An automated on-line turbulent flow liquid-chromatography technology coupled to a high resolution mass spectrometer LTQ-Orbitrap for suspect screening of antibiotic transformation products during microalgae wastewater treatment.

The evaluation of wastewater treatment capabilities in terms of removal of water pollutants is crucial when assessing water mitigation issues. Not only the monitoring of target pollutants becomes a critical point, but also the transformation products (TPs) generated. Since these TPs are very often unknown compounds, their study in both wastewater and natural environment is currently recognized as a tedious task and challenging research field. In this study, a novel automated suspect screening methodology was developed for a comprehensive assessment of the TPs generated from nine antibiotics during microalgae water treatment. Three macrolides (azithromycin, erythromycin, clarithromycin), three fluoroquinolones (ofloxacin, ciprofloxacin, norfloxacin) and three additional antibiotics (trimethoprim, pipemidic acid, sulfapyridine) were selected as target pollutants. The analysis of samples was carried out by direct injection in an on-line turbulent flow liquid chromatography-high resolution mass spectrometry (TFC-LC-LTQ-Orbitrap-MS/MS) system, followed by automatic data processing for compound identification. The screening methodology allowed the identification of 40 tentative TPs from a list of software predicted intermediates created automatically. Once known and unknown TPs were identified, degradation pathways were suggested considering the different mechanisms involved on their formation (biotic and abiotic). Results reveal microalgae ability for macrolide biotransformation, but not for other antibiotics such as for fluoroquinolones. Finally, the intermediates detected were included into an in-house library and applied to the identification of tentative TPs in real toilet wastewater treated in a microalgae based photobioreactor (PBR). The overall approach allowed a comprehensive overview of the performance of microalgae water treatment in a fast and reliable manner: it represents a useful tool for the rapid screening of wide range of compounds, reducing time invested in data analysis and providing reliable structural identification.

Screening and prioritization of micropollutants in wastewaters from on-site sewage treatment facilities.

A comprehensive screening of micropollutants was performed in wastewaters from on-site sewage treatment facilities (OSSFs) and urban wastewater treatment plants (WWTPs) in Sweden. A suspect screening approach, using high resolution mass spectrometry, was developed and used in combination with target analysis. With this strategy, a total number of 79 micropollutants were successfully identified, which belong to the groups of per- and polyfluoroalkyl substances (PFASs), pesticides, phosphorus-containing flame retardants (PFRs) and pharmaceuticals and personal care products (PPCPs). Results from this screening indicate that concentrations of micropollutants are similar in influents and effluents of OSSFs and WWTPs, respectively. Removal efficiencies of micropollutants were assessed in the OSSFs and compared with those observed in WWTPs. In general, removal of PFASs and PFRs was higher in package treatment OSSFs, which are based on biological treatments, while removal of PPCPs was more efficient in soil bed OSSFs. A novel comprehensive prioritization strategy was then developed to identify OSSF specific chemicals of environmental relevance. The strategy was based on the compound concentrations in the wastewater, removal efficiency, frequency of detection in OSSFs and on in silico based data for toxicity, persistency and bioaccumulation potential.

Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase - Degradation of compounds does not always eliminate toxicity.

In this study, the performance of immobilised laccase (Trametes versicolor) was investigated in combination with the mediator syringaldehyde (SYR) in removing a mixture of 38 antibiotics in an enzymatic membrane reactor (EMR). Antibiotics were spiked in osmosed water at concentrations of 10µg·L(-1) each. Laccase without mediator did not reduce the load of antibiotics significantly. The addition of SYR enhanced the removal: out of the 38 antibiotics, 32 were degraded by >50% after 24h. In addition to chemical analysis, the samples' toxicity was evaluated in two bioassays (a growth inhibition assay and the Microtox assay). Here, the addition of SYR resulted in a time-dependent increase of toxicity in both bioassays. In cooperation with SYR, laccase effectively removes a broad range of antibiotics. However, this enhanced degradation induces unspecific toxicity. If this issue is resolved, enzymatic treatment may be a valuable addition to existing water treatment technologies.

Identification of markers of cancer in urban sewage through the use of a suspect screening approach.

The administration of anticancer drugs during chemotherapy treatments has increased considerably in recent years, and based on the growing incidence of cancer worldwide there is a foreseen increase in their use over the coming years. Many anticancer drugs are not removed by conventional wastewater treatment plants (WWTPs) and can therefore reach the aquatic environment and potentially threaten aquatic life. The objective of this work was to apply a suspect screening methodology to detect chemotherapy and radiotherapy drugs and their related compounds such metabolites and/or biomarkers in wastewater. The use of logical pre-determined criteria to refine the suspect list down to a relatively small number of relevant compounds greatly improved the efficiency of the analysis. Mass accuracy, isotopic patterns and predicted retention time were used to tentatively identify the suspects. Successful identification of cancer-related suspects included two antineoplastic hormones, two X-ray contrast agents and a pyrrolizidine alkaloid related to an herbal medicine. This is the first time that a suspect screening paradigm has been successfully applied to the identification of pharmaceuticals and biomarkers related to chemotherapy in wastewater.

Characterization of ciprofloxacin-resistant isolates from a wastewater treatment plant and its receiving river.

In this study we characterised the ciprofloxacin-resistant strains isolated in biofilm and sediments from a wastewater treatment plant (WWTP) discharge point and its receiving river. We also examined the prevalence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes in these isolates and determined whether they harbour plasmid-encoded ß-lactamases such as TEM, SHV and CTX-M. Moreover, antibiotic concentrations were also measured to evaluate the level of contamination of these pharmaceuticals in the sampling area. Antibiotics were found in the range of ng L(-1) in WWTP effluents, but most of them were no longer found in downstream river. However, some fluoroquinolones were detected in sediment downstream demonstrating their high persistence and their capacity to be retained in the river sediments. Most of the ciprofloxacin-resistant isolates belonged to the Gammaproteobacteria class and 17 of them, 8 (7.6%) from the first sampling and 9 (6.1%) from the second sampling, carried a qnr gene. In particular, 15 isolates carried the qnrS gene and 2 carried the qnrB gene. Among the qnr-positive isolates, 12 harboured the aac(6')-lb-cr gene and 2 of them also carried a ß-lactamase on the same plasmid, indicating that they may be transferred simultaneously. It is also noteworthy that all qnr-positive isolates identified as Aeromonas species harboured the same qnrS allele, namely the qnrS2. This study reinforces the importance of environmental bacteria as vehicles for dissemination of antibiotic resistance genes.