The Toxin-Antitoxin System DarTG Catalyzes Reversible ADP-Ribosylation of DNA.

The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

Occurrence and Behavior of Macrolide Antibiotics in Municipal Wastewater Treatment: Possible Importance of Metabolites, Synthesis Byproducts, and Transformation Products.

A one-year study on the occurrence and fate of macrolide antibiotics and their metabolites, synthesis byproducts, and transformation products (TPs) was performed in the wastewater treatment plant of the city of Zagreb (Croatia). The target compounds were found in all analyzed influent and effluent samples with the total concentrations of azithromycin-, clarithromycin-, and erythromycin-related compounds reaching up to 25, 12, and 0.25 µg/L, respectively. The most prominent individual constituents were the parent macrolides azithromycin and clarithromycin. However, a substantial contribution of their derivatives, formed by deglycolysation and microbial phosphorylation, was also detected. In addition, widespread presence of several linearized nontarget TPs was confirmed for the first time in real wastewater samples by suspect screening analysis. Complex characterization of macrolide-derived compounds enabled decoupling of industrial and therapeutic sources from the in situ transformations. Due to the high inputs and incomplete removal and/or formation of several TPs during the conventional wastewater treatment, the average mass load of azithromycin-related compounds in secondary effluents exceeded 3.0 g/day/1000 inhabitants. This is the first study to reveal the importance of metabolites, byproducts, and TPs for the overall mass balance of macrolide antibiotics in urban wastewater systems.

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor.

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.

The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase.

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Catabolism of terbuthylazine by mixed bacterial culture originating from s-triazine-contaminated soil.

The s-triazine herbicide terbuthylazine (TERB) has been used as the main substitute of atrazine in many EU countries for more than 10 years. However, the ecological consequences of this substitution are still not fully understood. Since the fate of triazine herbicides is primarily dependent on microbial degradation, in this paper, we investigated the ability of a mixed bacterial culture, M3-T, originating from s-triazine-contaminated soil, to degrade TERB in liquid culture and soil microcosms. The M3-T culture grown in mineral medium with TERB as the N source and citrate as the C source degraded 50 mg L(-1) of TERB within 3 days of incubation. The culture was capable of degrading TERB as the sole C and N source, though at slower degradation kinetics. A thorough LC-MS analysis of the biodegradation media showed the formation of hydroxyterbuthylazine (TERB-OH) and N-t-butylammelide (TBA) as major metabolites, and desethylterbuthylazine (DET), hydroxydesethylterbuthylazine (DET-OH) and cyanuric acid (CA) as minor metabolites in the TERB degradation pathway. TBA was identified as a bottleneck in the catabolic pathway leading to its transient accumulation in culture media. The supplementation of glucose as the exogenous C source had no effect on TBA degradation, whereas citrate inhibited its disappearance. The addition of M3-T to sterile soil artificially contaminated with TERB at 3 mg kg(-1) of soil resulted in an accelerated TERB degradation with t 1/2 value being about 40 times shorter than that achieved by the native microbial community. Catabolic versatility of M3-T culture makes it a promising seed culture for accelerating biotransformation processes in s-triazine-contaminated environment.

Uptake/depuration kinetics, bioaccumulation potential and metabolic transformation of a complex pharmaceutical mixture in zebrafish (Danio rerio).

Uptake and elimination kinetics, bioconcentration factors (BCFs), and metabolic transformation of 20 different pharmaceutically active compounds (PhACs), covering a wide range of therapeutic categories and physico-chemical properties, were studied using zebrafish (Danio rerio). The fish were exposed to the mixture of the selected PhACs at environmentally relevant concentrations similar to 10 µg L-1. The experiments were performed in semi-static conditions and comprised a 7-day uptake period followed by a 7-day depuration period. Most of the PhACs reached a concentration plateau within the 7-day uptake-phase which was followed by an efficient depuration, with the observed uptake (ku) and depuration rate constants (kd,) ranging between 0.002 and 3.752 L kg-1 h-1, and 0.010 to 0.217 h-1, respectively. The investigated PhACs showed low to moderate BCFs. The highest BCFs of 47.8, 28.6 and 47.6 L kg-1 were determined for sertraline, diazepam and desloratadine, respectively. A high contribution of metabolic products to the total internal concentration was observed for some PhACs such as codeine (69%), sulfamethoxazole (51%) and verapamil (87%), which has to be taken into account when assessing the bioconcentration potential. Moreover, most of the metabolites exhibited significantly longer half-lives in zebrafish than their parent compounds and affected the overall depuration kinetics.

