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.

Effects of industrial pollution on the reproductive biology of Squalius laietanus (Actinopterygii, Cyprinidae) in a Mediterranean stream (NE Iberian Peninsula).

Mediterranean rivers are severely affected by pollutants from industry, agriculture and urban activities. In this study, we examined how industrial pollutants, many of them known to act as endocrine disruptors (EDCs), could disturb the reproduction of the Catalan chub (Squalius laietanus). The survey was conducted throughout the reproductive period of S. laietanus (from March to July 2014) downstream an industrial WWTP located in the River Ripoll (NE Iberian Peninsula). Eighty fish (28 females and 52 males) were caught by electrofishing upstream and 77 fish (33 females and 44 males) downstream a WWTP. For both sexes, the gonadosomatic index (GSI) and gonadal histology were examined and related to water chemical analysis and fish biomarkers. Female fecundity was assessed using the gravimetric method. Fish from the polluted site showed enhanced biomarker responses involved in detoxification. Also, in the polluted site, lower GSI values were attained in both sexes and females displayed lower numbers of vitellogenic oocytes. Gonadal histology showed that all maturation stages of testicles and ovaries were present at the two study sites but fish males from the polluted site had smaller diameter seminiferous tubules. Water chemical analysis confirmed greater presence of EDCs in the river downstream the industrial WWTP. The chemicals benzotriazole and benzothiazole could be partially responsible for the observed alterations in the reproductive biology of S. laietanus.

Ranking of crop plants according to their potential to uptake and accumulate contaminants of emerging concern.

The reuse of treated wastewater (TWW) for irrigation and the use of biosolids and manures as soil amendment constitute significant pathways for the introduction of the contaminants of emerging concern (CECs) to the agricultural environment. Consequently, CECs are routinely detected in TWW-irrigated agricultural soils and runoff from such sites, in biosolids- and manure-amended soils, and in surface and groundwater systems and sediments receiving TWW. Crop plants grown in such contaminated agricultural environments have been found to uptake and accumulate CECs in their tissues, constituting possible vectors of introducing CECs into the food chain; an issue that is presently considered of high priority, thus needing intensive investigation. This review paper aims at highlighting the responsible mechanisms for the uptake of CECs by plants and the ability of each crop plant species to uptake and accumulate CECs in its edible tissues, thus providing tools for mitigating the introduction of these contaminants into the food chain. Both biotic (e.g. plants' genotype and physiological state, soil fauna) and abiotic factors (e.g. soil pore water chemistry, physico-chemical properties of CECs, environmental perturbations) have been proven to influence the ability of crop plants to uptake and accumulate CECs. According to authors' estimates, based on the thorough elaboration of knowledge produced by existing relevant studies, the ability of crop plants to uptake and accumulate CECs decrease in the order of leafy vegetables > root vegetables > cereals and fodder crops > fruit vegetables; though, the uptake of CECs by important crop plants, such as fruit trees, is not yet evaluated. Overall, further studies must be performed to estimate the potential of crop plants to uptake and accumulate CECs in their edible tissues, and to characterize the risk for human health represented by their presence in human and livestock food products.

Implications of the use of organic fertilizers for antibiotic resistance gene distribution in agricultural soils and fresh food products. A plot-scale study.

The spread of antibiotic resistance genes (ARG) into agricultural soils, products, and foods severely limits the use of organic fertilizers in agriculture. In order to help designing agricultural practices that minimize the spread of ARG, we fertilized, sown, and harvested lettuces and radish plants in experimental land plots for two consecutive agricultural cycles using four types of fertilizers: mineral fertilization, sewage sludge, pig slurry, or composted organic fraction of municipal solid waste. The analysis of the relative abundances of more than 200,000 ASV (Amplicon Sequence Variants) identified a small, but significant overlap (<10%) between soil's and fertilizer microbiomes. Clinically relevant ARG were found in higher loads (up to 100 fold) in fertilized soils than in the initial soil, particularly in those treated with organic fertilizers, and their loads grossly correlated to the amount of antibiotic residues found in the corresponding fertilizer. Similarly, low, but measurable ARG loads were found in lettuce (tetM, sul1) and radish (sul1), corresponding the lowest values to samples collected from minerally fertilized fields. Comparison of soil samples collected along the total period of the experiment indicated a relatively year-round stability of soil microbiomes in amended soils, whereas ARG loads appeared as unstable and transient. The results indicate that ARG loads in soils and foodstuffs were likely linked to the contribution of bacteria from organic fertilizer to the soil microbiomes, suggesting that an adequate waste management and good pharmacological and veterinarian practices may significantly reduce the presence of these ARGs in agricultural soils and plant products.

