Evaluation of contaminants of emerging concern attenuation through a vegetation filter managed using different operating conditions.

In wastewater treatment using Vegetation Filters (VFs), natural processes reduce contaminants present in water although some of them can reach the environment. In this study, 39 contaminants of emerging concern (CECs) are evaluated in a pilot VF under different operating conditions during almost four years. The use of woodchip amendments and the change from surface irrigation through furrows to drip irrigation (and from weekly to daily water application) provide CEC concentration reductions in the water infiltrating through the vadose zone. Biodegradation is the main process taking place and has been favoured mainly by woodchip soil amendments and the increased residence. Median attenuation percentages of the CECs most frequently detected with highest concentrations in applied wastewater vary between 52% and 100% at the end of the study (at 45 cm depth). Among targeted CECs, caffeine, and its transformation product paraxanthine are the most attenuated. Flecainide and venlafaxine show a persistent behaviour. However, their leaching concentrations are very low (< 31 ng/L). Concerning the underlying aquifer, the groundwater quality in terms of CEC concentrations is conditioned by the surrounding area rather than the operation of the VF. Levels in groundwater are always below those in wastewater and infiltrating water.

Untargeted metabolomic study by liquid chromatography-mass spectrometry in brain tissues on the effects of combined cocaine and ethanol self-administration in male and female young rats.

The combined use of ethanol and cocaine is frequent among drug-abuse users and leads to further exacerbation of health consequences compared to individual consumption and this is of special concern during the transition to adulthood. Despite its high prevalence, the effect of combined consumption of cocaine and ethanol has been scarcely studied. In this work, we report the first untargeted metabolomic study in brain tissues to contribute to the advancement in the knowledge of the possible neurobiological effects of this polysubstance dependence. Liquid Chromatography coupled to high resolution Mass Spectrometry was employed to analyze three different brain tissues samples, prefrontal cortex, striatum and hippocampus, from male and female young rats exposed intravenously to a self-administration of these drugs. After optimizing the best sample treatment and selecting the chromatographic and detection conditions to find the maximum number of significant features (possible biomarker metabolites), the high resolution of the Orbitrap analyzer used in this work has made it possible to find up to 761 significant features with assigned molecular formula, of which up to 190 were tentatively identified and 44 unequivocally confirmed. The results demonstrated that the altered metabolic pathways are involved in multiple functions: receptor systems, such as the Glutamine-Glutamic acid-GABA axis or the catecholamine pathway, purinergic and pyrimidine pathways, fatty acids or oxidative stress, among others.

Untargeted metabolomic analysis to explore the impact of soil amendments in a non-conventional wastewater treatment.

As non-conventional wastewater treatment, vegetation filters make the most of the natural attenuation processes that occur in soil to remove contaminants, while providing several environmental benefits. However, this practice may introduce contaminants of emerging concern (CECs) and their transformation products (TPs) into the environment. A potential improvement to the system was tested using column experiments containing soil (S) and soil amended with woodchips (SW) or biochar (SB) irrigated with synthetic wastewater that included 11 selected CECs. This study evaluated: i) known CECs attenuation and ii) unknown metabolites formation. Known CECs attenuation was assessed by total mass balance by considering both water and soil media. An untargeted metabolomic strategy was developed to assess the formation of unknown metabolites and to identify them in water samples. The results indicated that SB enhanced CECs attenuation and led to the formation of fewer metabolites. Sorption and biodegradation processes were favored by the bigger surface area of particles in SB column, especially for compounds with negative charges. Incorporating woodchips into soil shortened retention times in the column, which reduced attenuation phenomena and resulted in the formation of significantly more metabolites. Incomplete biodegradation reactions, fostered by shorter retention times in SW column could mainly explain these results.

Metabolic fingerprinting of chemotherapy-resistant prostate cancer stem cells. An untargeted metabolomic approach by liquid chromatography-mass spectrometry.

