Determination of Multiclass Pharmaceutical Residues in Milk Using Modified QuEChERS and Liquid-Chromatography-Hybrid Linear Ion Trap/Orbitrap Mass Spectrometry: Comparison of Clean-Up Approaches and Validation Studies.

A multi-residue method was developed to identify and quantify pharmaceutical drug residues in full-fat milk, using a modified QuEChERS extraction procedure and sonication combined with Ultra-High-Performance Liquid Chromatography-High-Resolution Orbitrap Mass Spectrometry (UHPLC-HR-Orbitrap-MS). Sample preparation involves three different QuEChERS extraction procedures and sorbents for the purification step. The optimized modified extraction method, combined with the clean-up approaches using C18 and the EMR-Lipid sorbent, has been validated in terms of linearity, recovery, precision, LOD and LOQ, matrix effects (ME) and expanded uncertainty. The optimized method showed a linearity >0.9903, recoveries within the range 65.1-120.1%, precision (expressed as %RSD) <17.5%, medium (<39.9%) to low (<16.7%) matrix effects and acceptable expanded uncertainty (<33.1%). Finally, the proposed method was applied to representative real samples of milk (by local markets), revealing the existence of one pharmaceutical drug (imidocarb) in one sample.

Beneficial properties of Drimia numidica leaf methanolic extract against the cytogenotoxic effects of mitomycin C on human lymphocytes.

This study investigated the phytochemical profile of Drimia numidica leaf methanolic extract, as well as its cyto-genotoxic and cyto/genoprotective potential against mitomycin C (MMC) mediated effects on healthy human lymphocytes. Photosynthetic pigments, trace elements, and secondary metabolites were estimated and/or identified in methanolic extract of mature leaves, and the latter was further used for assessing its in vitro biological effects on MMC-free and/or MMC-treated human lymphocytes (at low, non-toxic concentrations of 0.001 and 0.01% v/v). The results showed that D. numidica leaf methanolic extract, being rich in carotenoids, phenolics, flavonoids, organic acids and bufadienolides, could be protective against MMC mediated cyto/genotoxic potential in healthy human lymphocytes. Biomolecules possessing antioxidant and antitumor potential, such as beta-carotene and lutein among others, chlorogenic acid, caffeic acid and their derivatives, minerals such as Si, as well as apigenin- and luteolin-derived glycosides, either individual or in a mixture, could be beneficial rather than harmful, at least at the extract concentrations tested. Although further in vitro and in vivo studies are still needed for elucidating the beneficial (individual and/or additive/synergistic) role of those compounds, the results of the present study are quite promising, thus encouraging new challenges for the appropriate utilization of D. numidica leaf extract.

Enhancing bioactive compounds accumulation in red beet (Beta Vulgaris L.) plants by managing N nutrition. The identification of the 'critical' zone as a cultivation technique.

The increasing interest in natural health-promoting compounds, which are mostly plant secondary metabolites, inspired attempts to stimulate mechanisms strengthening their bioaccumulation in crop plants via abiotic stress while maintaining the yield potential. This study investigates the long-term effects of limiting nitrogen (N) supply on the concentration of total phenolics, free radical activity of natural antioxidants, betacyanin content, biomass production, net photosynthetic rate, total chlorophyll content, and plant water relations in red beetroot plants (Beta vulgaris L.) grown hydroponically. Depending on fertilization, the range of N supply for evaporative demand comprises two contrasted nutrient zones, in which N is limiting (zone-1) or non-limiting (zone-2). Based on the carbon-nutrient-balance hypothesis, at the transition from 1st-zone to 2nd-zone, there is a narrow transition zone in which the plant nutrient status is considered 'critical'. Herein, to determine the 'critical' zone, a modified Michaelis-Menten (M-M) model was used using a piecewise linear regression on two indexes: net photosynthetic rates and free radical-scavenging capacity of phenolic antioxidants. The model showed that the 'critical' transition points of net photosynthetic rate and phenolic free radical content are located in a narrow zone ranging between 196.70 ± 8.75 and 271.54 ± 75.50 ppm NO3-, while the cropping season appears to affect slightly the range of 'critical' (transition) zone. Thus, supplying N to red beetroot plants to levels ranging within this 'critical' zone may be an efficient, profitable and sustainable way to increase the accumulation of health-promoting plant bioactive compounds (total phenolic compounds with radical activity and betacyanins) in hydroponically cultivated reed beetroot plants.

