Although published literature provides a clear demonstration of widespread occurrence of opioid analgesics (OAs) in the aquatic environment, analytical methods suitable for a systematic study of this pharmaceutical class, which would include a broad spectrum of opioid analgesics and their metabolites, are still missing. In this work, a comprehensive multiresidue method for quantitative analysis of 27 opioid analgesics and their metabolites, including 2 morphine glucuronide conjugates, was developed and validated for three matrices: raw wastewater (RW), secondary effluent (SE) and river water. The method comprised different classes of opioid analgesics, including natural opiates (morphine and codeine), their semi-synthetic derivatives (hydrocodone, hydromorphone, oxycodone, oxymorphone and buprenorphine) as well as fully synthetic opioids such as methadone, fentanyl, sufentanil, propoxyphene and tramadol. The optimized enrichment procedure involved mixed-mode, strong cation-exchange sorbent in combination with a sequential elution procedure. The extracts were analyzed by reversed-phase liquid chromatography using a Synergy Polar column coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS). Accurate quantification of target OAs was achieved using 19 deuterated analogues as surrogate standards. Method accuracies for RW, SE and river water varied in the range from 91 to 126%, 74 to 120% and 75 to 116%, respectively. Careful optimization of the procedure allowed reliable determination of OAs with method quantification limits in the low ng/L range (RW: 0.3-3.5 ng/L; SE: 0.2-1.9 ng/L, river water: 0.1-0.8 ng/L. The developed method was applied for analysis of RW, SE and river water samples from Croatia. The concentrations of individual OAs in municipal wastewater varied in a wide range (from < QL to 859 ng/L) and the most prevalent representatives were tramadol, codeine, morphine and methadone and their derivatives. Elevated concentrations of morphine glucuronides (up to 370 ng/L) found in raw municipal wastewater indicated their importance in the overall morphine mass balance.
A one-year study on the occurrence and fate of macrolide antibiotics and their metabolites, synthesis by-products 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 non-target 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 insitu transformations. Due to the high inputs, 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, by-products and TPs for the overall mass balance of macrolide antibiotics in urban wastewater systems.
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 ± 18 to 156 ± 37 doses/day/1000 inhabitants 15-64 years), followed by heroin (from 11 ± 10 to 71 ± 19 doses/day/1000 inhabitants 15-64 years), cocaine (from 8.3 ± 0.9 to 23 ± 4.0 doses/day/1000 inhabitants 15-64 years) and amphetamine (from 1.3 ± 0.9 to 21 ± 6.1 doses/day/1000 inhabitants 15-64 years) whereas the consumption of MDMA was comparatively lower (from 0.18 ± 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.
Methodology details Preparation of the phosphate-buffer minimal salts mediumGrowth media composed of K2HPO4 (4.4 g/L), KH2PO4 (1.7 g/L) and yeast extract (50 mg/L) was sterilized by autoclaving (121 o C, 20 min.) after which it was supplemented with 10% salts medium. Filter sterilized (0.20 μm) 100x salts stock contained MgSO4x7H2O (19.5 g/L), MnSO4x4H20 (6.6 g/L), FeSO4x7H20 (1 g/L), CaCl2x6H20 (0.447 g/L) and several drops of concentrated H2SO4 to prevent precipitation of basic salts. Depending of the experiment organic carbon source in the form of glucose and nitrogen source, in the form of NH4Cl, were filter sterilized and added to the growth medium. DNA extraction procedure, PCR amplifications and analysis detailsCultures were centrifuged (10 000 g, 10 min) and pellets were resuspended in the elution buffer.DNA was extracted from the harvested cells by combining chemical (addition of Lysis buffer and Proteinase K) and mechanical lysis steps (agitation for 12 min at max. speed, Vortex-Genie (MoBio). After centrifugation (11 000 g, 30 s) supernatant containing DNA was collected and transferred onto the Nucleospin Microbial DNA Column. DNA was purified by washing silica membrane with 2 different buffers following centrifugation steps (11 000 g, 30 s). DNA was eluted from the column after the addition of the elution buffer, incubation at room temperature (1 min) and subsequent centrifugation step (11 000 g, 30 s). Quality and quantity of the extracted DNA was assessed by NanoDrop spectrophotometer (BioSpec-nano, Shimadzu). PCR program used for amplification of the V4 16S rRNA region with primers 515/806 included: 94°C (for 3 min), 30 cycles (5 cycle used on PCR products) of 94°C (for 30 s), 53°C (for 40 s) and 72°C (for 1 min), after which a final elongation step at 72°C (5 min) was performed. After amplification,
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 and 1.5 days. A significantly faster elimination (t 1/2 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 (t 1/2 = 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.
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