Resolution and quantification of complex mixtures of polycyclic aromatic hydrocarbons in heavy fuel oil sample by means of GC × GC-TOFMS combined to multivariate curve resolution.

Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) combined to multivariate curve resolution-alternating least-squares (MCR-ALS) is proposed for the resolution and quantification of very complex mixtures of compounds such as polycyclic aromatic hydrocarbons (PAHs) in heavy fuel oil (HFO). Different GC × GC-TOFMS data slices acquired during the analysis of HFO samples and PAH standards were simultaneously analyzed using the MCR-ALS method to resolve the pure component elution profiles in the two chromatographic dimensions as well as their pure mass spectra. Outstandingly, retention time shifts within and between GC × GC runs were not affecting the results obtained using the proposed strategy and proper resolution of strongly coeluted compounds, baseline and background contributions was achieved. Calibration curves built up with standard samples of PAHs allowed the quantification of ten of them in HFO aromatic fractions. Relative errors in their estimated concentrations were in all cases below 6%. The obtained results were compared to those obtained by commercial software provided with GC × GC-TOFMS instruments and to Parallel Factor Analysis (PARAFAC). Inspection of these results showed improvement in terms of data fitting, elution process description, concentration relative errors and relative standard deviations.

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.

Effects of prescription antibiotics on soil- and root-associated microbiomes and resistomes in an agricultural context.

The use of treated wastewater for crop irrigation is rapidly increasing to respond to the ever-growing demands for water and food resources. However, this practice may contribute to the spread of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in agricultural settings. To evaluate this potential risk, we analyzed microbiomes and resistomes of soil and Lactuca sativa L. (lettuce) root samples from pots irrigated with tap water spiked with 0, 20, or 100 µg L-1 of a mixture of three antibiotics (Trimethoprim, Ofloxacin, Sulfamethoxazole). The presence of antibiotics induced changes in bacterial populations, particularly in soil, as revealed by 16S rDNA sequence analysis. Parallel shotgun sequencing identified a total of 56 different ARGs conferring resistance against 14 antibiotic families. Antibiotic -treated samples showed increased loads of ARGs implicated in mutidrug resistance or in both direct and indirect acquired resistance. These changes correlated with the prevalence of Xantomonadales species in the root microbiomes. We interpret these data as indicating different strategies of soil and root microbiomes to cope with the presence of antibiotics, and as a warning that their presence may increase the loads of ARBs and ARGs in edible plant parts, therefore constituting a potential risk for human consumers.

Development of an analytical procedure for the determination of emerging and priority organic pollutants in leafy vegetables by pressurized solvent extraction followed by GC-MS determination.

A new multiresidue method for the determination of 13 emerging and priority pollutants in lettuce, including pesticides, pharmaceuticals, personal care products, polycyclic aromatic hydrocarbons (PAHs), and phenolic estrogens, has been developed using matrix solid-phase dispersion combined to pressurized fluid extraction (PFE) followed by gas chromatography coupled to mass spectrometry determination. A sequential optimization strategy based on solvent optimization first, followed by experimental design, was performed in order to maximize target analyte extraction with the aid of response surface methodology. Firstly, a full factorial design was applied to choose the significant variables in PFE; extraction time and temperature were found to have the biggest overall effect on response for most of analytes. They were later optimized performing a central composite design and the variable response of these factors was modeled for all analytes. It was found that marked differences in physicochemical nature exerted a strong influence on extraction conditions and yield. Therefore, the effect of parameters on the response was rather different for some compounds. To overcome this conflicting behavior, a multiple response simultaneous optimization was applied using the desirability function to achieve global optimal operating conditions. The optimal conditions were attained at 13.5 min (two extraction cycles) and 104 degrees C in the PFE by using hexane acetone mixture (1:1). Limit of detection and limit of quantitation values were found to be between 6.6 and 58 and 7.6 and 61.7 microg kg(-1), respectively.