Assessing the human risk and the environmental fate of pharmaceutical Tramadol.

Tramadol (TRA) is a widely used human pharmaceutical and a well-established emerging pollutant and its potential genotoxic and cytotoxic effects on humans as well as its fate in aqueous systems demand full investigation. The present study is a multidisciplinary approach and provides important insights on the potential risks of Tramadol on humans accompanied by its photolytic transformation under simulated solar irradiation. The present study revealed that Tramadol can induce genotoxic and cytotoxic effects under the specific experimental conditions, significantly depended on the tested concentration. In addition, the photolytic transformation of Tramadol was investigated in detail under simulated solar irradiation in two different water matrices: ultrapure water (UW) and treated wastewater (WW). Differences in the degradation rates were observed between UW and WW, being slower in WW. The results showed that more than 70% of Tramadol was removed after 240 min in UW ([TRA] = 10 mg L-1, I = 500 W m-2). After this period, TOC removal was found to be about 40%. Transformation of N atoms into NO3- and NH4+ followed a similar trend reaching up to 38% release. Τramadol degraded mainly by HO radicals and 1O2 through a self-sensitizing process while direct photolysis was also significant. Hydroxylation, demethylation and N-oxidation of the parent compound were found to be the main degradation pathways confirming the important role of HO and 1O2 in the photolytic process. Toxicity measurements showed a noticeable increase of the inhibition for Vibrio fischeri at the first stages which coincide with the formation of the major TPs.

Assessment of the toxic potential of rainwater precipitation: First evidence from a case study in three Greek cities.

Considering the necessity to monitor rainwater quality and the limited existing data regarding wet precipitations' toxic potential, the toxic potential of rainwater (RW) samples collected in three cities of Western Greece (Corfu, Agrinion and Patras) during May, October, November, and December of 2014 were investigated. The relationship of the adverse effects obtained, against cytokinesis block micronucleus (CBMN) and Vibrio fischeri bioluminescence (Microtox) assays with the physicochemical characteristics, the heavy metal concentration levels of rainwater samples and the meteorological parameters governing the climatic conditions in each region, were studied in detail. All samples showed pH values in the range of 5.37-7.17, high concentrations of ΝΗ4+ and the presence of SO42- and NO3-. The metals Al, Mn, Fe, Co, Ni, Cu, Zn, As, Sb, Cd, were detected in most of the samples, with maximum concentrations ranging from 0.16 µg L-1 (for Co) to 86.6 µg L-1 (for Fe). The CBMN assay showed statistically significant rainwater cytotoxic activity at 5% (v/v) concentration in all cities during all months studied and at 10% (v/v) in Agrinion and Corfu in May. No genotoxic effect was induced at 5% (v/v) concentration of rainwater, except for Corfu city during November, whereas 10% (v/v) of rainwater led to statistically significant increase in micronuclei frequencies in all three cities. The highest (100%) and the lowest (14%) inhibitory effect exhibited by the rainwater in the Vibrio test were noticed at Corfu city during May and December, respectively. Considering the inter-relations obtained among the abiotic and biotic variables measured in all cases, conductivity, SO42-, NH4+, and total metal burden could be related with both CBMN and Microtox assays data, regardless of the site- and month-specific differences. The multidisciplinary approach presented here showed that the physicochemical profile of rainwater precipitation could be significantly related with the induction of adverse effects on biota.

Dissipation and runoff transport of metazachlor herbicide in rapeseed cultivated and uncultivated plots in field conditions.

