Investigating the Transformation Products of Selected Antibiotics and 17 α-Ethinylestradiol under Three In Vitro Biotransformation Models for Anticipating Their Relevance in Bioaugmented Constructed Wetlands.

The degradation of three antibiotics (sulfamethoxazole, trimethoprim, and ofloxacin) and one synthetic hormone (17 α-ethinylestradiol) was investigated in three in-vitro biotransformation models (i.e., pure enzymes, hairy root, and Trichoderma asperellum cultures) for anticipating the relevance of the formation of transformation products (TPs) in constructed wetlands (CWs) bioaugmented with T. asperellum fungus. The identification of TPs was carried out employing high-resolution mass spectrometry, using databases, or by interpreting MS/MS spectra. An enzymatic reaction with ß-glucosidase was also used to confirm the presence of glycosyl-conjugates. The results showed synergies in the transformation mechanisms between these three models. Phase II conjugation reactions and overall glycosylation reactions predominated in hairy root cultures, while phase I metabolization reactions (e.g., hydroxylation and N-dealkylation) predominated in T. asperellum cultures. Following their accumulation/degradation kinetic profiles helped in determining the most relevant TPs. Identified TPs contributed to the overall residual antimicrobial activity because phase I metabolites can be more reactive and glucose-conjugated TPs can be transformed back into parent compounds. Similar to other biological treatments, the formation of TPs in CWs is of concern and deserves to be investigated with simple in vitro models to avoid the complexity of field-scale studies. This paper brings new findings on the emerging pollutants metabolic pathways established between T. asperellum and model plants, including extracellular enzymes.

Use of Passive and Grab Sampling and High-Resolution Mass Spectrometry for Non-Targeted Analysis of Emerging Contaminants and Their Semi-Quantification in Water.

Different groups of organic micropollutants including pharmaceuticals and pesticides have emerged in the environment in the last years, resulting in a rise in environmental and human health risks. In order to face up and evaluate these risks, there is an increasing need to assess their occurrence in the environment. Therefore, many studies in the past couple of decades were focused on the improvements in organic micropollutants' extraction efficiency from the different environmental matrices, as well as their mass spectrometry detection parameters and acquisition modes. This paper presents different sampling methodologies and high-resolution mass spectrometry-based non-target screening workflows for the identification of pharmaceuticals, pesticides, and their transformation products in different kinds of water (domestic wastewater and river water). Identification confidence was increased including retention time prediction in the workflow. The applied methodology, using a passive sampling technique, allowed for the identification of 85 and 47 contaminants in the wastewater effluent and river water, respectively. Finally, contaminants' prioritization was performed through semi-quantification in grab samples as a fundamental step for monitoring schemes.

Comparison of sunlight-AOPs for levofloxacin removal: kinetics, transformation products, and toxicity assay on Escherichia coli and Micrococcus flavus.

Levofloxacin (LFX) is a widely used antibiotic medication. Persistent traces of LFX in water and wastewater may induce bacterial resistance. Photon-driven advanced oxidation processes (AOPs) can assist in attaining complete abatement of LFX for environmental protection. This work benchmarks different solar AOPs based on hydroxyl radical (OH•) and sulphate radical (SO4•-) chemistry. Other oxidant precursors, as radical sources, were used to selectively control the generation of either hydroxyl radical (i.e., H2O2), sulphate radical (i.e., peroxydisulphate (PDS)), or a controlled mixture ratio of both OH•/SO4•- (i.e., peroxymonosulphate (PMS)). The influence of pH on degradation performance was evaluated using unbuffered and buffered solutions. Simulated irradiation/PMS process exhibited a strong pH-dependence attaining partial degradation of ca. 56% at pH 5 up to complete degradation at pH 7. Despite the similitudes on the abatement of target pollutant LFX in pristine solutions, only simulated irradiation/PDS treatment achieved effective abatement of LFX in wastewater samples given the higher selectivity of SO4•-. Toxicity tests were conducted with Escherichia coli (LMG2092) and Micrococcus flavus (DSM1790), demonstrating successful inhibition of the antibiotic character of polluted waters, which would contribute to preventing the development of resistant bacterial strains. Finally, a degradative pathway was suggested from the by-products and intermediates identified by LC-MS. Results demonstrate that the degradation of specific functional groups (i.e., piperazine ring) is associated with the loss of antibacterial character of the molecule.

Untargeted Metabolomics Studies on Drug-Incubated Phragmites australis Profiles.