Identification of P-glycoprotein inhibitors in contaminated freshwater sediments.

P-glycoprotein (P-gp, ABCB1) is an important part of the multixenobiotic resistance (MXR) defense system in aquatic organisms. The main goal of this study was identification of P-gp inhibitors in contaminated sediments using the effect-directed analysis (EDA) approach. The samples were collected from the Gorjak creek (Zagreb, Croatia), a recipient of wastewater effluents from the pharmaceutical industry. Sediment samples were extracted and fractionated using a two-tiered approach. Resulting nonpolar, medium polar, and polar fractions were tested on the inhibition of P-gp activity using P-gp overexpressing PLHC-1/dox cells and calcein-AM as model substrate. The obtained EC50 values (up to 757 µg/g, expressed in toxicity equivalents of model P-gp inhibitor cyclosporine A) revealed high inhibitory potential of polar fractions of investigated sediments and clearly reflected the impact of pharmaceutical wastewater. P-gp specific ATPase assay and the cytotoxicity modulation experiments with colchicine indicated that most of the observed P-gp inhibition was due to the presence of noncompetitive inhibitors. A detailed chemical analysis by ultrahigh-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOFMS) revealed nonionic surfactants, including alcohol polyethoxylates (LAEOs) and polypropylene glycols (PPGs), as the major components of the most active subfractions. Testing of several LAEO and PPG commercial mixtures confirmed their potential to inhibit the fish P-glycoprotein and modulate toxicity of other xenobiotics present in complex environmental samples.

Integrated procedure for multiresidue analysis of dissolved and particulate drugs in municipal wastewater by liquid chromatography-tandem mass spectrometry.

For the first time, an integrated procedure for a quantitative multiresidue analysis of dissolved and particulate illicit drug target residues was developed and validated in three different wastewater matrices. The procedure consists of a comprehensive sample enrichment, fractionation and cleanup followed by the determination of target analytes by triple quadrupole liquid chromatography-tandem mass spectrometry in both positive and negative ionisation polarities. The enrichment of illicit drugs from suspended solids and aqueous samples was performed using pressurised liquid extraction and solid phase extraction (SPE), respectively. The performance of different SPE cartridges was investigated in order to optimise the overall recovery and to reduce the matrix effects. The optimal results were obtained by combining mixed cation exchange (Oasis MCX) cartridges for fractionated enrichment, weak anion exchange for an additional extract cleanup and optimised chromatographic separation to minimise the impact from co-extracted interferences. The method was applied for the analysis of raw wastewater (RW), activated sludge (AS) and secondary effluent (SE) samples collected at four different wastewater treatment plants. The average contributions of the particulate drugs in the RW and AS were 1-28 and 23-65 %, respectively. This suggested that the total mass loads of some drugs might be underestimated by neglecting the particulate fraction. Moreover, relatively high distribution coefficients, determined for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (RW = 1211 L/kg) and 11-hydroxy-Δ(9)-tetrahydrocannabinol (RW = 1,786 L/kg) implied that adsorption might play a significant role in their overall removal during wastewater treatment.

Multiclass determination of drug residues in water and fish for bioaccumulation potential assessment.

In this work, a wide-scope liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of environmental levels of multiclass drugs and their metabolites in water and fish samples was developed. The method allowed the reliable determination of 44 drugs, covering a rather wide range of chemistries and physicochemical characteristics. In order to obtain a reliable and robust analytical protocol, different combinations of extraction and cleanup techniques were systematically examined. Aqueous samples were extracted using a simple Oasis HLB SPE enrichment protocol with pH-optimized sample percolation (pH 3). The extraction of cryo-homogenized biota samples was performed using double extraction with MeOH basified with 0.5% NH3, which allowed high extraction recoveries for all target analytes. The problem of the coextracted lipid matrix, which is known to be the key obstacle for reliable biota analysis, was systematically examined in a series of model cleanup experiments. A combination of cryo-precipitation, filtration, and HLB SPE cleanup was proposed as a protocol, which allowed reliable and robust analysis of all target compounds at low ng/g levels. At the final conditions, the method which was validated at three concentration levels showed high extraction recoveries (68-97%), acceptable matrix effects (12 to -32%), accuracies (81-129%), and reproducibilities (3-32%) for all analytes. The developed method was used to determine drug concentrations in river water and in feral freshwater fish, including whole fish and muscle tissue, from the Sava River (Croatia), in order to estimate their corresponding bioaccumulation potential. With respect to bioaccumulation potential in whole fish and fish muscle, the most relevant drugs were lisinopril, sertraline, terbinafine, torsemide, diazepam, desloratadine, and loratadine with estimated bioaccumulation factors ranging from 20 to 838 and from 1 to 431, respectively.

