biofilms develop nitrification in brackish water in comparable time as in freshwater, and brackish start-up can be a strategy for bioreactors with varying salinity.

Chemical assessment of marine sediments in vicinity of Norwegian fish farms - A pilot study.

While aquaculture is growing rapidly all over the world and generating many economic benefits, so have the environmental concerns about the externalities posed by the fish-farming industry. The distribution profiles of organic compounds and inorganic elements were explored in marine surface sediments collected in proximity of two active Norwegian fish farms, Hestøya and Nørholmen (<200-1100 m from the perimeter edge of the installations). Overall, the sediment organic matter (SOM) content was 7.3 ± 4.9%, with 7.9 ± 5.1% and 4.0 ± 0.5% for Hestøya and Nørholmen, respectively. A non-targeted analysis was performed for screening organic compounds in marine sediments, and the presence of 60 compounds was detected. Among suspect compounds were alkanes, alkenes, aromatics, aldehydes, ketones, esters, alcohols, diols, polycyclic aromatic hydrocarbons (PAHs), terpenes and terpenoids. Heptanal, benzaldehyde, 4-oxoisophorone, 1,7-dimethylnaphthalene and 3-bromophenol were the most abundant compounds in marine sediments. In total, concentrations of 47 elements were measured, concentrations of As, Cd, Cr, Cu, Hg, Mo, Ni, Sn and Zn were strongly influenced by anthropogenic inputs, while concentrations of Ce, Co, Al, Fe and Ti were related to the geology of the local bedrock. The chemical composition of marine sediments was different at Hestøya and Nørholmen, indicating different anthropogenic inputs in these areas. In general, concentrations of toxic elements were below the proposed guidelines for Norwegian marine sediment quality and can be characterised as background pollution. Overall, fish-farming activities had only a minor or negligible influence on marine sediments and are unlikely to cause any harm to local aquatic life in the studied area.

Biofilms grown in aquatic microcosms affect mercury and selenium accumulation in Daphnia.

Experiments examining mercury (Hg) toxicity in Daphnia are usually conducted in highly standardized conditions that prevent the formation of biofilm. Although such standardization has many advantages, extrapolation of results to natural conditions and inference of ecological effects is challenging. This is especially true since biofilms can accumulate metals/metalloids and play a key role in their transfer to higher trophic level organisms. In this study, we experimentally tested the effects of spontaneously appearing biofilm in Daphnia cultures on accumulation of Hg and its natural antagonist selenium (Se) in Daphnia magna. We added Hg (in the form of mercury (II) chloride) at two concentrations (0.2 µg/L and 2 µg/L) to experimental microcosms and measured the uptake of Hg and Se by D. magna in the presence and absence of biofilm. To test for consistent and replicable results, we ran two identical experimental sets one week apart. Biofilm presence significantly reduced the accumulation of Hg, while increasing the tissue Se content in D. magna, and these findings were reproducible across experimental sets. These findings indicate that highly standardized tests may not be adequate to predict the bioaccumulation and potential toxicity of metals/metalloids under natural conditions.

Biofilms remember: Osmotic stress priming as a microbial management strategy for improving salinity acclimation in nitrifying biofilms.

With increasing freshwater scarcity and greater use of seawater, fluctuating salinities are becoming common in water treatment systems. This can be challenging for salinity-sensitive processes like nitrification, especially in recirculating aquaculture systems (RAS), where maintaining nitrification efficiency is crucial for fish health. This study was undertaken to determine if prior exposure to seawater (priming) could improve nitrification in moving bed biofilm reactors (MBBR) under salinity increase from freshwater to seawater. The results showed that seawater-primed freshwater MBBRs had less than 10% reduction in nitrification activity and twice the ammonia oxidation capacity of the unprimed bioreactors after seawater transfer. The primed biofilms had different microbial community composition but the same nitrifying taxa, suggesting that priming promoted physiological adaptation of the nitrifiers. Priming may also have strengthened the extrapolymeric matrix protecting the nitrifiers. In MBBRs started up in brackish water (12‰ salinity), seawater priming had no significant impact on the nitrification activity and the microbial community composition. These bioreactors were inherently robust to salinity increase, likely because they were already primed to osmotic stress by virtue of their native salinity of 12‰. The results show that osmotic stress priming is an effective strategy for improving salinity acclimation in nitrifying biofilms and can be applied to water treatment systems where salinity variations are expected.

