High-performance liquid chromatography-tandem mass spectrometry with post-column pH modification: Independent pH optimization for chromatographic separation and electrospray ionization.

RATIONALE: In recent environmental research, multi-methods using high-performance liquid chromatography-mass spectrometry (HPLC/MS/MS) have become more and more important for the analysis of organic micropollutants in environmental matrices. As the targeted compounds usually have different physicochemical properties, the optimization of these methods is challenging. METHODS: The pH in the electrospray of the mass spectrometer ion source was modified independently of the one used for the HPLC separation, using a post-column HPLC flow to make the spray acidic or alkaline. The method development was carried out manually in a systematic multistep way. RESULTS: The method used for the analysis of organic biocides (e.g., terbutryn, propiconazole, and isothiazolinones) was optimized. It was shown that the use of a neutral as well as an acidified gradient could not be optimized for all target compounds, whereas a neutral gradient with post-column acidification was optimum for all target compounds. CONCLUSIONS: Acidic or alkaline post-column infusion allows pH optimization for ionization conditions, independent of the pH optimization for chromatographic separation. The introduction of an additional modifier might also allow the minimization of matrix effects or directed formation of sodium adducts, without affecting the chromatographic separation.

Degradation of triclosan by environmental microbial consortia and by axenic cultures of microorganisms with concerns to wastewater treatment.

Triclosan is an antimicrobial agent, which is widely used in personal care products including toothpaste, soaps, deodorants, plastics, and cosmetics. Widespread use of triclosan has resulted in its release into wastewater, surface water, and soils and has received considerable attention in the recent years. It has been reported that triclosan is detected in various environmental compartments. Toxicity studies have suggested its potential environmental impacts, especially to aquatic ecosystems. To date, removal of triclosan has attracted rising attention and biodegradation of triclosan in different systems, such as axenic cultures of microorganisms, full-scale WWTPs, activated sludge, sludge treatment systems, sludge-amended soils, and sediments has been described. In this study, an extensive literature survey was undertaken, to present the current knowledge of the biodegradation behavior of triclosan and highlights the removal and transformation processes to help understand and predict the environmental fate of triclosan. Experiments at from lab-scale to full-scale field studies are shown and discussed.

Biocide Runoff from Building Facades: Degradation Kinetics in Soil.

Biocides are common additives in building materials. In-can and film preservatives in polymer-resin render and paint, as well as wood preservatives are used to protect facade materials from microbial spoilage. Biocides leach from the facade material with driving rain, leading to highly polluted runoff water (up to several mg L-1 biocides) being infiltrated into the soil surrounding houses. In the present study the degradation rates in soil of 11 biocides used for the protection of building materials were determined in laboratory microcosms. The results show that some biocides are degraded rapidly in soil (e.g., isothiazolinones: T1/2 < 10 days) while others displayed higher persistence (e.g., terbutryn, triazoles: T1/2 ≫ 120 days). In addition, mass balances of terbutryn and octylisothiazolinone were determined, including nine (terbutryn) and seven (octylisothiazolinone) degradation products, respectively. The terbutryn mass balance could be closed over the entire study period of 120 days and showed that relative persistent metabolites were formed, while the mass balances for octylisothiazolinone could not be closed. Octylisothiazolinone degradation products did not accumulate over time suggesting that the missing fraction was mineralized. Microtox-tests revealed that degradation products were less toxic toward the bacterium Aliivibrio fischeri than their parent compounds. Rain is mobilizing these biocides from the facades and transports them to the surrounding soils; thus, rainfall events control how often new input to the soil occurs. Time intervals between rainfall events in Northern Europe are shorter than degradation half-lives even for many rapidly degraded biocides. Consequently, residues of some biocides are likely to be continuously present due to repeated input and most biocides can be considered as "pseudo-persistent"-contaminants in this context. This was verified by (sub)urban soil screening, where concentrations of up to 0.1 µg g-1 were detected for parent compounds as well as terbutryn degradation products in soils below biocide treated facades.

Leaching of Terbutryn and Its Photodegradation Products from Artificial Walls under Natural Weather Conditions.

