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.

Impact of organic soil amendments in antibiotic levels, antibiotic resistance gene loads, and microbiome composition in corn fields and crops.

The potential spreading of antibiotic resistance genes (ARG) into agricultural fields and crops represent a fundamental limitation on the use of organic fertilization in food production systems. We present here a study of the effect of spreading four types of organic soil amendments (raw pig slurry, liquid and solid fractions, and a digested derivative) on demonstrative plots in two consecutive productive cycles of corn harvest (Zea mays), using a mineral fertilizer as a control, following the application of organic amendments at 32-62 T per ha (150 kg total N/ha) and allowing 5-8 months between fertilization and harvest. A combination of qPCR and high-throughput 16S rDNA sequencing methods showed a small, but significant impact of the fertilizers in both ARG loads and microbiomes in soil samples, particularly after the second harvesting cycle. The slurry solid fraction showed the largest impact on both ARG loads and microbiome variation, whereas its digestion derivatives showed a much smaller impact. Soil samples with the highest ARG loads also presented increased levels of tetracyclines, indicating a potential dual hazard by ARG and antibiotic residues linked to some organic amendments. Unlike soils, no accumulation of ARG or antibiotics was observed in corn leaves (used as fodder) or grains, and no grain sample reached detection limits for neither parameter. These results support the use of organic soil amendments in corn crops, while proposing the reduction of the loads of ARGs and antibiotics from the fertilizers to greatly reduce their potential risk.

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.

Systematic identification of trimethoprim metabolites in lettuce.

Antibiotics are some of the most widely used drugs. Their release in the environment is of great concern since their consumption is a major factor for antibiotic resistance, one of the most important threats to human health. Their occurrence and fate in agricultural systems have been extensively investigated in recent years. Yet whilst their biotic and abiotic degradation pathways have been thoroughly researched, their biotransformation pathways in plants are less understood, such as in case of trimethoprim. Although trimethoprim has been reported in the environment, its fate in higher plants still remains unknown. A bench-scale experiment was performed and 30 trimethoprim metabolites were identified in lettuce (Lactuca sativa L.), of which 5 belong to phase I and 25 to phase II. Data mining yielded a list of 1018 ions as possible metabolite candidates, which was filtered to a final list of 87 candidates. Molecular structures were assigned for 19 compounds, including 14 TMP metabolites reported for the first time. Alongside well-known biotransformation pathways in plants, additional novel pathways were suggested, namely, conjugation with sesquiterpene lactones, and abscisic acid as a part of phase II of plant metabolism. The results obtained offer insight into the variety of phase II conjugates and may serve as a guideline for studying the metabolization of other chemicals that share a similar molecular structure or functional groups with trimethoprim. Finally, the toxicity and potential contribution of the identified metabolites to the selective pressure on antibiotic resistance genes and bacterial communities via residual antimicrobial activity were evaluated.

Metabolomic and phenotypic implications of the application of fertilization products containing microcontaminants in lettuce (Lactuca sativa).

Cultivation practice using organic amendments is plausible to ensure global food security. However, plant abiotic stress due to the presence of metals and organic microcontaminants (OMCs) in fertilization products cannot be overlooked. In this study, we monitored lettuce metabolism and phenotypic response following the application of either sewage sludge (SS), the organic fraction of municipal solid waste, swine manure (SM), chemical fertilizers (CF), or no amendment (C) in a greenhouse facility. The experimental set-up consisted of five treatments with five replicates (25 experimental units randomly distributed). All fertilizers were supplied at the equivalent agronomic total nitrogen dose, but the occurrence of trace metals and/or OMCs was greater in the SS and SM than the rest. Non-target metabolomic analysis (high-resolution mass spectrometry coupled with partial least squares regression) identified more than 300 plant metabolites (amino acids, organic acids, sugar alcohols, and sugars), 55 of which showed significant changes in their relative abundances depending on the type of amendment. Functional analysis indicated that the use of CF or SS increased the levels of metabolites involved in carbohydrate and nitrogen metabolism. Therefore, although SS and SM fertilizers had a greater presence of heavy metals and/or OMCs, our results indicate that they did not induce measurable adverse effects in the lettuce phenotype or metabolism. Metabolic changes between fertilizers (CF and SS vs. C and SM) were mainly due to nitrogen availability.