Plants produce a huge number of functionally and chemically different natural products that play an important role in linking the plant with the adjacent environment. Plants can also absorb and transform external organic compounds (xenobiotics). Currently there are only a few studies concerning the effects of xenobiotics and their transformation products on plant metabolites using a mass spectrometric untargeted screening strategy. This study was designed to investigate the changes of the Phragmites australis metabolome following/after diclofenac or carbamazepine incubation, using a serial coupling of reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) combined with accurate high-resolution time-of-flight mass spectrometer (TOF-MS). An untargeted screening strategy of metabolic fingerprints was developed to purposefully compare samples from differently treated P. australis plants, revealing that P. australis responded to each drug differently. When solvents with significantly different polarities were used, the metabolic profiles of P. australis were found to change significantly. For instance, the production of polyphenols (such as quercetin) in the plant increased after diclofenac incubation. Moreover, the pathway of unsaturated organic acids became more prominent, eventually as a reaction to protect the cells against reactive oxygen species (ROS). Hence, P. australis exhibited an adaptive mechanism to cope with each drug. Consequently, the untargeted screening approach is essential for understanding the complex response of plants to xenobiotics.

Nitric oxide reactivity accounts for N-nitroso-ciprofloxacin formation under nitrate-reducing conditions.

The formation of N-nitroso-ciprofloxacin (CIP) was investigated both in wastewater treatment plants including nitrification/denitrification stages and in sludge slurry experiments under denitrifying conditions. The analysis of biological wastewater treatment plant effluents by Kendrick mass defect analysis and liquid chromatography - high resolution - mass spectrometry (LCHRMS) revealed the occurrence of N-nitroso-CIP and N-nitroso-hydrochlorothiazide at concentration levels of 34 ± 3 ng/L and 71 ± 6 ng/L, respectively. In laboratory experiments and dark conditions, produced N-nitroso-CIP concentrations reached a plateau during the course of biodegradation experiments. A mass balance was achieved after identification and quantification of several transformation products by LCHRMS. N-nitroso-CIP accounted for 14.3% of the initial CIP concentration (20 µg/L) and accumulated against time. The use of 4,5-diaminofluorescein diacetate and superoxide dismutase as scavengers for in situ production of nitric oxide and superoxide radical anion respectively, revealed that the mechanisms of formation of N-nitroso-CIP likely involved a nitrosation pathway through the formation of peroxynitrite and another one through codenitrification processes, even though the former one appeared to be prevalent. This work extended the possible sources of N-nitrosamines by including a formation pathway relying on nitric oxide reactivity with secondary amines under activated sludge treatment.

Lemna minor studies under various storage periods using extended-polarity extraction and metabolite non-target screening analysis.

Plant metabolomic studies cover a broad band of compounds, including various functional groups with different polarities and other physiochemical properties. For this reason, specific optimized methods are needed in order to enable efficient and non-destructive extraction of molecules over a large range of LogD values. This study presents a simple and efficient extraction procedure for Lemna minor samples demonstrating polarity extension of the molecular range. The Lemna samples chosen were kept under the following storage conditions: 1) fresh, 2) stored for a few days at -80 °C, and 3) stored for 6 months at -80 °C. The samples were extracted using five specifically chosen solvents: 100 % ethanol, 100 % methanol (MeOH), acidic 90 % MeOH (MeOH-water-formic acid (FAC) (90:9.5:0.5, v/v/v), MeOH-water (50:50, v/v), and 100 % water. The final extraction procedure was conducted subject to three solvent conditions, and the subsequent polarity-extended analysis was applied for Lemna minor samples using RPLC-HILIC-ESI-TOF-MS. The extraction yield is in descending order (acidic 90 % MeOH), 50 % MeOH, 100 % water and 100 % MeOH. The results displayed significant molecular differences, both in the extracts investigated and in the fresh Lemna samples, compared to stored samples, in terms of the extraction yield and reducing contents as well as the number of features. The storage of Lemna minor resulted in changes to the fingerprint of its metabolites as the reducing contents increased. The comparisons enable a direct view of molecule characterizations, in terms of their polarity, molecular mass, and signal intensity. This parametric information would appear ideal for further statistical data analysis. Consequently, the extraction procedure and the analysis/data evaluation are highly suitable for the so-called extended-polarity non-target screening procedure.

Enrichment of endophytic Actinobacteria in roots and rhizomes of Miscanthus × giganteus plants exposed to diclofenac and sulfamethoxazole.

This study investigates how wastewater containing 2 mg l-1 of sulfamethoxazole (SMX) and 2 mg l-1 of diclofenac (DCF) affects the composition of bacterial communities present in the roots and rhizomes of Miscanthus × giganteus plants grown in laboratory-scale constructed wetlands. Bacterial communities in plant roots and rhizomes were identified in treated and control samples by 16S rRNA amplicon sequencing. Moreover, bacterial endophytes were isolated in R2A and 1/10 869 media and screened for their ability to metabolize SMX and DCF in liquid medium by HPLC. Our results show significant changes in the abundance of main genera, namely Sphingobium and Streptomyces between control and treated plants. Around 70% of the strains isolated from exposed plants belonged to the phylum Actinobacteria and were classified as Streptomyces, Microbacterium, and Glycomyces. In non-exposed plants, Proteobacteria represented 43.5% to 63.6% of the total. We identified 17 strains able to remove SMX and DCF in vitro. From those, 76% were isolated from exposed plants. Classified mainly as Streptomyces, they showed the highest SMX (33%) and DCF (41%) removal efficiency. These isolates, alone or in combination, might be used as bio-inoculants in constructed wetlands to enhance the phytoremediation of SMX and DCF during wastewater treatment.