Chemoresistance is one of the most important challenges in cancer therapy. The presence of cancer stem cells within the tumor may contribute to chemotherapy resistance since these cells express high levels of extrusion pumps and xenobiotic metabolizing enzymes that inactivate the therapeutic drug. Despite the recent advances in cancer cell metabolism adaptations, little is known about the metabolic adaptations of the cancer stem cells resistant to chemotherapy. In this study, we have undertaken an untargeted metabolomic analysis by liquid chromatography-high-resolution spectrometry combined with cytotoxicity assay, western blot, quantitative real-time polymerase chain reaction (qPCR), and fatty acid oxidation in a prostate cancer cell line resistant to the antiandrogen 2-hydroxiflutamide with features of cancer stem cells, compared to its parental androgen-sensitive cell line. Metabolic fingerprinting revealed 106 out of the 850 metabolites in ESI+ and 67 out of 446 in ESI- with significant differences between the sensitive and the resistant cell lines. Pathway analysis performed with the unequivocally identified metabolites, revealed changes in pathways involved in energy metabolism as well as posttranscriptional regulation. Validation by enzyme expression analysis indicated that the chemotherapy-resistant prostate cancer stem cells were metabolically dormant with decreased fatty acid oxidation, methionine metabolism and ADP-ribosylation. Our results shed light on the pathways underlying the entry of cancer cells into dormancy that might contribute to the mechanisms of drug resistance.

Improved methods for the detection and quantification of SARS-CoV-2 RNA in wastewater.

Since the start of the COVID-19 pandemic, different methods have been used to detect the presence of genetic material of SARS-CoV-2 in wastewater. The use of wastewater for SARS-CoV-2 RNA detection and quantification showed different problems, associated to the complexity of the matrix and the lack of standard methods used to analyze the presence of an enveloped virus, such as coronavirus. Different strategies for the concentration process were selected to carry out the detection and quantification of SARS-CoV-2 RNA in wastewater: (a) aluminum hydroxide adsorption-precipitation, (b) pre-treatment with glycine buffer and precipitation with polyethylene-glycol (PEG) and (c) ultrafiltration (Centricon). Our results showed that the reduction of organic matter, using the pre-treatment with glycine buffer before the concentration with Centricon or aluminum hydroxide adsorption-precipitation, improved the recovery percentage of the control virus, Mengovirus (MgV) (8.37% ± 5.88 n = 43; 6.97% ± 6.51 n = 20, respectively), and the detection of SARS-CoV-2 in comparison with the same methodology without a pre-treatment. For the concentration with Centricon, the use of 100 mL of wastewater, instead of 200 mL, increased the MgV recovery, and allowed a positive detection of SARS-CoV-2 with N1 and N2 targets. The quantity of SARS-CoV-2 RNA detected in wastewater did not show a direct correlation with the number of confirmed cases, but the study of its upwards or downwards trend over time enabled the detection of an increase of epidemiological data produced in September 2020, January 2021 and April 2021.

Influence of microplastics on the bioconcentration of organic contaminants in fish: Is the "Trojan horse" effect a matter of concern?

Microplastics (MPs) have been shown to act as sorbent phases and thus carriers of organic chemicals in the aquatic environment. Therefore, concerns exist that MP ingestion increases the uptake and accumulation of organic chemicals by aquatic organisms. However, it is unclear if this pathway is relevant compared to other exposure pathways. Here we compared the bioconcentration capacity of two hydrophobic organic chemicals (i.e., chlorpyrifos and hexachlorobenzene) in a freshwater fish (Danio rerio) when exposed to chemicals through water only and in combination with contaminated polyethylene MPs. Additionally, a suite of biomarker analyses (acetylcholine esterase, glutathione S-transferase, alkaline phosphatase, catalase) was carried out to test whether MPs can enhance the toxic stress caused by chemicals. Two 14-day semi-static experiments (one for each chemical) were carried out with adult fish. Each experiment consisted of (1) a control treatment (no chemicals, no MPs); (2) a treatment in which fish were exposed to chlorpyrifos or hexachlorobenzene only through water; (3) a treatment in which fish were exposed to the chemicals through water and contaminated polyethylene MPs (100 mg MP/L). Two additional treatments were included for the biomarker analysis. These contained MPs at two different concentrations (5 and 100 mg MP/L) but no chemicals. The presence of contaminated MPs in contaminated water did not enhance but rather decreased the bioconcentration of both chemicals in fish compared to the treatment that contained contaminated water in absence of MPs. This was more pronounced for hexachlorobenzene, which is more hydrophobic than chlorpyrifos. Enzyme activity levels in fish were only significantly altered in the presence of MPs for alkaline phosphatase. This study indicates that MP presence in freshwater ecosystems is not expected to increase the risks associated with chemical bioconcentration in aquatic organisms and that other exposure pathways (i.e., uptake via respiration, skin permeability) may be of higher importance.