Toxicity and phototoxicity of water-accommodated fraction obtained from Prestige fuel oil and Marine fuel oil evaluated by marine bioassays.

Acute toxicity and phototoxicity of heavy fuel oil extracted directly from the sunken tanker Prestige in comparison to a standard Marine fuel oil were evaluated by obtaining the water-accommodated fraction (WAF) and using mussel Mytilus galloprovincialis and sea urchin Paracentrotus lividus embryogenesis bioassays, and copepod Acartia tonsa and fish Cyprinodon variegatus survival bioassays. Aromatic hydrocarbon (AH) levels in WAF were measured by gas chromatography. Prestige WAF was not phototoxic, its median effective concentrations (EC50) were 13% and 10% WAF for mussel and sea urchin respectively, and maximum lethal threshold concentrations (MLTC) were 12% and 50% for copepod and fish respectively. Marine WAF resulted phototoxic for mussel bioassay. EC50s of Marine WAF were 50% for sea urchin in both treatments and 20% for mussel under illumination. Undiluted Marine WAF only caused a 20% decrease in mussel normal larvae. Similar sensitivities were found among sea urchins, mussels and copepods, whilst fish were less sensitive. Unlike Marine WAF, Prestige WAF showed EC50 values at dilutions below 20%, and its toxicity was independent of lighting conditions. The differences in toxicity between both kinds of fuel could not be explained on the basis of total AH content.

Distribution of polycyclic aromatic hydrocarbons (PAHs) and tributyltin (TBT) in Barcelona harbour sediments and their impact on benthic communities.

Sediments have long been recognised as a sink for many contaminants like polycyclic aromatic hydrocarbons (PAHs) and tributyltin (TBT), which by virtue of their nature can strongly adsorb onto sediments affecting the benthic community inhabiting them. Using geographical information systems, this study reports and combines the results of several already existing studies along Barcelona harbour in order to assess the potential ecological impacts of these contaminants on the benthos of the harbour ecosystem. Chemical analysis indicated low to moderate contents of PAHs and high contents of TBT in sediments in Barcelona harbour. Comparison against existing sediment quality guidelines (SQGs) indicated that acutely toxic effects would not be expected for PAHs but for TBT, which represents a serious environmental threat for the benthic community. Benthos surveys revealed a deterioration of the benthic community throughout the harbour, especially in the inner port.

Two important limitations relating to the spiking of environmental samples with contaminants of emerging concern: How close to the real analyte concentrations are the reported recovered values?

Occurrence and effects of contaminants of emerging concern pose a special challenge to environmental scientists. The investigation of these effects requires reliable, valid, and comparable analytical data. To this effect, two critical aspects are raised herein, concerning the limitations of the produced analytical data. The first relates to the inherent difficulty that exists in the analysis of environmental samples, which is related to the lack of knowledge (information), in many cases, of the form(s) of the contaminant in which is present in the sample. Thus, the produced analytical data can only refer to the amount of the free contaminant ignoring the amount in which it may be present in other forms; e.g., as in chelated and conjugated form. The other important aspect refers to the way with which the spiking procedure is generally performed to determine the recovery of the analytical method. Spiking environmental samples, in particular solid samples, with standard solution followed by immediate extraction, as is the common practice, can lead to an overestimation of the recovery. This is so, because no time is given to the system to establish possible equilibria between the solid matter-inorganic and/or organic-and the contaminant. Therefore, the spiking procedure need to be reconsidered by including a study of the extractable amount of the contaminant versus the time elapsed between spiking and the extraction of the sample. This study can become an element of the validation package of the method.

The potential implications of reclaimed wastewater reuse for irrigation on the agricultural environment: The knowns and unknowns of the fate of antibiotics and antibiotic resistant bacteria and resistance genes - A review.