Phytoplankton pigment dynamics in marine lake fluctuating between stratified and holomictic euxinic conditions.

Biomass dynamics in the marine lake are strongly dependent on seasonal variability in vertical stratification, indicating rapid adaptation of phytoplankton to short-term changes in the water column. A small marine lake (Rogoznica Lake, Croatia), which fluctuates between stably stratified and holomictic euxinic conditions, was used as a model to study the phytoplankton responses to environmental perturbations, in particular the anoxic stress, caused by periodic holomixia. The epilimnion showed significant temporal and vertical variability with a chlorophyll a subsurface maximum with the highest biomass near the chemocline. Fucoxanthin-containing biomass (diatoms) dominated in the epilimnion in colder seasons and was first to recover after holomictic euxinic events. The shift towards the smaller groups prevailed during highly stratified water column conditions in warmer seasons. Results for the hypolimnion were more enigmatic, with high concentrations of alloxanthin, zeaxanthin, and violaxanthin indicating the presence of a viable small-size mixotrophic community under extreme conditions.

Assessing the impact of a major electronic music festival on the consumption patterns of illicit and licit psychoactive substances in a Mediterranean city using wastewater analysis.

The consumption patterns of five categories of psychoactive substances (PS), including "conventional" illicit drugs, new psychoactive substances (NPS), therapeutic opioids, alcohol and nicotine, were studied in the city of Split, Croatia, using wastewater-based epidemiology (WBE), with an emphasis on the impact of a large electronic music festival. The study involved the analysis of 57 urinary biomarkers of PS in raw municipal wastewater samples collected in three characteristic periods, including the festival week in the peak-tourist season (July) and reference weeks in the peak-tourist season (August) and the off-tourist season (November). Such a large number of biomarkers allowed the recognition of distinct patterns of PS use associated with the festival, but also revealed some subtle differences between summer and autumn seasons. The festival week was characterized by markedly increased use of illicit stimulants (MDMA: 30-fold increase; cocaine and amphetamine: 1.7-fold increase) and alcohol (1.7-fold increase), while consumption of other common illicit drugs (cannabis and heroin), major therapeutic opioids (morphine, codeine and tramadol) and nicotine remained rather constant. Interestingly, NPS and methamphetamine clearly contributed to the festival PS signature in wastewater, but their prevalence was rather low compared to that of common illicit drugs. Estimates of cocaine and cannabis use were largely consistent with prevalence data from national surveys, whereas differences were found for typical amphetamine-type recreational drugs, particularly MDMA, and for heroin. The WBE data suggest that the largest proportion of morphine came from heroin consumption and that the percentage of heroin users seeking treatment in Split is probably rather low. The prevalence of smoking calculated in this study (30.6 %) was consistent with national survey data for 2015 (27.5-31.5 %), while the average alcohol consumption per capita >15 years (5.2 L) was lower than sales statistics suggest (8.9 L).

Identification of biotransformation products of macrolide and fluoroquinolone antimicrobials in membrane bioreactor treatment by ultrahigh-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was applied for the identification of transformation products (TPs) of fluoroquinolone (norfloxacin and ciprofloxacin) and macrolide (azithromycin, erythromycin, and roxitromycin) antimicrobials in wastewater effluents from a Zenon hollow-fiber membrane bioreactor (MBR). The detected TPs were thoroughly characterized using the accurate mass feature for the determination of the tentative molecular formulae and MS-MS experiments for the structural elucidation of unknowns. Several novel TPs, which have not been previously reported in the literature, were identified. The TPs of azithromycin and roxithromycin, identified in MBR effluent, were conjugate compounds, which were formed by phosphorylation of desosamine moiety. Transformation of fluoroquinolones yielded two types of products: conjugates, formed by succinylation of the piperazine ring, and smaller metabolites, formed by an oxidative break-up of piperazine moiety to form the 7-[(2-carboxymethyl)amino] group. A semi-quantitative assessment of these TPs suggested that they might have contributed significantly to the overall balance of antimicrobial residues in MBR effluents and thus to the overall removal efficiency. Determination of TPs during a period of 2 months indicated a conspicuous dynamics, which warrants further research to identify microorganisms involved and treatment conditions leading to their formation.