Analytical method to monitor contaminants of emerging concern in water and soil samples from a non-conventional wastewater treatment system.

Nonconventional wastewater treatments, such as vegetation filters (VFs), are propitious systems to attenuate contaminants of emerging concern (CECs) in small municipalities. The development of standardised multiresidue and multimatrix methods suitable for measuring a reliable number of CEC in environmental samples is crucial for monitoring infiltrating concentrations and for ensuring these systems' treatment capacity. The objective of this study is to develop and validate an analytical method for the simultaneous determination of CECs, including transformation products (TPs), with diverse physico-chemical properties, in environmental samples. The optimised method is based on sample clean-up and preconcentration by solid-phase extraction (SPE), followed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The method is able to detect and quantify 40 target CECs, including pharmaceuticals of different classes (analgesics, antibiotics, antihypertensives, lipid regulators, anticonvulsants, antidepressants, antiarrhythmics, beta-blockers, amongst others), hormones and lifestyle products with good reproducibility (variations below 23%), in different water matrices, and 28 CECs, in soil samples. Acceptable recoveries (65-120%) were obtained for most of the CECs in all the matrices. However in the soil samples, as complexity required a prior extraction treatment, the recovery of some analytes was affected, which reduced the number of target CECs. The achieved methodological quantification limits (0.05-5 ng/L and 0.04-1.1 ng/g levels for the water and the soil matrices, respectively) were reasonably low for most CECs. The proposed method was successfully applied to monitor CECs in a VF. The CECs detected at higher concentrations are some of the world's most widely used products (e.g. acetaminophen or caffeine and its main TP, paraxanthine). The results showed an almost 70% reduction in CEC concentrations during infiltration. The groundwater data indicated that the VF treatment operation did not affect the underlying aquifer (Cmax found in GW <1 µg/L).

Pharmaceutical and transformation products during unplanned water reuse: Insights into natural attenuation, plant uptake and human health impact under field conditions.

In urban and periurban areas, agricultural soils are often irrigated with surface water containing a complex mixture of contaminants due to wastewater treatment plant (WWTP) effluent discharges. The unplanned water reuse of these resources for crop irrigation can represent a pathway for contaminant propagation and a potential health risk due to their introduction in the food chain. The aim of this study is to provide data about the magnitude of attenuation processes and plant uptake, allowing for a reliable assessment of contaminant transfer among compartments and of the human health risk derived from unplanned water reuse activities. Target compounds are 25 pharmaceuticals, including transformation products (TPs). The field site is an agricultural parcel where maize is irrigated by a gravity-fed surface system supplied by the Jarama river, a water course strongly impacted by WWTP effluents. Throughout the 3-month irrigation period, irrigation water and water infiltrating through the vadose zone were sampled. The agricultural soil was collected before and after the irrigation campaign, and maize was sampled before harvesting. All selected compounds are detected in irrigation water (up to 12,867 ng L-1). Metformin, two metamizole TPs and valsartan occur with the highest concentrations. For most pharmaceuticals, results demonstrate a high natural attenuation during soil infiltration (>60%). However, leached concentrations of some compounds can be still at concern level (>400 ng L-1). A persistent behavior is observed for carbamazepine, carbamazepine epoxide and sulfamethoxazole. Pharmaceutical soil contents are in the order of ng g-1 and positively ionized compounds accumulate more effectively. Results also indicate the presence of a constant pool of drugs in soils. Only neutral and cationic pharmaceuticals are taken up in maize tissues, mainly in the roots. There is an insignificant threat to human health derived from maize consumption however, additional toxicity tests are recommended for 4AAA and acetaminophen.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Identification of contaminants of concern in the upper Tagus river basin (central Spain). Part 1: Screening, quantitative analysis and comparison of sampling methods.