Terbutryn is a commonly used biocide in construction materials. Especially polymer-resin-based renders and paints, used in external thermal insulation composite systems, are very susceptible to microbial deterioration. Previous studies have shown that biocides leach out of the material when contacted with rainwater; thus, they reach surface waters where they might have adverse effects on aquatic organisms. The knowledge on the long-term leaching performance and especially the formation and fate of degradation products is rare. In the present study, the leaching of terbutryn from artificial walls equipped with two types of render was observed for 19 months. In addition to concentration and mass load determinations for terbutryn, photodegradation products were identified and studied in the leachate and render. The results show that terbutryn leached mainly within the first 6-12 months. During the exposure, only 3% of the initial terbutryn was emitted to the runoff, while 64-80% remained in the coating. The overall mass balance could be closed by including several degradation products. Contrary to expectations, the major fraction of transformation products remained in the material and was not washed off immediately, which is of high importance for the long-term assessment of biocides in coating materials.

Biofilm Thickness Influences Biodiversity in Nitrifying MBBRs-Implications on Micropollutant Removal.

In biofilm systems for wastewater treatment (e.g., moving bed biofilms reactors-MBBRs) biofilm thickness is typically not under direct control. Nevertheless, biofilm thickness is likely to have a profound effect on the microbial diversity and activity, as a result of diffusion limitation and thus substrate penetration in the biofilm. In this study, we investigated the impact of biofilm thickness on nitrification and on the removal of more than 20 organic micropollutants in laboratory-scale nitrifying MBBRs. We used novel carriers (Z-carriers, AnoxKaldnes) that allowed controlling biofilm thickness at 50, 200, 300, 400, and 500 µm. The impact of biofilm thickness on microbial community was assessed via 16S rRNA gene amplicon sequencing and ammonia monooxygenase (amoA) abundance quantification through quantitative PCR (qPCR). Results from batch experiments and microbial analysis showed that (i) the thickest biofilm (500 µm) presented the highest specific biotransformation rate constants (kbio, L g(-1) d(-1)) for 14 out of 22 micropollutants; (ii) biofilm thickness positively associated with biodiversity, which was suggested as the main factor for the observed enhancement of kbio; (iii) the thinnest biofilm (50 µm) exhibited the highest nitrification rate (gN d(-1) g(-1)), amoA gene abundance and kbio values for some of the most recalcitrant micropollutants (i.e., diclofenac and targeted sulfonamides). Although thin biofilms favored nitrification activity and the removal of some micropollutants, treatment systems based on thicker biofilms should be considered to enhance the elimination of a broad spectrum of micropollutants.

Degradation of PPCPs in activated sludge from different WWTPs in Denmark.

Pharmaceuticals and Personal care products (PPCPs) are often found in effluents from wastewater treatment plants (WWTPs) due to insufficient removal during wastewater treatment processes. To understand the factors affecting the removal of PPCPs in classical activated sludge WWTPs, the present study was performed to assess the removal of frequently occurring pharmaceuticals (Naproxen, Fenoprofen, Ketoprofen, Dichlofenac, Carbamazepine) and the biocide Triclosan in activated sludge from four different Danish WWTPs. The respective degradation constants were compared to operational parameters previous shown to be of importance for degradation of micropollutants such as biomass concentration, and sludge retention time (SRT). The most rapid degradation, was observed for NSAID pharmaceuticals (55-90% for Fenoprofen, 77-94% for Ketoprofen and 46-90% for Naproxen), followed by Triclosan (61-91%), while Dichlofenac and Carbamazepine were found to be persistent in the systems. Degradation rate constants were calculated as 0.0026-0.0407 for NSAID pharmaceuticals and 0.0022-0.0065 for triclosan. No relationships were observed between degradation rates and biomass concentrations in the diverse sludges. However, for the investigated PPCPs, the optimal SRT was within 14-20 days (for these values degradation of these PPCPs was the most efficient). Though all of these parameters influence the degradation rate, none of them seems to be overall decisive. These observations indicate that the biological composition of the sludge is more important than the design parameters of the respective treatment plant.

Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (Douchi) in Yunan, China.

Three Bacillus cereus strains isolated from an outbreak of food poisoning caused by the consumption of fermented black beans (douchi) containing B. cereus is described. The outbreak involved 139 persons who had nausea, vomiting, and diarrhea. The strains were isolated from vomit and the unprepared douchi. Two of the strains produced the emetic toxin cereulide, as evidenced by polymerase chain reaction analysis for the presence of the nonribosomal synthetase cluster responsible for the synthesis of cereulide and by chemical analysis by high-performance liquid chromatography-mass spectrometry. These two strains belong to genetic group III of B. cereus, and multiple locus sequence typing revealed that the type was ST26, as a major part of B. cereus emetic strains. One of these strains produced significantly more cereulide at 37°C than the type cereulide producer (F4810/72), and it was also able to produce the toxin at 40°C and 42°C. The third strain belongs to genetic group IV, and it is a new multiple locus sequence type closely related to strains that are cytotoxic and enterotoxigenic. It possesses genes for hemolysin BL, nonhemolytic enterotoxin, and cytotoxin K2; however, it varies from the majority of strains possessing genes for hemolysin BL by not being hemolytic. Thus, two B. cereus strains producing the emetic toxin cereulide and a strain producing enterotoxins might have been involved in this food-poisoning incident caused by the consumption of a natural fermented food. The ability of one of the strains to produce cereulide at ≥37°C makes it possible that it is produced in the human gut in addition to occurring in the food.

