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.

Wastewater analysis to monitor spatial and temporal patterns of use of two synthetic recreational drugs, ketamine and mephedrone, in Italy.

Wastewater analysis was applied in a four-year monitoring study to assess temporal and spatial patterns of ketamine and mephedrone use in the general population in Italy. Composite raw wastewater samples were collected from sewage treatment plants (STPs) in 17 cities. Target analytes were measured using a validated method based on solid phase extraction and liquid chromatography coupled to tandem mass spectrometry analysis. Mass loads were use to assess ketamine and mephedrone use and were normalized to the population served by the plants. Ketamine was detected in wastewater in all except one (Palermo) of the cities investigated, while mephedrone was detected only in Bologna and Florence. Ketamine mass loads progressively increased from 2010 to 2013, and in Milan rose from 1 to 1.5 g/day in 2008-2010 to 3.4-3.6 g/day in 2013-2014. Mass loads were higher in north and central Italy than in the south, and in larger rather than small cities. Wastewater analysis was suitable to provide objective and up-to-date information on the use of ketamine in Italy, to identify ketamine spatial and temporal changes, and to confirm the low use of mephedrone. These results can complement information from population surveys which often provide only scant and incomplete figures for these substances.

A novel approach for monitoring tobacco use in local communities by wastewater analysis.

OBJECTIVE: We propose a novel approach for measuring tobacco use in a community through the chemical analysis of nicotine metabolites in urban wastewater. It offers frequent monitoring and 'real-time', 'evidence-based' estimates of tobacco consumption which may complement epidemiological surveillance systems normally repeated only every few years. METHODS: Two urinary metabolites of nicotine, namely cotinine and trans-3'-hydroxycotinine, were selected as biomarkers of tobacco consumption in urban wastewater. During smoking, a known amount of nicotine is absorbed and after metabolism excreted as metabolites in urine, ending up in the wastewater; quantitative analysis of the metabolites in the wastewater allows back-calculation of the nicotine collectively absorbed by the population producing the sewage and, indirectly, their tobacco use. Representative samples of wastewater were collected from sewage treatment plants in eight Italian cities and analysed by mass spectrometry. Mass loads of the metabolites were used to estimate nicotine consumption. RESULTS: Wastewater analysis in the cities under study was used to estimate the number of cigarettes smoked, in order to compare the results of this study with those obtained from population surveys. The number of cigarettes calculated with the two methods were closely comparable and wastewater analysis was sufficiently sensitive to confirm the differences in tobacco consumption between northern and southern Italy, previously described in population surveys. CONCLUSIONS: The described approach can serve as a supplementary indicator of tobacco consumption in local communities. This approach can provide objective and updated information, which are useful to assess the efficacy of tobacco-control interventions, with the aim of designing and implementing effective tobacco control plans.

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.

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).

Applicability of an on-line solid-phase extraction liquid chromatography - tandem mass spectrometry for the wastewater-based assessment of human exposure to chemicals from personal care and household products.

Wastewater-based epidemiology (WBE) can be a useful complementary approach to assess human exposure to potentially harmful chemicals, including those from personal care and household products. In this work, a fully automated multiresidue method, based on on-line solid-phase extraction liquid chromatography - tandem mass spectrometry, was developed for the determination of 27 biomarkers of human exposure to selected chemicals from personal care and household products, including parabens, UV filters, phthalates and alternative plasticizers, phosphorous flame retardants/plasticizers (PFRs), and bisphenols. These biomarkers include both the parent compounds and their human metabolites. In addition, two oxidative stress biomarkers, 8-epi-prostaglandin F2α and 4-hydroxy nonenal mercapturic acid, were also considered in the study. The method was carefully optimized to tackle the challenges of analyzing compounds with different physico-chemical properties in a highly complex raw wastewater matrix, while model experiments were performed to investigate filtration losses and analyte stability. The applicability of the developed method was tested by analyzing raw wastewater from four European cities: Antwerp, Brussels (Belgium), Girona (Spain), and Zagreb (Croatia). Twenty-one biomarkers (10 parent compounds and 11 metabolites) were detected in all analyzed wastewater samples. The parent compounds with the highest mass loads were PFRs, parabens, and bisphenol S, while phthalate monoesters were the most prominent metabolites. The mass loads of most compounds were quite similar across cities, but geographic differences were observed for some biomarkers, such as metabolites of phthalates and alternative plasticizers. Exposure was then assessed for seven substances for which quantitative urinary excretion data are known. Our results indicate that safe reference values were exceeded for several contaminants, including butylated phthalates, bisphenol A, and tris(2-butoxyethyl) phosphate, particularly for toddlers. With this relatively simple method, which requires less sample manipulation, it is possible to promptly identify and monitor exposure to harmful chemicals at the population level using the WBE approach.