Pesticides and point source contaminants (primarily pharmaceuticals) were monitored in 16 sampling sites of the upper Tagus river basin during spring, summer and autumn of 2016. A qualitative screening analysis was performed using a library of 430 compounds. Next, a novel method was implemented for the selection and quantification of contaminants with LC-MS/MS. The method is based on the frequency of detection in the screening, ecotoxicity data and the potential use in the watershed. Moreover, the efficacy of grab samples and passive samples (POCIS) in detecting compound-specific exposure patterns was compared during the summer sampling campaign. The screening method detected the presence of 268 compounds in the study area, out of which 52 were selected for the quantitative analysis (20 pesticides and 32 point source chemicals). Although very helpful in the prioritization exercise, the qualitative screening demonstrated some biases and the need for improvement by using more effective instruments for confirming positive results. Grab samples proved not to be fully suitable for contaminants with discontinuous exposure such as pesticides, which may be underestimated, but offer a sufficient basis for the characterization of contaminants coming from urban wastewaters. All selected chemicals showed a very high concentration variability due to differences among sampling sites, which are related to agricultural intensity and demographic pressure. Some insecticides (chlorpyrifos, dimethoate, imidacloprid), herbicides (diuron, metribuzine, simazine, terbuthylazine), and fungicides (carbendazim) were measured at concentrations exceeding 100 ng/L; while paracetamol, ibuprofen, some antibiotics (azithromycin, sulfamethoxazole, trimethoprim) and life-style compounds (caffeine, paraxanthine, nicotine) were found at very high concentrations (up to several µg/L). The results of this work represent the basis for the development of an ecological risk assessment for the aquatic ecosystem in the upper Tagus river basin and for the identification of basin-specific contaminant mixtures of environmental concern.

Identification of contaminants of concern in the upper Tagus river basin (central Spain). Part 2: Spatio-temporal analysis and ecological risk assessment.

This study provides a description of the water quality status in the tributaries of the upper Tagus River and a preliminary risk assessment for freshwater organisms. A wide range of physico-chemical parameters, nutrients, metals and organic contaminants (20 pesticides, and 32 point source chemicals, mainly pharmaceuticals) were monitored during spring, summer and autumn of 2016. Monitoring of organic contaminants was performed using conventional grab sampling and passive samples (POCIS). The variation of the different groups of parameters as regards to land use and sampling season was investigated. The prioritization of organic and inorganic contaminants was based on the toxic unit (TU) approach, using toxicity data for algae, invertebrates and fish. Finally, the compliance with the Environmental Quality Standards (EQS) set as part of the Water Framework Directive (WFD) was evaluated for the listed substances. This study shows that the land use characteristics had a large influence on the spatial distribution of the contaminants and other water quality parameters, while temporal trends were only significant for physico-chemical parameters, and marginally significant for insecticides. Acute toxicity is likely to occur for some metals (copper and zinc) in the most impacted sites (TU values close to or above 1). Low acute toxicity was determined for organic contaminants (individual compounds and mixtures) on the basis of grab samples. However, the assessment performed with POCIS samples identified diuron, chlorpyrifos and imidacloprid as potentially hazardous compounds. Several contaminant mixtures that may cause chronic toxicity and that should be considered in future regional chemical monitoring plans were identified. Our study also shows that some metals and pesticides exceeded the WFD regulatory thresholds and that only 30% of the sampled sites had a good chemical status. Further research is needed to identify chemical emission sources and to design proper abatement options in the Tagus river basin.