Assessment of toxicological profiles of the municipal wastewater effluents using chemical analyses and bioassays.

The hazardous chemical contamination of untreated wastewater and secondary effluent from the wastewater treatment plant (WWTP) of the city of Zagreb, Croatia was comprehensively characterized using large-volume solid-phase extraction (SPE) and silica gel fractionation, followed by a detailed analysis of the resulting extracts by a combination of chemical and bioassay methods. Over 100 individual contaminants or closely related-contaminant groups were identified by high-resolution gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF). Ecotoxicity profiling of the investigated samples, including cytotoxicity, chronic toxicity and EROD activity; inhibition of the multixenobiotic resistance (MXR), genotoxicity and estrogenic potential, revealed the most significant contribution of toxic compounds to be present in polar fractions. Wastewater treatment using conventional activated sludge process reduced the initial toxicity of raw wastewater to various extents, ranging from 28% for algal toxicity to 73.2% for an estrogenic activity. The most efficient toxicity removal was observed for the polar compounds.

Analysis and occurrence of macrolide residues in stream sediments and underlying alluvial aquifer downstream from a pharmaceutical plant.

Macrolide antibiotics azithromycin (AZI), erythromycin (ERY) and clarithromycin (CLA) have been recently included in the EU Watch List of contaminants of emerging concern in the aquatic environment. However, their comprehensive assessment in different environmental compartments, by including synthesis intermediates, by-products and transformation products, is still missing. In this work, a novel method, based on pressurized liquid extraction and liquid chromatography-tandem mass spectrometry, was developed and validated for the determination of such an extended range of macrolide residues in sediment and soil samples at low ng/g levels. The method was applied to determine distribution of 13 macrolides in surface and alluvial aquifer sediments collected in a small stream with a history of chronic exposure to wastewater discharges from AZI production. The total concentrations of the target macrolide compounds in surface sediments were up to 29 µg/g and the most prominent individual macrolides were parent AZI, its synthesis intermediate N-demethyl AZI and transformation products decladinosyl AZI and N'-demethyl AZI. Some ERY-related compounds, originating from AZI synthesis, were also frequently detected, though at lower concentration levels (up to 0.31 ng/g in total). The distribution of macrolide residues in surface sediments indicated their active longitudinal transport by resuspension and redeposition of the contaminated sediment particles. The vertical concentration profiles in stream sediments and the underlying alluvial aquifer revealed that macrolide residues reached deeper alluvial sediments (up to 5 m). Moreover, significant levels of macrolides were found in groundwater samples below the streambed, with the total concentrations reaching up to 1.7 µg/L. This study highlights the importance of comprehensive chemical characterization of the macrolide residues, which were shown to persist in surface and alluvial aquifer sediment more than ten years after their discharge into the aquatic environment.

Selective interaction of microcystin congeners with zebrafish (Danio rerio) Oatp1d1 transporter.

Microcystins (MCs) are the most studied cyanotoxins. The uptake of MCs in cells and tissues of mammals and fish species is mostly mediated by organic anion-transporting polypeptides (OATPs in humans and rodents; Oatps in other species), and the Oatp1d1 appears to be a major transporter for MCs in fish. In this study, six MC congeners of varying physicochemical properties (MC-LR, -RR, -YR, -LW, -LF, -LA) were tested by measuring their effect on the uptake of model Oatp1d1 fluorescent substrate Lucifer yellow (LY) in HEK293T cells transiently or stably overexpressing zebrafish Oatp1d1. MC-LW and -LF showed the strongest interaction resulting in an almost complete inhibition of LY transport with IC50 values of 0.21 and 0.26 µM, while congeners -LR, -YR and -LA showed lower inhibitory effects. To discern between Oatp1d1 substrates and inhibitors, results were complemented by Michaelis-Menten kinetics and chemical analytical determinations of MCs uptake, along with molecular docking studies performed using the developed zebrafish Oatp1d1 homology model. Our study showed that Oatp1d1-mediated transport of MCs could be largely dependent on their basic physicochemical properties, with log POW being the most obvious determinant. Finally, apart from determination of the chemical composition of cynobacterial blooms, a reliable risk assessment should take into account the interaction of identified MC congeners with Oatp1d1 as their primary transporter, and herewith we demonstrated that such a comprehensive approach could be based on the use of highly specific in vitro models, accompanied by chemical assessment and in silico molecular docking studies.