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.

Wastewater-based epidemiology to assess human exposure to personal care and household products - A review of biomarkers, analytical methods, and applications.

Humans are nowadays exposed to numerous chemicals in our day-to-day life, including parabens, UV filters, phosphorous flame retardants/plasticizers, bisphenols, phthalates and alternative plasticizers, which can have different adverse effects to human health. Estimating human's exposure to these potentially harmful substances is, therefore, of paramount importance. Human biomonitoring (HBM) is the existing approach to assess exposure to environmental contaminants, which relies on the analysis of specific human biomarkers (parent compounds and/or their metabolic products) in biological matrices from individuals. The main drawback is its implementation, which involves complex cohort studies. A novel approach, wastewater-based epidemiology (WBE), involves estimating exposure from the analysis of biomarkers in sewage (a pooled urine and feces sample of an entire population). One of the key challenges of WBE is the selection of biomarkers which are specific to human metabolism, excreted in sufficient amounts, and stable in sewage. So far, literature data on potential biomarkers for estimating exposure to these chemicals are scattered over numerous pharmacokinetic and HBM studies. Hence, this review provides a list of potential biomarkers of exposure to more than 30 widely used chemicals and report on their urinary excretion rates. Furthermore, the potential and challenges of WBE in this particular field is discussed through the review of pioneer WBE studies, which for the first time explored applicability of this novel approach to assess human exposure to environmental contaminants. In the future, WBE could be potentially applied as an "early warning system", which could promptly identify communities with the highest exposure to environmental contaminants.

Effects of industrial effluents containing moderate levels of antibiotic mixtures on the abundance of antibiotic resistance genes and bacterial community composition in exposed creek sediments.

Environmental discharges of very high (mg/L) antibiotic levels from pharmaceutical production contributed to the selection, spread and persistence of antibiotic resistance. However, the effects of less antibiotic-polluted effluents (µg/L) from drug-formulation on exposed aquatic microbial communities are still scarce. Here we analyzed formulation effluents and sediments from the receiving creek collected at the discharge site (DW0), upstream (UP) and 3000 m downstream of discharge (DW3000) during winter and summer season. Chemical analyses indicated the largest amounts of trimethoprim (up to 5.08 mg/kg) and azithromycin (up to 0.39 mg/kg) at DW0, but sulfonamides accumulated at DW3000 (total up to 1.17 mg/kg). Quantitative PCR revealed significantly increased relative abundance of various antibiotic resistance genes (ARGs) against ß-lactams, macrolides, sulfonamides, trimethoprim and tetracyclines in sediments from DW0, despite relatively high background levels of some ARGs already at UP site. However, only sulfonamide (sul2) and macrolide ARG subtypes (mphG and msrE) were still elevated at DW3000 compared to UP. Sequencing of 16S rRNA genes revealed pronounced changes in the sediment bacterial community composition from both DW sites compared to UP site, regardless of the season. Numerous taxa with increased relative abundance at DW0 decreased to background levels at DW3000, suggesting die-off or lack of transport of effluent-originating bacteria. In contrast, various taxa that were more abundant in sediments than in effluents increased in relative abundance at DW3000 but not at DW0, possibly due to selection imposed by high sulfonamide levels. Network analysis revealed strong correlation between some clinically relevant ARGs (e.g. blaGES, blaOXA, ermB, tet39, sul2) and taxa with elevated abundance at DW sites, and known to harbour opportunistic pathogens, such as Acinetobacter, Arcobacter, Aeromonas and Shewanella. Our results demonstrate the necessity for improved management of pharmaceutical and rural waste disposal for mitigating the increasing problems with antibiotic resistance.

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.

Pollution from azithromycin-manufacturing promotes macrolide-resistance gene propagation and induces spatial and seasonal bacterial community shifts in receiving river sediments.