Quantifying measurement uncertainty in full-scale compost piles using organic micro-pollutant concentrations.

Reductions in measurement uncertainty for organic micro-pollutant concentrations in full scale compost piles using comprehensive sampling and allowing equilibration time before sampling were quantified. Results showed that both application of a comprehensive sampling procedure (involving sample crushing) and allowing one week of equilibration time before sampling reduces measurement uncertainty by about 50%. Results further showed that for measurements carried out on samples collected using a comprehensive procedure, measurement uncertainty was associated exclusively with the analytic methods applied. Application of statistical analyses confirmed that these results were significant at the 95% confidence level. Overall implications of these results are (1) that it is possible to eliminate uncertainty associated with material inhomogeneity and (2) that in order to reduce uncertainty, sampling procedure is very important early in the composting process but less so later in the process.

Transformation mechanisms of the antidepressant citalopram in a moving bed biofilm reactor: Substrate-depended pathways, eco-toxicities and enantiomeric profiles.

Citalopram (CIT) is one of the most consumed antidepressants and frequently detected in aquatic environments worldwide. Conventional wastewater treatment cannot remove this neuronal active pharmaceutical efficiently. Past studies showed that moving bed biofilm reactors (MBBRs) can degrade CIT but the exact transformation pathways and toxicity reduction remained unclear. In this study, the effects of substrate stimulation on CIT transformation in an MBBR were systematically investigated. The results showed that a co-metabolic stimulation by acetate increased the transformation rate by 54 % and 24 % at high (300 µg/L) and environmental concentration (1.8 µg/L) of CIT, respectively. Conversely, the complex substrates in raw wastewater reduced the reaction rates by 44 %, suggesting a competitive inhibition on the enzymatic sites. The substrate stimulation changed the enantiomeric fraction (EF) of CIT from racemic (EF=0.5) to 0.60 at the high CIT concentrations, while those at lower concentrations resulted in an EF of 0.33, indicating that probably different enantioselective enzymes degraded CIT at high concentrations than at low concentrations, i.e., the presence of 300 µg/L CIT was possibly sufficient to induce the synthesis of different enantioselective enzymes, than those originally present. Through non-target and target analysis, in total 19 transformation products (TPs) including 7 TPs that were hitherto not mentioned in the literature were identified. Among these were quaternary amines, alkenes and conjugate TPs. The major transformation pathways were a) nitrile hydrolysis (up to 43 %), b) amide hydrolysis, and c) N-oxidation. Dosing acetate up-regulated significantly the amide hydrolysis, N-oxidation and conjugation pathways but inhibited the N-demethylation and α-carbon hydroxylation pathways. The in-silico toxicity assessment of CIT and its TPs suggested the overall eco-toxic potential of TPs was reduced by MBBR. Furthermore, the degradation under carbon-limited (famine) conditions favored the formation of the more toxic carboxamide, N-desmethyl and alkene TPs, while carbon-rich conditions, promoted the production of the less toxic carboxylic acid, N-oxide and ester TPs. Therefore, this study demonstrated that a) the co-metabolic stimulation of CIT metabolization by dosing a simple carbon source or b) inhibition of CIT metabolization by complex substrates; c) substrate stimulation made a difference on CIT transformation rates, enantiomeric profiles, pathways and toxic potentials. Overall, a simple-carbon co-metabolic stimulated MBBR was an efficient up-regulation strategy to minimize hazardous CIT and CIT-TPs as much as possible.

Microbial biofilm metabolization of benzalkonium compounds (benzyl dimethyl dodecyl ammonium & benzyl dimethyl tetradecyl ammonium chloride).