The use of reclaimed wastewater (RWW) for the irrigation of crops may result in the continuous exposure of the agricultural environment to antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In recent years, certain evidence indicate that antibiotics and resistance genes may become disseminated in agricultural soils as a result of the amendment with manure and biosolids and irrigation with RWW. Antibiotic residues and other contaminants may undergo sorption/desorption and transformation processes (both biotic and abiotic), and have the potential to affect the soil microbiota. Antibiotics found in the soil pore water (bioavailable fraction) as a result of RWW irrigation may be taken up by crop plants, bioaccumulate within plant tissues and subsequently enter the food webs; potentially resulting in detrimental public health implications. It can be also hypothesized that ARGs can spread among soil and plant-associated bacteria, a fact that may have serious human health implications. The majority of studies dealing with these environmental and social challenges related with the use of RWW for irrigation were conducted under laboratory or using, somehow, controlled conditions. This critical review discusses the state of the art on the fate of antibiotics, ARB and ARGs in agricultural environment where RWW is applied for irrigation. The implications associated with the uptake of antibiotics by plants (uptake mechanisms) and the potential risks to public health are highlighted. Additionally, knowledge gaps as well as challenges and opportunities are addressed, with the aim of boosting future research towards an enhanced understanding of the fate and implications of these contaminants of emerging concern in the agricultural environment. These are key issues in a world where the increasing water scarcity and the continuous appeal of circular economy demand answers for a long-term safe use of RWW for irrigation.

Comparative characterization of a wool-wax extract by two complementary chromatographic techniques.

A detailed characterization of lipids present in a wool-wax extract, obtained by supercritical fluid extraction, is presented. Thin-layer chromatography coupled to a flame ionization detector (TLC-FID) has been used as a screening technique to identify the different chemical classes of complex mixtures. Furthermore, to confirm peak identification obtained by TLC-FID and to check its suitability as a quality control technique, preparative TLC fractionation has been carried out by re-analyzing the different fractions obtained by TLC-FID and by gas chromatography coupled to mass spectrometry in the electron impact and in the chemical ionization modes. By means of this comparative study, the TLC-FID identification has been confirmed. Also, the complementarity of the different techniques used confirmed the identification of different lipid classes, namely fatty acids, alcohols, sterols, sterane diol, stanone, steryl sulphate, hydroxyacids, alkane diols, aliphatic and steryl esters, hydroxy steryl esters, triacylglycerol, diesters, and ceramides types II and VI.

Predicting single and mixture toxicity of petrogenic polycyclic aromatic hydrocarbons to the copepod Oithona davisae.

In the present study, the acute toxicity of 10 polycyclic aromatic hydrocarbons (PAHs) associated with the Prestige fuel oil spill (Spain, 2002) were evaluated, either as single substances or in mixtures, in adults of the copepod Oithona davisae. All but dimethylphenanthrene had negative effects on O. davisae survival at concentrations below their water solubility, with 48-h median lethal concentrations for naphthalene and pyrene of 56.1 and 0.8 micromol/L, respectively, making these the least and most toxic compounds. Polycyclic aromatic hydrocarbons had narcotic effects on copepods, as evidenced by the lack of motility at lower concentrations than those causing death. Naphthalene showed the greatest narcotic effects, and phenanthrene showed minor effects. Acute toxicity of the tested PAHs was inversely related (r2 = 0.9) with their octanol-water partition coefficient, thereby confirming the validity of the baseline quantitative structure-activity regression models for predicting the toxicity of PAH compounds in copepod species. When supplied in mixtures, the toxic effect of PAHs was additive. These results indicate that the many PAHs in an oil spill can be considered unambiguous baseline toxicants (class 1) acting additively as nonpolar narcotics in copepods; hence, their individual and combined toxicity can be predicted using their octanol-water partition coefficient.

Trace level determination of organochlorine, organophosphorus and pyrethroid pesticides in lanolin using gel permeation chromatography followed by dual gas chromatography and gas chromatography-negative chemical ionization mass spectrometric confirmation.

A methodology for multi-class pesticide determination at trace level in lanolin is presented. Gel permeation chromatography on a Bio-Beads SX-3 column followed by a dual GC chromatographic determination has been developed. The effluent of the analytical column (50% diphenyl-methyl- or 14% cyanopropyl-phenylpolysiloxane) was split into an electron-capture and a nitrogen-phosphorus detection system. The chromatographic system was optimised for 28 pesticides commonly used to control sheep pests and corresponding to organochlorine, organophosphorus and pyretroid classes. Identification has been carried out by gas chromatography coupled to negative chemical ionization mass spectrometry. Recoveries ranged from 72 to 94% and the detection limits from 20 to 97 ng/g depending on the pesticide class, the RSDs were below 10%. Finally, the developed analytical methodology has been successfully applied to the determination of pesticides in several lanolin samples.