Effluents from antibiotic manufacturing may contain high concentrations of antibiotics, which are the main driving force behind the selection and spread of antibiotic resistance genes in the environment. However, our knowledge about the impact of such effluent discharges on the antibiotic resistome and bacterial communities is still limited. To gain insight into this impact, we collected effluents from an azithromycin-manufacturing industry discharge site as well as upstream and downstream sediments from the receiving Sava river during both winter and summer season. Chemical analyses of sediment and effluent samples indicated that the effluent discharge significantly increased the amount of macrolide antibiotics, heavy metals and nutrients in the receiving river sediments. Quantitative PCR revealed a significant increase of relative abundances of macrolide-resistance genes and class 1 integrons in effluent-impacted sediments. Amplicon sequencing of 16S rRNA genes showed spatial and seasonal bacterial community shifts in the receiving sediments. Redundancy analysis and Mantel test indicated that macrolides and copper together with nutrients significantly correlated with community shift close to the effluent discharge site. The number of taxa that were significantly increased in relative abundance at the discharge site decreased rapidly at the downstream sites, showing the resilience of the indigenous sediment bacterial community. Seasonal changes in the chemical properties of the sediment along with changes in effluent community composition could be responsible for sediment community shifts between winter and summer. Altogether, this study showed that the discharge of pharmaceutical effluents altered physicochemical characteristics and bacterial community of receiving river sediments, which contributed to the enrichment of macrolide-resistance genes and integrons.

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.

Biotransformation of macrolide antibiotics using enriched activated sludge culture: Kinetics, transformation routes and ecotoxicological evaluation.

The biotransformation of three prominent macrolide antibiotics (azithromycin, clarithromycin and erythromycin) by an activated sludge culture, which was adapted to high concentrations of azithromycin (10 mg/L) was investigated. The study included determination of removal kinetics of the parent compounds, identification of their major biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, which enabled a tentative identification of TPs formed during the experiments. The ecotoxicological evaluation included two end-points, residual antibiotic activity and toxicity to freshwater algae. The enriched activated sludge culture was capable of degrading all studied macrolide compounds with high removal efficiencies (>99%) of the parent compounds at elevated concentrations (10 mg/L). The elimination of all three macrolide antibiotics was associated with the formation of different TPs, including several novel compounds previously unreported in the literature. Some of the TPs were rather abundant and contributed significantly to the overall mass balance at the end of the biodegradation experiments. Biodegradation of all investigated macrolides was associated with a pronounced reduction of the residual antibiotic activity and algal toxicity, indicating a rather positive ecotoxicological outcome of the biotransformation processes achieved by the enriched sludge culture.

Comprehensive determination of macrolide antibiotics, their synthesis intermediates and transformation products in wastewater effluents and ambient waters by liquid chromatography-tandem mass spectrometry.

Macrolide antibiotics are a prominent group of emerging contaminants frequently found in wastewater effluents and wastewater-impacted aquatic environments. In this work, a novel analytical method for simultaneous determination of parent macrolide antibiotics (azithromycin, erythromycin, clarithromycin and roxithromycin), along with their synthesis intermediates, byproducts, metabolites and transformation products in wastewater and surface water was developed and validated. Samples were enriched using solid-phase extraction on Oasis HLB cartridges and analyzed by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The target macrolide compounds were separated on an ACE C18 PFP column and detected using multiple reaction monitoring in positive ionization polarity. The optimized method, which included an additional extract clean-up on strong anion-exchange cartridges (SAX), resulted in high recoveries and accuracies, low matrix effects and improved chromatographic separation of the target compounds, even in highly complex matrices, such as raw wastewater. The developed method was applied to the analysis of macrolide compounds in wastewater and river water samples from Croatia. In addition to parent antibiotics, several previously unreported macrolide transformation products and/or synthesis intermediates were detected in municipal wastewater, some of them reaching µg/L levels. Moreover, extremely high concentrations of macrolides up to mg/L level were found in pharmaceutical industry effluents, indicating possible importance of this source to the total loads into ambient waters. The results revealed a significant contribution of synthesis intermediates and transformation products to the overall mass balance of macrolides in the aquatic environment.

Illicit drug consumption in school populations measured by wastewater analysis.