Aerobic biodegradation of tramadol by pre-adapted activated sludge culture: Cometabolic transformations and bacterial community changes during enrichment.

The biodegradation of biorecalcitrant opioid drug tramadol (TRAM) was studied in a model biodegradation experiment performed with an enriched activated sludge culture pre-adapted to high concentration of TRAM (20 mg/L). TRAM and its transformation products (TPs) were determined by applying ultrahigh-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS), the sludge culture was characterized using a 16S rRNA gene amplicon sequencing, whereas ecotoxicological evaluation was performed based on determination of toxicity to freshwater algae. Tramadol removal was much faster (t1/2 = 1.3 days) and more efficient in glucose-containing mineral medium (cometabolic conditions) than in a medium without glucose. The elimination of the parent compound resulted in the formation of five TPs, two of which (TP 249 and TP 235) were identified as N-desmethyltramadol (N-DM TRAM) and N,N-didesmethyltramadol (N,N-diDM TRAM). The remaining 3 TPs (TP 277a-c) were isomeric compounds with an elemental composition of protonated molecules C16H24NO3 and a putative structure which involved oxidative modification of the dimethylamino group. Pronounced changes in the taxonomic composition of the activated sludge were observed during the enrichment, especially regarding an enhanced percentage of 8 genera (Bacillus, Mycobacterium, Enterobacter, Methylobacillus, Pedobacter, Xanthobacter, Leadbetterella and Kaistia), which might be related to the observed transformations. The removal of TRAM resulted in proportional reduction of algal toxicity, implying a positive result of the accomplished transformation processes.

The occurrence of contaminants of emerging concern in Slovenian and Croatian wastewaters and receiving Sava river.

This study investigated the occurrence of 48 contaminants of emerging concern (CECs) in wastewater effluents from three Slovenian and three Croatian waste water treatment plants (WWTPs) representing the major inputs into the upper and middle course of the Sava River and simultaneously in the Sava River itself. Two sampling campaigns were carried out (May and July 2017). Samples were extracted using solid-phase extraction and analysed by gas chromatography - mass spectrometry. In effluents, 23 CECs were >LOQ with caffeine and the UV-filter 4-hydroxybenzophenone (H-BP) present in the highest concentrations (<49,600 ng L-1 and <28,900 ng L-1, respectively) and most frequently detected (detection frequency; DFr > 83.3%). Bisphenol B and E were detected for the first time in WW from Velika Gorica (May) and Zapresic (July), respectively. In surface water (SW), 19 CECs were detected >LOQ with CAF again being the most abundant and most frequently detected (DFr = 92.9%). Bisphenols AP, CL2, P and Z were detected >LOQ for the first time in European SW. Active pharmaceutical ingredients naproxen, ketoprofen, carbamazepine and diclofenac; the preservative methyl paraben; CAF and UV-filter HM-BP were the most abundant CECs in SW and WW. An increasing trend in the total CEC load downstream was observed, indicating the cumulative effects of individual sources along the river. The Croatian Zapresic, Zagreb and Velika Gorica WWTP effluents contributed the most towards the enhanced loads of the CECs studied probably due to their size or insufficient treatment. HM-BP was the only compound found at a levels exhibiting high environmental risk (RQ = 1.13) downstream from Ljubljana and Domzale-Kamnik WWTPs. Other SW samples that contained HM-BP, ibuprofen (API) and/or benzyl paraben (preservative) posed a medium risk to the environment. The results suggest the need for further monitoring of CECs in the Sava River Basin.

Biodegradation study of methadone by adapted activated sludge: Elimination kinetics, transformation products and ecotoxicological evaluation.