Trace elements and polychlorinated biphenyls (PCBs) in terrestrial compartments of Svalbard, Norwegian Arctic.

Despite being a remote location, the Arctic is a major receptor for anthropogenic pollution transported from the mid-latitudes. Vegetation and underlying organic soils in the Norwegian Arctic, Svalbard were used to study the occurrences of polychlorinated biphenyls (PCBs) and trace elements. In this study, current concentrations of PCBs and trace elements, namely, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, S, Sb, U and Zn in the terrestrial compartments of Svalbard are presented. Samples were collected from Adventdalen near Longyearbyen and from areas in proximity to Ny-Ålesund. There was significant variability in soil organic matter (SOM) among the soils analysed (5.0%-72.1%), with the highest values detected in Ny-Ålesund. The concentrations of Al, As, Cr Cu, Fe, Pb and Ni were associated with the geology of the local bedrock. The concentrations of all elements, except for Cd, Hg and Zn, were higher in soils than those in the overlying vegetation layers. Mean concentrations of ∑PCBs were significantly higher in vegetation (6.90 ±â€¯0.81 ng g-1 dw) than the underlying organic soils (3.70 ±â€¯0.36 ng g-1 dw). An inverse correlation of PCBs with the elements originating from the local bedrock indicated that their concentrations were potentially impacted by atmospheric deposition. PCBs and Cd were strongly associated, proposing a potential concomitant source of origin in Svalbard. Concentrations of PCBs and trace elements measured herein were below the proposed guidelines for Norwegian soil quality.

The presence of mercury and other trace metals in surface soils in the Norwegian Arctic.

Svalbard is an important study area for investigating the long-range transport of mercury (Hg) and other trace elements to the Arctic. Few studies have focused on their concentrations in Arctic soils. With ongoing climate change leading to thawing permafrost ground the soil compartment is of increasing importance in the Arctic. In this study, elemental composition and soil organic matter (SOM) content of surface and mineral soils in Svalbard are presented. The aim is to provide new data on soils in the Arctic and to gain more knowledge about the role of the soil in the biogeochemical cycle of mercury (Hg). Concentrations are reported for Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, S and Zn. Samples were taken in Adventdalen and in the area near Ny-Ålesund. We obtained a mean Hg concentration of 0.111 ± 0.036 µg/g in surface soils (range 0.041-0.254 µg/g). Hg levels in mineral soils (mean: 0.025 ± 0.013 µg/g; range: 0.004-0.060 µg/g) were substantially lower than in the corresponding surface soils. Hg strongly accumulates in the surface soil layer (upper 3 cm) and is associated with SOM (surface soil: 59 ± 14%). Hg concentrations in the surface soil were slightly lower than those in the humus layer in mainland Norway and were comparable to levels in soils elsewhere in the Arctic. An inverse association of Hg was found with elements attributed to the mineral soil, indicating that Hg is predominantly derived from atmospheric deposition.

Dynamic behaviour of trace metals in the Deûle River impacted by recurrent polluted sediment resuspensions: from diel to seasonal evolutions.

An automatic trace metal monitoring station (ATMS) have been implemented in the river bank of the Deûle canal in the city of Auby (northern France), in the vicinity of metallurgical industries that have heavily polluted sediments, in Cd, Pb and Zn. Additionally, the fluvial traffic in this river is intense and surface sediments are resuspended several times per day. This paper is the following part of that published recently (Superville et al., 2014) in this journal. The main purpose of this paper is to present new information on diel and seasonal variations of trace metals in the Deûle River and highlight the potential role of sediments in the contamination of the water column. To this end, Cd, Zn and Pb were monitored at a high frequency (~1h(-1)) by voltammetry, distinguishing an electrolabile and an acid-leachable fraction. Our measurements revealed that (i) acid-leachable Cd is mainly bound to light particles such as clays and carbonates and its daily behaviour does not really depend on the intensity of sediment remobilisation process; (ii) acid-leachable Pb and Zn exhibit some seasonal variations which are explained here, suggesting they should at least partly depend on the ageing of hydrous ferric oxides in the sediments; (iii) in spring, the electrolability of Zn increases in agreement with the biological activity that may break some strong association between Zn and ligands and/or particles (e.g., organic matter, hydrous ferric oxides, etc.). This trend is observed for electrolabile Pb later in summer, but on a smaller scale since this metal is generally more strongly scavenged and less labile than Zn; and (iv) electrolabile Mn concentrations increase during the day due to desorption process of resuspended sediment particles in the water.