Effects of increased temperature, drought, and an insecticide on freshwater zooplankton communities.

In the present study we performed a microcosm experiment to assess the effects of the insecticide lufenuron on zooplankton communities exposed to increased temperature and drought in (semi-)arid regions. The experiment consisted of 3 environmental scenarios, assessed in 2 parts. Firstly, we assessed how water temperature (20 and 28 °C) affects the sensitivity and resilience of the zooplankton community to lufenuron. Secondly, we investigated the influence of drought on the structure of the zooplankton community at a high water temperature (28 °C) and evaluated its possible interaction with lufenuron. The results show that the community exposed to lufenuron at 28 °C had a faster lufenuron-related response and recovery than the community at 20 °C. The combined effects of lufenuron and temperature resulted in a synergistic effect on some taxa (Daphnia sp., Cyclopoida, and Copepoda nauplii). The tested zooplankton community had a high resilience to drought, although some particular taxa were severely affected after desiccation (Calanoida). Interactions between drought and lufenuron were not statistically significant. However, rewetting after desiccation contributed to lufenuron remobilization from sediments and resulted in a slight Cyclopoida population decline at high exposure concentrations. The study shows how environmental conditions related to global change in (semi-)arid regions may influence chemical fate and the vulnerability of zooplankton communities to chemical stress. Environ Toxicol Chem 2019;38:396-411. © 2018 SETAC.

Effects of imidacloprid and a neonicotinoid mixture on aquatic invertebrate communities under Mediterranean conditions.

Neonicotinoid insecticides are considered contaminants of concern due to their high toxicity potential to non-target terrestrial and aquatic organisms. In this study we evaluated the sensitivity of aquatic invertebrates to a single application of imidacloprid and an equimolar mixture of five neonicotinoids (imidacloprid, acetamiprid, thiacloprid, thiamethoxam, clothianidin) using mesocosms under Mediterranean conditions. Cyclopoida, Cloeon dipterum and Chironomini showed the highest sensitivity to neonicotinoids, with calculated NOECs below 0.2 µg/L. The sensitivity of these taxa was found to be higher than that reported in previous studies performed under less warm conditions, proving the high influence of temperature on neonicotinoid toxicity. The short-term responses of the zooplankton and the macroinvertebrate communities to similar imidacloprid and neonicotinoid mixture concentrations were very similar, suggesting that the concentration addition model can be used as a plausible hyphotesis to assess neonicotinoid mixture effects in aquatic ecosystems. Long-term mixture toxicity assessments, however, should consider the fate of the evaluated substances in the environment of concern. As part of this study, we also demonstrated that Species Sensitivity Distributions constructed with chronic laboratory toxicity data and calculated (multi-substance) Potentially Affected Fractions provide an accurate estimation to asssess the ecotoxicologial risks of imidacloprid and neonicotinoid mixtures to aquatic invertebrate species assemblages.

An optimized sample treatment method for the determination of antibiotics in seawater, marine sediments and biological samples using LC-TOF/MS.