Determination of perfluorocarboxylic acids in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gas chromatography-negative ion chemical ionization mass spectrometry.

A rapid, selective and simple analytical procedure using tetrabutylammonium as ion pair in conjunction with solid-phase microextraction followed by in-port derivatization-GC-negative ion chemical ionization mass spectrometry was developed. The procedure allows an accurate determination of perfluoroalkylcarboxylic acids in aqueous samples at ng L(-1) levels (i.e. method detection limit 20 ng L(-1) forperfluorodecanoic acid) improving previous GC methods in terms of analysis time and sensitivity. Ammonia as reagent gas in the negative ion chemical ionization mass spectrometry increased the sensitivity at least 3-fold compared to methane for perfluorocarboxylic acid butyl esters. The developed procedure was successfully applied to effluents from wastewater treatment plants (i.e. 0.05-8.2 microg L(-1)) and harbor seawaters.

Complete characterisation of lanolin steryl esters by sub-ambient pressure gas chromatography-mass spectrometry in the electron impact and chemical ionisation modes.

Steryl esters occurring in lanolin have been characterised by sub-ambient pressure gas chromatography coupled to mass spectrometry. Electron impact and chemical ionisation modes with different reagent gases have been evaluated in order to carry out unambiguous peak identification. Steryl esters with different sterol (i.e. cholesterol, lanosterol and dihydrolanosterol) and acid moieties either according to carbon number (i.e C10-C23) or isomeric forms (i.e. normal, iso and anteiso) have been identified. Identification of the sterol and acid moieties has been carried out by means of the mass spectral information obtained in the electron impact, chemical ionisation mode either in the positive or negative modes using methane, isobutane and ammonia as reagent gases. Isomeric identification has been achieved by chromatographic retention parameters (i.e. entire-chain length and fractional-chain length) and by the free fatty acid profile also present in lanolin.

Determination of volatile alkyl sulfides in wastewater by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry.

An analytical procedure based on headspace solid-phase microextraction (SPME) followed by gas chromatography coupled to mass spectrometry in the electron impact mode has been developed for the determination of low-molecular-mass sulfides and disulfides in wastewater. Parameters affecting to the extraction of these volatile alkyl sulfides (VASs) with the SPME, such as the extraction temperature, sample volume, pH and the NaCl addition to the matrix, have been optimised using a polydimethylsiloxane-Carboxen fibre. The linear dynamic range was close to three orders of magnitude for all the studied compounds. Detection limits of 4 ng l(-1) for dimethyl sulfide, 0.7 ng l(-1) for ethylmethyl sulfide, 5 ng l(-1) for diethyl sulfide and 1 ng l(-1) for dimethyl disulfide were achieved, with a relative standard deviation between 4 and 6%. The developed analytical methodology was applied to determine those VASs in different wastewaters.

Determination of linear alkylbenzensulfonates in aqueous matrices by ion-pair solid-phase microextraction-in-port derivatization-gas chromatography-mass spectrometry.

Trace determination (low ng/ml) of linear alkylbenzensulfonates (LASs) in water was achieved by solid-phase microextraction (SPME) of ion-pairs formed with tetrabutylammonium. This ion-pairing reagent served two purposes. First, it allowed the extraction of LAS with the polydimethylsiloxane fiber by counterion association and second, the derivatization of the formed LAS ion pairs in the GC injection port at 300 degrees C to form the corresponding sulfonated butyl esters. The methodology developed allows the isomer specific determination of LAS at low detection limits (0.16-0.8 ng/ml), depending on the alkyl chain lengths of LASs with RSDs of 10-12%. Furthermore, the developed methodology was applied to urban wastewater and sea water and compared with a solid-phase extraction (SPE) method (e.g. C18 and strong anion-exchange sorbent) to obtain concordant values for urban wastewater. Moreover, the developed SPME methodology overcame the procedural blank and matrix-dependent recoveries found in the SPE methodologies at low LAS concentrations.