Antibiotic and antibiotic-resistant gene loads in swine slurries and their digestates: Implications for their use as fertilizers in agriculture.

The spread of antibiotic resistance in bacteria is a matter of global concern, and the identification of possible sources of the associated genetic elements (antibiotic resistance genes -ARGs-, components of the horizontal gene transfer mechanism), is becoming an urgent need. While the transmission of ARGs in medical settings have been adequately characterized, ARG propagation in agroecosystems remains insufficiently studied. Particularly crucial is the determination of potential risks associated to the use of swine slurries and related products as component of organic fertilizers, an increasingly used farming practice. We determined ARGs and antibiotic loads analysed from swine slurries and digestates from eight farms from Catalonia (NE Spain), and compared the results with their microbiome composition. Both ARGs and antibiotic were conspicuous in farm organic wastes, and the levels of some antibiotics exceeded currently accepted minimum inhibitory concentrations. Particularly, the presence of high loads of fluoroquinolones was directly correlated to the prevalence of the related qnrS1 ARG in the slurry. We also found evidence that ARG loads were directly correlated to the prevalence of determined bacterial taxa (Actinobacteria, Proteobacteria, Spirochaeta), a parameter that could be potentially modulated by the processing of the raw slurry prior to their use as fertilizer.

Occurrence and human health risk assessment of antibiotics and their metabolites in vegetables grown in field-scale agricultural systems.

The occurrence of antibiotics (ABs) in four types of commercially grown vegetables (lettuce leaves, tomato fruits, cauliflower inflorescences, and broad bean seeds) was analyzed to assess the human exposure and health risks associated with different agronomical practices. Out of 16 targeted AB residues, seven ABs belonging to three groups (i.e., benzyl pyrimidines, fluoroquinolones, and sulfonamides) were above the method detection limit in vegetable samples ranging from 0.09 ng g-1 to 3.61 ng g-1 fresh weight. Data analysis (quantile regression models, principal component and hierarchical cluster analysis) showed manure application, irrigation with river water (indirect wastewater reuse), and vegetable type to be the most significant factors for AB occurrence in the targeted crops. Metabolites were detected in 70 of the 80 vegetable samples analyzed, and their occurrence was both plant- and compound-specific. In 73 % of the total samples, the concentration of AB metabolites was higher than the concentration of their parent compound. Finally, the potential human health risk estimated using the hazard quotient approach, based on the acceptable daily intake and the estimated daily intake, showed a negligible risk for human health from vegetable consumption. However, canonical-correspondence analysis showed that detected ABs explained 54 % of the total variation in AB resistance genes abundance in the vegetable samples. Thus, further studies are needed to assess the risks of antibiotic resistance promotion in vegetables and the significance of the occurrence of their metabolites.

Elucidating biotransformation pathways of ofloxacin in lettuce (Lactuca sativa L).

Antibiotics can be uptaken by plants from soil desorption or directly from irrigation water, but their metabolization pathways in plants are largely unknown. In this paper, an analytical workflow based on high-resolution mass spectrometry was applied for the systematic identification of biotransformation products of ofloxacin in lettuce. The targeted metabolites were selected by comparing the mass chromatograms of exposed with control samples using an advanced spectra-processing method (Fragment Ion Search). The innovative methodology presented allowed us to identify a total of 11 metabolites, including 5 ofloxacin metabolites that are being reported for the first time in plants. Accordingly, major transformation pathways were proposed revealing insight into how ofloxacin and related chemicals are metabolized in lettuce. Furthermore, the influence of biotransformation on potential residual antimicrobial activity of identified compounds was discussed. Human exposure to antibiotics at doses below the minimum inhibitory concentrations is crucial in human risk assessment, including food ingestion; however, in the case of ofloxacin presented results reveal that plant metabolites should also be considered so as not to underestimate their risk.

Simultaneous determination of multiclass antibiotics and their metabolites in four types of field-grown vegetables.