Antibiotics used in marine aquaculture have been reported to accumulate in sediments and non-target aquatic organisms, modifying the biodiversity and the environmental conditions in areas close to the fish farms. Improved analytical methods are required to assess the spread and the impacts of aquaculture antibiotics in the marine environment, as well as to estimate resistance development risks. In this study, we have optimized a method for simultaneous quantitative determination of oxytetracycline, florfenicol and flumequine in marine samples using liquid chromatography coupled to time-of-flight high resolution mass spectrometry (LC-TOF/MS). The method optimization was carried out for seawater, sediment and biological samples (biofilm and two benthic invertebrate species: Gammarus aequicauda and Monodonta articulata). Special attention was paid to the optimization of the extraction and purification steps, testing: liquid-liquid and solid-liquid extractions, the use of silica and other commercial sorbents' clean-up, and single and tandem solid phase extraction procedures. The limits of quantification (MQLs) achieved with the developed method are 0.1-0.5 µg L-1 in seawater; 1-5 µg kg-1 in marine sediments; 5-25 µg kg-1 in biofilm; and 100-500 µg kg-1 in invertebrates, with good accuracy and precision. Method recoveries in spiked samples are 65-120% in seawater and sediment samples, and 63-110% in the biological samples. The method has been successfully implemented for the determination of antibiotic concentrations in sediment and invertebrate samples collected from a Mediterranean bay in south-east Spain. These represent significant advances in the analysis of antibiotics in environmental samples, especially for wild marine taxa, and attend for a proper assessment of the environmental fate and side effects of aquaculture antibiotics in the marine environment.

A Tricin Derivative from Deschampsia antarctica Desv. Inhibits Colorectal Carcinoma Growth and Liver Metastasis through the Induction of a Specific Immune Response.

In colorectal carcinoma patients, distant metastatic disease is present at initial diagnosis in nearly 25% of them. The majority of patients with metastatic colorectal carcinoma have incurable disease; therefore, new therapies are needed. Agents derived from medicinal plants have already demonstrated therapeutic activities in human cancer cells. Antartina is an antitumor agent isolated from Deschampsia antarctica Desv. This study aimed to evaluate the antitumor properties of Antartina in colorectal carcinoma models. We used human and murine colorectal carcinoma cell lines for investigating proliferation, apoptosis, and cell-cycle effects of Antartina therapy in vitro Avatar and immunocompetent colorectal carcinoma animal models were applied for evaluating the effects of Antartina in vivo Immune response against colorectal carcinoma model was investigated using CTL assay, analyzing dendritic cell activation and intratumor T-cell subpopulation, and by tumor rechallenge experiments. Antartina inhibits in vitro human colorectal carcinoma cell proliferation; however, in vivo experiments in Avatar colorectal carcinoma model Antartina display a limited antitumor effect. In an immunocompetent colorectal carcinoma mice model, Antartina potently inhibited tumor growth and liver metastases, leading to complete tumor regressions in >30% of mice and increased animal survival. In addition, Antartina induced a potent specific cytotoxic T-cell response against colorectal carcinoma and a long-lasting antitumor immunity. Interestingly, Antartina increased tumor immunogenicity and stimulated dendritic cell activation. No toxic effects were observed at the doses employed. Our findings showed that Antartina has the ability to induce antitumor immunity against colorectal carcinoma and can be used to develop new tools for the treatment of colorectal carcinoma. Mol Cancer Ther; 17(5); 966-76. ©2018 AACR.

Recent contributions for improving sensitivity in chiral CE.

The flexibility and versatility of the chiral CE are unrivaled and the same instrumentation can be used to separate a diverse range of analytes, both large and small molecules, whether charged or uncharged. However, one of the disadvantages is generally thought to be the poor sensitivity of ultraviolet (UV) detection, which is the most popular among CE detectors. This review focuses on methodologies and applications regarding improvements of sensitivity in chiral CE published in the last 2 years (June 2015 until May 2017). This contribution continues to update this series of biannual reviews, first published in Electrophoresis in 2006. The main body of the review brings a survey of publications organized according to different approaches to detect a low amount of analytes, either by sample treatment procedures or by in-capillary sample preconcentration techniques, both using UV detection, or even by employing detection systems more sensitive than UV absorption, such as LIF or MS. This review provides comprehensive tables listing the new approaches in sensitive chiral CE with categorizing by the fundamental mechanism to enhance the sensitivity, which provide relevant information on the strategies employed. The concluding remarks in the final part of the review evaluate present state of art and the trends for sensitivity enhancement in chiral CE.

Presence or Absence of mlr Genes and Nutrient Concentrations Co-Determine the Microcystin Biodegradation Efficiency of a Natural Bacterial Community.