Benzalkonium chlorides (BACs) are quaternary ammonium compounds (QUATs) that are used as biocides. The degradation of these compounds in wastewater treatment plants is essential to reduce their spread into the environment and thus prevent the development of QUAT-resistant genes. The biodegradation of two BACs (BAC-12 and BAC-14) was investigated in moving bed biofilm reactors (MBBRs). Degradation half-lives of 12 and 20 h for BAC-12 and - 14, respectively, were detected as well as the formation of 42 metabolites. Two new degradation pathways for the BACs were identified in this study: 1) one involving an ω-oxidation, followed by ß-oxidation and 2) one via an ω-oxidation followed by an α-oxidation that was succeeded by ß-oxidation. Similar metabolites were detected for both BAC-12 and BAC-14. Additional metabolites were detected in the study, that could not be assigned to the above-mentioned pathways, revealing even more metabolic pathways in the MBBR which is probably due to the complexity of the microbial community in the biofilm. Interestingly, both TP194 (Benzyl-(carboxymethyl)-dimethylazanium) and TP208B (Benzyl-(2-carboxyethyl)-dimethylazanium) were identified as end products of the ω/ß-pathway and the α/ß-pathway. TP208B, TP152 and TP250 that were identified in this study, as well as the known BDMA were discovered in the effluent of a wastewater treatment plant.

Eat seldom is better than eat frequently: Pharmaceuticals degradation kinetics, enantiomeric profiling and microorganisms in moving bed biofilm reactors are affected by feast famine cycle times.

Feast-famine (FF) regimes improved the removal of recalcitrant pharmaceuticals in moving bed biofilm reactors (MBBRs), but the optimal FF cycle remained unresolved. The effects of FF cycle time on the removal of bulk substrates (organic carbon and nitrogen) and trace pharmaceuticals by MBBR are systematically evaluated in this study. The feast to famine ratio was fixed to 1:2 to keep the same loading rate, but the time for the FF cycles varied from 18 h to 288 h. The MBBR adapted to the longest FF cycle time (288 h equaling 48 × HRT) resulted in significantly higher degradation rates (up to +183%) for 12 out of 28 pharmaceuticals than a continuously fed (non-FF) reactor. However, other FF cycle times (18, 36, 72 and 144 h) only showed a significant up-regulation for 2-3 pharmaceuticals compared to the non-FF reactor. Enantioselective degradation of metoprolol and propranolol occurred in the second phase of a two phase degradation, which was different for the longer FF cycle time. N-oxidation and N-demethylation pathways of tramadol and venlafaxine differed across the FF cycle time suggestin the FF cycle time varied the predominant transformation pathways of pharmaceuticals. The abundance of bacteria in the biofilms varied considerably between different FF cycle times, which possibly caused the biofilm to remove more recalcitrant bulk organic C and pharmaceuticals under long cycle times.

A two-dimensional HPLC separation for the enantioselective determination of hexabromocyclododecane (HBCD) isomers in biota samples.

A new method for enantioselective analysis of isomers of hexabromocyclododecane (HBCD) is described, using a two-dimensional high-performance liquid chromatography (HPLC) approach to avoid coelution, in particular between (+) α-HBCD, (+) ß-HBCD, or (+) γ-HBCD. After isomer separation on a conventional column, the single isomers are transferred to an enantioselective HPLC column using heart cuts. Two enantioseparations are conducted in two separate partial chromatograms: one for α-HBCD and one for ß- and γ-HBCD. The result is a completely undisturbed enantioselective separation for α-HBCD at a resolution of 4.11. A peak capacity of 107 was achieved. This peak capacity is utilized by the six peaks of the three isomers with two enantiomers each by 6%. This method was applied to samples of sand eel oil, glaucous gull, and ringed seal. The calibration was performed by treating each enantiomer as a single analyte using a multilevel internal standard calibration. Enantiomeric fractions of 0.495-0.501 with standard deviations (SDs) of 0.056-0.071 were determined for racemic standards of α-HBCD, while the values for fish oil were 0.548-0.562 with SD of 0.018-0.041, depending on the respective mass spectrometric transition.

Optimized sampling strategy for measurement of biomass properties during full-scale composting.

Biomass to be composted is often very heterogeneous and collection of representative samples for determination of compost properties is therefore difficult, especially under full-scale conditions. During full-scale composting different biomasses in the amount of 10-100 tons are mixed, yielding a very heterogeneous mixture. Final sample size for compost property determination is usually a few grams compared with compost pile masses of hundreds of tons. Desired sample particle size is about 1 mm, while compost particle size ranges from 5 to 50 cm. This study focuses on the development of a strategy for sampling under full-scale conditions for minimum measurement uncertainty based on selected material properties. Optimization was conducted considering multiple parameters, such as number of pile turnings before sampling, number of samples collected, sample mass, sample homogenization, particle size reduction and number of replicate measurements. Measurement uncertainty was evaluated using water content, inorganic matter content and nutrient (nitrogen, phosphorus) content. For each parameter measurement variability was determined as a function of sampling strategy and used to identify optimal sampling strategy.