The developed method was evaluated for the determination of 10 antibiotics belonging to four chemical classes (fluoroquinolones, sulfonamides, lincosamides, and metoxybenzylpyrimidines) and six of their metabolites in four vegetable matrices (lettuce, tomato, cauliflower, and broad beans). The reported method detection limits were sufficiently low (0.1-5.8 ng/g dry weight) to detect target compounds in vegetables under real agricultural practices. Absolute and relative recovery values ranged from 40 to 118% and from 70 to 118%, respectively, for all targeted compounds at the spike level of 100 ng/g dry weight. Regarding method precision, the highest relative standard deviation (RSD) was obtained for enrofloxacin in lettuce (20%), while for the rest of the compounds in all matrices, the RSD values were below 20% for the same spike level. Matrix effects, due to electrospray ionization, ranged from - 26 to 29% for 85% of all estimated values. In a field study, four of the 10 targeted antibiotics were detected in tested vegetables. For the first time, antibiotic metabolites were quantified in vegetables grown under real field conditions. More specifically, decarboxyl ofloxacin and TMP304 were detected in tomato fruits (1.5 ng/g dry weight) and lettuce leaves (21.0-23.1 ng/g dry weight), respectively. It is important to remark that the concentration of TMP304 was five times higher than that from the parental compound, emphasizing the importance of metabolite analysis in monitoring studies. Therefore, the method provided a robust, reliable, and simple-to-use tool that could prove useful for routine multiclass analysis of antibiotics and their metabolites in vegetable samples. Graphical abstract.

Seasonal occurrence and cancer risk assessment of polycyclic aromatic hydrocarbons in street dust from the Novi Sad city, Serbia.

This is the first investigation that identified seasonal occurrence, distribution and sources of 16 polycyclic aromatic hydrocarbons (PAHs) in 60 street dust samples collected within urban zone of Novi Sad, the second largest city in Serbia. The obtained results were further used for comprehensive assessment of carcinogenic risk of Serbian inhabitants exposed to PAHs present in street dust by the incremental lifetime cancer risk method. The total level of 16 PAHs ranged between 35 µg kg-1 and 2422 µg kg-1 in samples taken in summer and between 35 µg kg-1 and 587 µg kg-1 in samples taken in winter. In both seasons, 4-ring PAHs were the most dominant compounds and high molecular weight (HMW) PAHs had similar contribution (55% in summer and 65% in winter). The highest content was determined for fluoranthene (Fly) in both seasons (597 µg kg-1 in winter, 301 µg kg-1 in summer). The PAHs source apportionment was analyzed by principal component analysis (PCA) and diagnostic ratios, and combustion of petroleum seemed to be the main sources of the PAHs in street dust. The cancer risk level for children and adult were comparable for dermal contact and by ingestion, and ranged from 10-6 to 10-4 indicating a potential risk. Additionally, the total incremental life time cancer risk (ILCR) was assessed for children and adult population taking into account three possible exposure routs and the median total cancer risk was ˃10-5, with 7% of the samples having the risk ˃10-4 that should be considered of high concern with potential health problem. These results are the first of this kind for the whole Serbia and the Western Balkan region and can be considered as the base line for future research.

Polycyclic aromatic hydrocarbons in urban soil of Novi Sad, Serbia: occurrence and cancer risk assessment.

Contents of 16 polycyclic aromatic hydrocarbons were analyzed in 30 soil samples from 15 locations in Novi Sad, Serbia, assessing for the first time the corresponding health risks in the Serbian urban zone. Total concentrations were in the range of 22-2247 µg kg-1, with a mean and median value of 363 and 200 µg kg-1, respectively. Comparison with the relevant maximum allowed contents proposed by the Serbian government and with the Dutch target values implied that soils from the urban area of Novi Sad were "suitable as residential soils" and that no intervention would be needed if the current levels were retained. Seven diagnostic ratios were calculated, indicating the pyrogenic sources of PAHs as the dominant. Cancer risks in humans via accidental ingestion, inhalation of soil particles, and dermal contact with soil were estimated. Cancer risk for soil ingestion by children was the highest. The total lifetime carcinogenic risk as sum of individual cancer risks for seven carcinogenic polycyclic aromatic hydrocarbons was within the range 10-4 to 10-6, indicating acceptable risks at 30 and 47% of sites for children and adults, respectively. However, for the rest of the samples, total lifetime cancer risk was >10-4 indicating over the acceptable risk, even though the contents in soil were not of concern as the comparison with the environmental guidance previously showed. This could be explained by (a) the dominant concentrations of higher molecular weight compounds with 4 to 6 rings, among which there are compounds with higher toxicity equivalents, but also with (b) the extreme conditions used for the conservative risk assessment under maximal exposure frequency, exposure time, and ingestion rates.