Discussion paper: Sustainable increase of crop production through improved technical strategies, breeding and adapted management - A European perspective.

During the next decade it will be necessary to develop novel combinations of management strategies to sustainably increase crop production and soil resilience. Improving agricultural productivity, while conserving and enhancing biotic and abiotic resources, is an essential requirement to increase global food production on a sustainable basis. The role of farmers in increasing agricultural productivity growth sustainably will be crucial. Farmers are at the center of any process of change involving natural resources and for this reason they need to be encouraged and guided, through appropriate incentives and governance practices, to conserve natural ecosystems and their biodiversity, and minimize the negative impact agriculture can have on the environment. Farmers and stakeholders need to revise traditional approaches not as productive as the modern approaches but more friendly with natural and environmental ecosystems values as well as emerging novel tools and approaches addressing precise farming, organic amendments, lowered water consumption, integrated pest control and beneficial plant-microbe interactions. While practical solutions are developing, science based recommendations for crop rotations, breeding and harvest/postharvest strategies leading to environmentally sound and pollinator friendly production and better life in rural areas have to be provided.

Metabolism of carbamazepine in plant roots and endophytic rhizobacteria isolated from Phragmites australis.

Carbamazepine (CBZ) is a pharmaceutical frequently categorized as a recalcitrant pollutant in the aquatic environment. Endophytic bacteria previously isolated from reed plants have shown the ability to promote growth of their host and to contribute to CBZ metabolism. In this work, a horseradish (Armoracia rusticana) hairy root (HR) culture has been used as a plant model to study the interactions between roots and endophytic bacteria in response to CBZ exposure. HRs could remove up to 5% of the initial CBZ concentration when they were grown in spiked Murashige and Skoog (MS) medium. Higher removal rates were observed when HRs were inoculated with the endophytic bacteria Rhizobium radiobacter (21%) and Diaphorobacter nitroreducens (10%). Transformation products resulting from CBZ degradation were identified using liquid chromatography-ultra high-resolution quadrupole time of flight mass spectrometry (LC-UHR-QTOF-MS). CBZ metabolism could be divided in four pathways. Metabolites involving GSH conjugation and 2,3-dihydroxylation, as well as acridine related compounds are described in plants for the first time. This study presents strong evidence that xenobiotic metabolism and degradation pathways in plants can be modulated by the interaction with their endophytic community. Hence it points to plausible applications for the elimination of recalcitrant compounds such as CBZ from wastewater in CWs.

Uptake of carbamazepine by rhizomes and endophytic bacteria of Phragmites australis.

Carbamazepine is an antiepileptic and mood-stabilizing drug which is used widely in Europe and North America. In the environment, it is found as a persistent and recalcitrant contaminant, being one of the most prominent hazardous pharmaceuticals and personal care products in effluents of wastewater treatment plants. Phragmites australis is one of the species with both, the highest potential of detoxification and phytoremediation. It has been used successfully in the treatment of industrial and municipal wastewater. Recently, the identification of endophytic microorganisms from different plant species growing in contaminated sites has provided a list of candidates which could be used as bio-inoculants for bioremediation of difficult compounds. In this study, Phragmites australis plants were exposed to 5 mg/L of carbamazepine. After 9 days the plants had removed 90% of the initial concentration. Endophytic bacteria were isolated from these plants and further characterized. Phylogenetic analysis based on 16S rDNA sequencing revealed that the majority of these isolates belong to three groups: Proteobacteria, Actinobacteria, and Bacteroidetes. Carbamazepine uptake and plant growth promoting (PGP) traits were analyzed among the isolates. Ninety percent of the isolates produce indole acetic acid (IAA) and all of them possess at least one of the PGP traits tested. One isolate identified as Chryseobacterium taeanense combines good carbamazepine uptake and all of the PGP traits. Rhizobium daejeonense can remove carbamazepine and produces 23 µg/mL of IAA. Diaphorobacter nitroreducens and Achromobacter mucicolens are suitable for carbamazepine removal while both, Pseudomonas veronii and Pseudomonas lini show high siderophore production and phosphate solubilization. Alone or in combination, these isolates might be applied as inoculates in constructed wetlands in order to enhance the phytoremediation of carbamazepine during wastewater treatment.