BACKGROUND: Analysis of student consumption of illicit drugs (ID) by school population surveys (SPS) provides information useful for prevention, but the results may be influenced by subjective factors. We explored wastewater (WW) analysis to improve the information. METHODS: We used WW analysis to measure ID consumption in eight secondary schools in Italy in 2010-13 (students aged 15-19). Samples were collected from the sewage pipes of the schools during lessons for one week each year. Samples were analysed by mass spectrometry to measure ID and consumption by students was compared to that of the general population. RESULTS: We found THCCOOH (human metabolite of THC) concentrations in 2010 indicating significant consumption of cannabis in all the schools and benzoylecgonine (human metabolite of cocaine) suggesting a limited consumption of cocaine in all but one school. Morphine was only found in traces, and amphetamine, methamphetamine, ecstasy, ketamine and mephedrone were not detectable. Repeated analysis showed cannabis stable until 2012 with increases in 2013, low cocaine and morphine levels, and none of the other ID. DISCUSSION: WW analysis suggested that students used amounts of cannabis comparable to the general population, with low, sporadic use of cocaine and opioids, but excluded the use of significant amounts of amphetamine, methamphetamine, ecstasy, ketamine and mephedrone. WW analysis was useful to confirm SPS figures and provides complementary findings for effective prevention strategies. This is the first time WW analysis has been used to investigate consumption of a large number of ID and new psychoactive substances (NPS) in schools.

Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries.

Effluents from pharmaceutical industries are recognized as significant contributors to aquatic pollution with antibiotics. Although such pollution has been mostly reported in Asia, knowledge on industrial discharges in other regions of the world, including Europe, and on the effects associated with such exposures is still limited. Thus, we performed chemical, microbiological and ecotoxicological analyses of effluents from two Croatian pharmaceutical industries during four seasons. In treated effluents of the company synthesizing macrolide antibiotic azithromycin (AZI), the total concentration of AZI and two macrolide by-products from its synthesis was 1-3 orders of magnitude higher in winter and springtime (up to 10.5 mg/L) than during the other two seasons (up to 638 µg/L). Accordingly, the highest total concentrations (up to 30 µg/L) in the recipient river were measured in winter and spring. Effluents from second company formulating veterinary antibiotics contained fluoroquinolones, trimethoprim, sulfonamides and tetracyclines ranging from low µg/L to approx. 200 µg/L. Low concentrations of these antibiotics, from below the limit of quantification to approx. few µg/L, have also been measured in the recipient stream. High frequency of culturable bacteria resistant to AZI (up to 83%) or sulfamethazine (up to 90%) and oxytetracycline (up to 50%) were also found in studied effluents. Finally, we demonstrated that toxicity to algae and water fleas often exceeded the permitted values. Most highly contaminated effluents induced multiple abnormalities in zebrafish embryos. In conclusion, using a wide array of analyses we have demonstrated that discharges from pharmaceutical industries can pose a significant ecological and public health concern due to their toxicity to aquatic organisms and risks for promoting development and spread of antibiotic resistance.

Improving wastewater-based epidemiology to estimate cannabis use: focus on the initial aspects of the analytical procedure.

Wastewater-based epidemiology is a promising and complementary tool for estimating drug use by the general population, based on the quantitative analysis of specific human metabolites of illicit drugs in urban wastewater. Cannabis is the most commonly used illicit drug and of high interest for epidemiologists. However, the inclusion of its main human urinary metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in wastewater-based epidemiology has presented several challenges and concentrations seem to depend heavily on environmental factors, sample preparation and analyses, commonly resulting in an underestimation. The aim of the present study is to investigate, identify and diminish the source of bias when analysing THC-COOH in wastewater. Several experiments were performed to individually assess different aspects of THC-COOH determination in wastewater, such as the number of freeze-thaw cycles, filtration, sorption to different container materials and in-sample stability, and the most suitable order of preparatory steps. Results highlighted the filtration step and adjustment of the sample pH as the most critical parameters to take into account when analysing THC-COOH in wastewater. Furthermore, the order of these initial steps of the analytical procedure is crucial. Findings were translated into a recommended best-practice protocol and an inter-laboratory study was organized with eight laboratories that tested the performance of the proposed procedure. Results were found satisfactory with z-scores ≤ 2.