Sartan blood pressure regulators in classical and biofilm wastewater treatment - Concentrations and metabolism.

Sartans are a group of pharmaceuticals widely used to regulate blood pressure. Their concentration levels were monitored in 80 wastewater treatment plants (WWTP) in the Baltic Sea Region, reached from limit of detection up to 6 µg/L. The concentrations were significantly different in different countries, but consistent within the respective country. The degradation of sartans (losartan, valsartan, irbesartan) in moving bed biofilm reactors (MBBRs) that utilize biofilms grown on mobile carriers to treat wastewater was investigated for the first time, and compared with the degradation in a conventional activated sludge (CAS) treatment plant. The results showed the formation of six microbial transformation products (TPs) of losartan, four of valsartan, and four of irbesartan in biological wastewater treatment. Four of these metabolites have not been described in the literature before. Chemical structures were suggested and selected TPs were verified and quantified depending on availability of true standards. Valsartan acid was a common TP of losartan, valsartan, and irbesartan. Losartan and irbesartan also shared one TP: losartan/irbesartan TP335. Based on the mass balance analysis, losartan carboxylic acid is the main TP of losartan, and valsartan acid is the main TP of valsartan during the biotransformation process. For irbesartan, TP447 is likely to be the main TP, as its peak areas were two orders of magnitude higher than those of all the other detected TPs of this compound. The effects of adapting biofilms to different biological oxygen demand (BOD) loading on the degradation of sartans as well as the formation of their TPs were investigated. Compared to feeding a poor substrate (pure effluent wastewater from a CAS), feeding with richer substrate (1/3 raw and 2/3 effluent wastewater) promoted the metabolism of most compounds (co-metabolization). However, the addition of raw wastewater inhibited some metabolic pathways of other compounds, such as from losartan/irbesartan to TP335 (competitive inhibition). The formation of irbesartan TP447 did not change with or without raw wastewater. Finally, the sartans and their TPs were investigated in a full-scale CAS wastewater treatment plant (WWTP). The removal of losartan, valsartan, and irbesartan ranged from 3.0 % to 72% and some of the transformation products (TPs) from human metabolism were also removed in the WWTP. However, some of the sartan TPs, i.e., valsartan acid, losartan carboxylic acid, irbesartan TP443 and losartan TP453, were formed in the WWTP. Relative high amounts of especially losartan carboxylic acid, which was detected with concentrations up to 2.27 µg/L were found in the effluent.

Mechanistic studies on the effect of easy degradable carbon on pharmaceuticals removal in intermittently fed moving bed biofilm reactors.

This study was conducted to provide for the first time systematic data on how intermittent feeding with carbon (ethanol) affects the kinetics of pharmaceuticals degradation in a moving bed biofilm reactor (MBBR). The relationship between the degradation rate constants (K) of 36 pharmaceuticals and the length of famine was tested with 12 different feast-famine ratios: For 17 pharmaceuticals, intermittent feeding increased K with a factor of 3-17, while for six other pharmaceuticals, it decreased K. Concerning intermittent loading, three dependencies were detected: 1) for some compounds (e.g., valsartan, ibuprofen, iohexol), the K decreased linearly with carbon loading, 2) for three compounds (2 sulfonamides and benzotriazole) K increased linearly with carbon loading 3) for most compounds (e.g., beta blockers, macrocyclic antibiotics, candesartan, citalopram, clindamycin, gabapentin) K had a maximum around 6 d famine (with 2 d feast). Optimizing processes on MBBRs need therefore be conducted based on a prioritization of compounds.

Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography.

Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants.

Species-specific time trends and enantiomer fractions of hexabromocyclododecane (HBCD) in biota from East Greenland.