The biotransformation study of difficult-to-degrade opioid analgesic methadone (MTHD) was performed by activated sludge culture adapted to high concentration of methadone (10 mg/L). The study included determination of elimination kinetics of the parent compound, taxonomic characterization of microbial culture, identification of biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation processes. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, whereas the ecotoxicological assessment was made based on determinations of toxicity to freshwater algae. Changes of the adapted sludge culture during the experiment were followed using the 16S rRNA gene amplicon sequencing. Depending on the experimental conditions, the elimination efficiency of methadone (10 mg/L) varied from 9% to 93% with the corresponding half-lives from 11.4 days to 1.5 days. A significantly faster elimination (t1/2 from 1.5 days to 5.8 days) was achieved at cometabolic conditions, using glucose-containing media, as compared to the experiments with MTHD as a single organic carbon source (t1/2 = 11.4 days). Moreover, increased biotransformation rate following the additional supplementation of ammonia, revealed a possible importance of nitrogen availability for the transformation at cometabolic conditions. The elimination of parent compound was associated with the formation of 3 different TPs, two of which were identical to main human metabolites of MTHD, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP). EDDP represented over 90% of the total TP concentration at the end of experiment. The biodegradation of MTHD was associated with a pronounced drop in algal toxicity, confirming a rather positive ecotoxicological outcome of the achieved biotransformation processes.

Long-term monitoring of drug consumption patterns in a large-sized European city using wastewater-based epidemiology: Comparison of two sampling schemes for the assessment of multiannual trends.

A comprehensive study aimed at monitoring of temporal variability of illicit drugs (heroin, cocaine, amphetamine, MDMA, methamphetamine and cannabis) and therapeutic opiate methadone in a large-sized European city using wastewater-based epidemiology (WBE) was conducted in the city of Zagreb, Croatia, during an 8-year period (2009-2016). The study addressed the impact of different sampling schemes on the assessment of temporal drug consumption patterns, in particular multiannual consumption trends and documented the possible errors associated with the one-week sampling scheme. The highest drug consumption prevalence was determined for cannabis (from 59 ±â€¯18 to 156 ±â€¯37 doses/day/1000 inhabitants 15-64 years), followed by heroin (from 11 ±â€¯10 to 71 ±â€¯19 doses/day/1000 inhabitants 15-64 years), cocaine (from 8.3 ±â€¯0.9 to 23 ±â€¯4.0 doses/day/1000 inhabitants 15-64 years) and amphetamine (from 1.3 ±â€¯0.9 to 21 ±â€¯6.1 doses/day/1000 inhabitants 15-64 years) whereas the consumption of MDMA was comparatively lower (from 0.18 ±â€¯0.08 to 2.7 doses ±0.7 doses/day/1000 inhabitants 15-64 years). The drug consumption patterns were characterized by clearly enhanced weekend and Christmas season consumption of stimulating drugs (cocaine, MDMA and amphetamine) and somewhat lower summer consumption of almost all drugs. Pronounced multiannual consumption trends were determined for most of the illicit drugs. The investigated 8-year period was characterized by a marked increase of the consumption of pure cocaine (1.6-fold), THC (2.7-fold), amphetamine (16-fold) and MDMA (15-fold) and a concomitant decrease (2.3-fold) of the consumption of pure heroin. The heroin consumption decrease was associated with an increase of methadone consumption (1.4-fold), which can be linked to its use in the heroin substitution therapy. The estimated number of average methadone doses consumed in the city of Zagreb was in a good agreement with the prescription data on treated opioid addicts in Croatia.

Occurrence and fate of emerging wastewater contaminants in Western Balkan Region.

This paper reports on a comprehensive reconnaissance of over seventy individual wastewater contaminants in the region of Western Balkan (WB; Bosnia and Herzegovina, Croatia and Serbia), including some prominent classes of emerging contaminants such as pharmaceuticals and personal care products, surfactants and their degradation products, plasticizers, pesticides, insect repellents, and flame retardants. All determinations were carried out using a multiresidue analytical approach, based on the application of gas chromatographic and liquid chromatographic techniques coupled to mass spectrometric detection. The results confirmed a widespread occurrence of the emerging contaminants in municipal wastewaters of the region. The most prominent contaminant classes, determined in municipal wastewaters, were those derived from aromatic surfactants, including linear alkylbenzene sulphonates (LAS) and alkylphenol polyethoxylates (APEO), with the concentrations in raw wastewater reaching into the mg/l range. All other contaminants were present in much lower concentrations, rarely exceeding few microg/l. The most abundant individual compounds belonged to several classes of pharmaceuticals (antimicrobials, analgesics and antiinflammatories, beta-blockers and lipid regulators) and personal care products (fragrances). Due to the rather poor wastewater management practices in WB countries, with less than 5% of all wastewaters being biologically treated, most of the contaminants present in wastewaters reach ambient waters and may represent a significant environmental concern.