The microcystin biodegradation potential of a natural bacterial community coexisting with a toxic cyanobacterial bloom was investigated in a water reservoir from central Spain. The biodegradation capacity was confirmed in all samples during the bloom and an increase of mlrA gene copies was found with increasing microcystin concentrations. Among the 24 microcystin degrading strains isolated from the bacterial community, only 28% showed presence of mlrA gene, strongly supporting the existence and abundance of alternative microcystin degradation pathways in nature. In vitro degradation assays with both mlr⁺ and mlr- bacterial genotypes (with presence and absence of the complete mlr gene cluster, respectively) were performed with four isolated strains (Sphingopyxis sp. IM-1, IM-2 and IM-3; Paucibacter toxinivorans IM-4) and two bacterial degraders from the culture collection (Sphingosinicella microcystinivorans Y2; Paucibacter toxinivorans 2C20). Differences in microcystin degradation efficiencies between genotypes were found under different total organic carbon and total nitrogen concentrations. While mlr⁺ strains significantly improved microcystin degradation rates when exposed to other carbon and nitrogen sources, mlr- strains showed lower degradation efficiencies. This suggests that the presence of alternative carbon and nitrogen sources possibly competes with microcystins and impairs putative non-mlr microcystin degradation pathways. Considering the abundance of the mlr- bacterial population and the increasing frequency of eutrophic conditions in aquatic systems, further research on the diversity of this population and the characterization and conditions affecting non-mlr degradation pathways deserves special attention.

Metabolomic fingerprinting of saffron by LC/MS: novel authenticity markers.

An untargeted metabolomic approach using liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry was developed in this work to identify novel markers for saffron authenticity which is an important matter related to consumer protection, quality assurance, active properties, and also economical impact (saffron is the most expensive spice). Metabolic fingerprinting of authentic and suspicious saffron samples from different geographical origin was obtained and analyzed. Different extracting protocols and chromatographic methodologies were evaluated to obtain the most adequate extracting and separation conditions. Using an ethanol/water mixture at pH 9.0 and an elution gradient with a fused core C18 column enabled obtaining the highest number of significant components between authentic and adulterated saffron. By using multivariate statistical analysis, predictive classification models for authenticity and geographical origin were obtained. Moreover, 84 and 29 significant metabolites were detected as candidates for markers of authenticity and geographical origin, respectively, from which only 34 metabolites were tentatively identified as authenticity markers of saffron, but none related to its geographical origin. Six characteristic compounds of saffron (kaempferol 3-O-glucoside, kaempferol 3-O-sophoroside, kaempferol 3,7-O-diglucoside, kaempferol 3,7,4'-O-triglucoside, kaempferol 3-O-sophoroside-7-O-glucoside, and geranyl-O-glucoside) were confirmed by comparing experimental MS/MS fragmentation patterns with those provided in scientific literature being proposed as novel markers of authenticity. Graphical Abstract Metabolomic fingerprinting of saffron.

Determination of nonprotein amino acids and betaines in vegetable oils by flow injection triple-quadrupole tandem mass spectrometry: a screening method for the detection of adulterations of olive oils.

A novel screening method using an automated flow injection electrospray ionization tandem mass spectrometry system is proposed for the simultaneous determination of five nonprotein amino acids (ß-alanine, alloisoleucine, ornithine, citrulline, pyroglutamic acid) and three betaines (glycine betaine, trigonelline, proline betaine) after derivatization with butanolic HCl. MS/MS experiments were carried out in a triple-quadrupole instrument using multiple reaction monitoring mode in <2 min. The proposed method provided high fingerprinting power to identify the presence of five of the studied compounds in different types of vegetable oils (soybean, sunflower, corn, olive) with LODs at parts per billion levels. The method was validated, and different mixtures of extra virgin olive oil with seed oils were analyzed, achieving the typification for the detection of adulterations in extra virgin olive oils up to 2% w/w. The nonprotein amino acid ornithine was confirmed as a marker for adulteration in the olive oils analyzed.