Time trends of hexabromocyclododecane (HBCD) isomers were studied for glaucous gull and ringed seal from East Greenland. The ringed seal results extended a previous time trend (1986-2008) to 2010. α-HBCD was the only isomer consistently above quantification limits. For glaucous gull liver, annual median values of α-HBCD (1994-2010) ranged from 22 to 120 ng/g lipid weight (lw) with no significant trend, while HBCD in ringed seal blubber from the same area showed a significant increase from 3.9 to 11 ng/g lw (1986-2010). Reasons for this difference are unknown, but might include different feeding habits and species-specific metabolisation processes. Concentrations of several organochlorine (OC) compounds were determined for glaucous gull and ringed seal samples collected from the same area in 2004. HBCD concentrations in glaucous gull liver appeared relatively low when compared to OC concentrations in the same tissue and to both HBCD and OCs in ringed seal blubber from the same area. Enantiomer fractions (EF) deviated significantly from racemic for all annual mean EFs in glaucous gull suggesting metabolisation processes toward an enrichment of (-)α-HBCD. For ringed seal, this enrichment was less pronounced and only significant for two of the ten years. For neither species, significant changes in EF were found over time.

Further transformation of the primary ozonation products of tramadol- and venlafaxine N-oxide: Mechanistic and structural considerations.

Ozonation has been used to effectively remove micropollutants from the secondary effluent in several wastewater treatment plants. It is known that ozonation transforms tertiary amine compounds into their respective N-oxides, however in an earlier study a mass balance could not be closed at elevated ozone concentrations, leading to the assumption that more ozonation products are possible. This study was conducted to elucidate which (hitherto unknown) ozonation products can be formed from venlafaxine and tramadol when ozonating wastewater. Ozonation experiments were performed with tramadol and venlafaxine N-oxide in two different set-ups. Both tramadol- and venlafaxine N-oxide degraded during ozonation in pure (deionized) water in both semi-continuous and batch mode ozonation set-ups. 13 and 17 new transformation products were detected from tramadol- and venlafaxine N-oxide respectively, using high resolution mass spectrometry with ESI(+) ionization. Empirical chemical formulas were proposed based on the determination of the exact masses and interpretation of the product ion spectra. These transformation products result from the addition of one to three oxygen atoms and removal of C, -CH2, C2H2, C3H6, etc., from the parent molecule, respectively. Quenching experiments suggested that most of the transformation products originated from the direct reaction with ozone (eight for tramadol N-oxide and ten for venlafaxine N-oxide), whereas fewer products originated from the reaction with OH radicals (three for tramadol N-oxide and three for venlafaxine N-oxide). Reaction mechanisms and chemical structures of products are proposed, based on the available active sites and past literature on ozone reaction mechanisms. The experimental results are compared to theory and literature on ozone reactive sites and ozone reaction mechanisms. All in all this shows that there can be multiple ozonation products, and ozonation pathways can be complex, even if initially only one ozonation product is formed.

Effects of substrate loading on co-metabolic transformation pathways and removal rates of pharmaceuticals in biofilm reactors.

This study focused on the effects of substrate (raw wastewater) on the biological removal of 20 pharmaceuticals in moving bed biofilm reactors. This is the first study discriminating experimentally between effects of adaptation (45 d) and stimulation (100 h) on the removal of micropollutants. The results presented in this paper show: i) Tramadol and venlafaxine are subject to microbial N-oxidation (besides the known demethylation). ii) Changes in substrate loading, changed the preferential degradation pathways, e.g., from N-oxidation (under starvation) to N-demethylation of both model compounds: tramadol and venlafaxine, during adaptation and stimulation to high substrate supply. iii) In starving biofilms, the effects of stimulation on removal rates are minor (-100 to +150 %) in comparison to those caused by adaptation (-100 to +700 %). iv) Adaptation to high loadings resulted in increased removal rates (up to 700 % in selected cases) v) Adaptation to high loadings followed by high loading of stimulation, resulted in the highest increase of removal rates (+49 % to +1800 %) for hard-to-degrade compounds (e.g., diclofenac). All in all, this study shows that the efficiency of biofilm reactors is heavily dependent on their adaptation to substrate.

Fungi and biochar applications in bioremediation of organic micropollutants from aquatic media.

The conventional wastewater treatment system such as bacteria, is not able to remove recalcitrant micropollutants effectively. While, fungi have shown high capacity in degradation of recalcitrant compounds. Biochar, on the other hand, has gained attention in water and wastewater treatment as a low cost and sustainable adsorbent. This paper aims to review the recent applications of three major fungal divisions including Basidiomycota, Ascomycota, and Mucoromycotina, in organic micropollutants removal from wastewater. Moreover, it presents an insight into fungal bioreactors, fungal biofilm and immobilization system. Biochar adsorption capacities for organic micropollutants removal under different operating conditions are summarized. Finally, few recommendations for further research are established in the context of the combination of fungal biofilm with the technologies relying on the adsorption by porous carbonaceous materials.