Evidence of highly dynamic geochemical behaviour of zinc in the Deûle river (northern France).

The monitoring of dissolved zinc in the Deûle river was undertaken at three different periods of the years 2008 and 2009. Electrolabile concentrations of Zn were estimated every 2 hours for several weeks by using an ATMS (Automatic Trace metal Monitoring System) based on voltammetric measurements using a solid Ag-Hg rotating disc working electrode. Complementary measurements were carried out with DGT (Diffusive Gradient in Thin films) pistons deployed directly in the river for 24 hours. Water samples filtrated at 0.45 µm were also analysed by HR-ICP-MS to estimate the total dissolved concentrations of zinc and other trace metals. High frequency monitoring of zinc over several weeks in the Deûle river indicated that the concentration could change significantly over short time periods. Resuspension of polluted sediment and biological activities are two main factors that control the behaviour of zinc in the Deûle river. Furthermore, in May 2009, daily cycles of the electrolabile zinc fraction have been observed at relatively constant total dissolved concentration. It is assumed that this particular behaviour of zinc is based on an exchange between colloids and/or nonelectrolabile forms and free cation and inorganic complexes at a daily time scale.

Automatic trace metal monitoring station use for early warning and short term events in polluted rivers: application to streams loaded by mining tailing.

An automatic trace metal monitoring station (ATMS) system was implemented to study seasonal and short time changes in selected metal concentrations in two river courses influenced by mine drainage. High frequency monitoring over periods of months revealed daily variations of zinc, iron and copper, and also proved the use of ATMS as an early warning system in such polluted environments. Complementary measurements with ICP-MS (inductively coupled plasma-mass spectrometry), ionic chromatography, and thermodynamic equilibrium calculations also gave some new insights into the geochemical behaviour of the metals in these two rivers.

PI3K-AKT-mTOR pathway is dominant over androgen receptor signaling in prostate cancer cells.

BACKGROUND: Androgen receptor (AR) and the phosphatidylinositol-3 kinase (PI3K) signaling are two of the most important pathways implicated in prostate cancer. Previous work has shown that there is crosstalk between these two pathways; however, there are conflicting findings and the molecular mechanisms are not clear. Here we studied the AR-PI3K pathway crosstalk in prostate cancer cells in vitro as well as in vivo. METHODS: Quantitative PCR, Western analysis, reporter assays, and proliferation analyses in vitro and in vivo were used to evaluate the effect of PI3K pathway inhibition on AR signaling and cell growth. RESULTS: Transcriptional activity of AR was increased when the PI3K pathway was inhibited at different levels. In the androgen responsive prostate cancer cell line LNCaP, androgen and the mTOR inhibitor rapamycin synergistically activated androgen target genes. Despite increased androgen signaling, rapamycin treatment reduced LNCaP cell growth; the AR antagonist bicalutamide potentiated this effect. Furthermore, the rapamycin derivative CCI-779 reduced the growth of CWR22 prostate cancer xenografts while increasing AR target gene expression. CONCLUSION: These findings suggest that inhibition of the PI3K pathway activates AR signaling. Despite the increase in AR signaling which has proliferative effects, the result of PI3K pathway inhibition is antiproliferative. These findings suggest that the PI3K pathway is dominant over AR signaling in prostate cancer cells which should be considered in developing novel therapeutic strategies for prostate cancer.

Electrochemical properties of silver-copper alloy microelectrodes for use in voltammetric field apparatus.

Microelectrodes of silver-copper alloys have been evaluated for use in voltammetric analyses. Increased overpotential towards the hydrogen overvoltage reaction (HER) was found as a function of increased copper content in the silver. A study of oxidizing products by cyclic voltammetry (CV) in NaOH solution showed ten anodic and eight cathodic peaks which are described in the present paper. The behaviour of these alloy electrodes is somewhere between pure silver and pure copper electrodes. Differential pulse anodic stripping voltammetry (DPASV) was used to measure zinc, cadmium and lead in ultrapure water only (18 MOmegacm), and good linearity was found for all metals (r (2)=0.998) in the range of 0.5 to 5 ppb with a 600- to 1,200-s plating time. It was additionally found that cadmium and lead were better separated on the alloy electrodes compared to pure silver electrodes. Measurements of nickel were carried out on alloy electrodes by use of adsorptive differential pulse cathodic stripping voltammetry (Ad-DPCSV), and good linearity (r (2)=1.000) was found in the range from 0.5 to 5 ppb with an adsorption time of 120 s. The alloy electrodes were also found to be sensitive to nitrate, and good linearity (r (2)=0.997) was found in the range from 1 mg L(-1) to 100 mg L(-1) using differential pulse voltammetry (DPV) scanning from -450 mV to -1,500 mV. Addition of nitrate in ultrapure water afforded two different peaks related to the successive reductions of nitrate and nitrite. In ammonium buffer solution (pH 8.6) only one peak resulting from reduction of nitrate was observed. Furthermore, the use of alloy electrodes containing 17% Cu was tested in real samples, by installing it in a voltammetric system for monitoring of zinc and lead in a polluted river, the river Deûle, near the town of Douai in northern France. Results were found to be in agreement with parallel measurements carried out by ICP-MS.

Evaluations of solid electrodes for use in voltammetric monitoring of heavy metals in samples from metallurgical nickel industry.

Evaluation of different solid electrode systems for detection of zinc, lead, cobalt, and nickel in process water from metallurgical nickel industry with use of differential pulse stripping voltammetry has been performed. Zinc was detected by differential pulse anodic stripping voltammetry (DPASV) on a dental amalgam electrode as intermetallic Ni-Zn compound after dilution in ammonium buffer solution. The intermetallic compound was observed at -375 mV, and a linear response was found in the range 0.2-1.2 mg L(-1) (r(2)=0.98) for 60 s deposition time. Simultaneous detection of nickel and cobalt in the low microg L(-1) range was successfully performed by use of adsorptive cathodic stripping voltammetry (AdCSV) of dimethylglyoxime complexes on a silver-bismuth alloy electrode, and a good correlation was found with corresponding AAS results (r(2)=0.999 for nickel and 0.965 for cobalt). Analyses of lead in the microg L(-1) range in nickel-plating solution were performed with good sensitivity and stability by DPASV, using a working electrode of silver together with a glassy carbon counter electrode in samples diluted 1:3 with distilled water and acidified with H(2)SO(4) to pH 2. A new commercial automatic at-line system was tested, and the results were found to be in agreement with an older mercury drop system. The stability of the solid electrode systems was found to be from one to several days without any maintenance needed.

Evolutionary profiling of the U49 snoRNA gene.

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modifications (2'-O-ribose methylation and pseudouridylation). Their genomic organization show great flexibility: some are individually or polycistronically transcribed, while others are encoded within introns of other genes. Here, we present an evolutionary analysis of the U49 gene in seven species. In all species analyzed, U49 contains the typical hallmarks of C and D box motifs, and a conserved 12-15 nt sequence complementary to rRNA that define them as homologs. In mouse, human, and Drosophila U49 is found encoded within introns of different genes, and in plants it is transcribed polycistronically from four different locations. In addition, U49 has two copies in two different introns of the RpL14 gene in Drosophila. The results indicate a substantial degree of duplication and translocation of the U49 gene in evolution. In light of its variable organization we discuss which of the two proposed mechanisms of rearrangement has acted upon the U49 snoRNA gene: chromosomal duplication or transposition through an RNA intermediate.