The environmental fate of metazachlor herbicide was investigated under field conditions in rapeseed cultivated and uncultivated plots, over a period of 225 days. The cultivation was carried out in silty clay soil plots with two surface slopes, 1 and 5 %. The herbicide was detectable in soil up to 170 days after application (DAA), while the dissipation rate was best described by first-order kinetics and its half-life ranged between 10.92 and 12.68 days. The herbicide was detected in the soil layer of 10-20 cm from 5 to 48 DAA, and its vertical movement can be described by the continuous stirred tank reactor (CSTR) in series model. Relatively low amounts of metazachlor (less than 0.31 % of the initial applied active ingredient) were transferred by runoff water. More than 80 % of the total losses were transferred at the first runoff event (12 DAA), with herbicide concentrations in runoff water ranging between 70.14 and 79.67 µg L-1. Minor amounts of the herbicide (less than 0.07 % of the initial applied active ingredient) were transferred by the sediment, with a maximum concentration of 0.57 µg g-1 (12 DAA), in plots with 5 % inclination. Finally, in rapeseed plants, metazachlor was detected only in the first sampling (28 DAA) at concentrations slightly higher than the limit of quantification; when in seeds, no residues of the herbicide were detected.

Environmental behavior of the fungicide metalaxyl in experimental tobacco field.

The loss of metalaxyl, a systemic fungicide, was determined in runoff water from loamy soil plots of various surface slopes cultivated with tobacco, over a period of 170 days. Conditions were selected to simulate agricultural practices employed in the Mediterranean region. The surface slopes of plots were 0, 2.5, 5, 7.5, 10% and both cultivated and uncultivated (control) areas were simultaneously monitored. The cumulative losses of metalaxyl in surface runoff from tilled and untilled plots with a slope of 10% were estimated at 0.469% and 0.740% of the initial applied active ingredient respectively, while for the plots with a slope 0% they were 0.033% and 0.044%. The dissipation in topsoil was studied for a period of 110 days. The half-lives that were calculated using first-order kinetics ranged from 13.7 to 16.6 days in tobacco soil and from 13.8 to 17 days in non-cropped soil. The dissipation of metalaxyl from the topsoil in cultivation of tobacco was higher in comparison with the untilled plots (46-62% and 52-69% respectively, of the applied dose in 23 days after the second application). The slope of soil surface, the compound solubility and sorption capacities are the main parameters that influenced the transport of metalaxyl residues via surface water in soil-water systems.

Spatiotemporal variation and risk assessment of pesticides in water of the lower catchment basin of Acheloos River, Western Greece.

A three-year monitoring survey (March 2005-February 2008) was conducted to investigate, on monthly basis, the presence of thirty pesticides belonging to various categories and metabolites, in Acheloos River (Western Greece), one of the most important water resources in Greece. Six sampling stations along the river were established. Water analyses were performed using solid-phase extraction combined with gas chromatography with flame thermionic detector and mass spectrometry. Statistical analysis using one-way ANOVA and Duncan's multiple range test (P < 0.05) was used to compare annual mean concentrations of pesticides, seasonal and spatial distribution. In general, the highest mean concentrations of the pesticides were recorded at the three stations downstream. The greatest average concentrations were determined during spring and summer in agreement with the pesticide application period. The observed lower concentrations after 2006 reflect the land-use change because of the elimination of tobacco, the main cultivation of the area for many decades. The compounds most frequently detected were diazinon (78.6%), DEA (69.3%), and fenthion (52.6%). Environmental risk assessment using risk quotient (RQ) approach showed high risk for six insecticides in 2005 and one in 2007. A compliance with the European Environmental Quality Standards (EQS) was observed for the priority pesticides.

Novel imazethapyr detoxification applying advanced oxidation processes.

Different degradation methods have been applied to assess the suitability of advanced oxidation process (AOPs) to promote mineralization of imazethapyr [(RS)-5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid], a widely used imidazolinone class herbicide, the persistence of which has been demonstrated in surface and ground waters destined to human uses. Independent of the oxidation process assessed, the decomposition of imazethapyr always followed a pseudo-first order kinetic. The direct UV-irradiation (UV) of the herbicide as well as its oxidation with ozone (O3), and hydrogen peroxide tied to UV-irradiation (H2O2/UV) were sufficiently slow to permit the identification of intermediate products, the formation pathway of which has been proposed. Ozonation joined to UV-irradiation (O3/UV), ozonation joined to titanium dioxide photo-catalysis (TiO2/UV+O3), sole photo-catalysis (TiO2/UV), and photo-catalysis reinforced with hydrogen peroxide-oxidation (TiO2/UV+H2O2) were characterized by a faster degradation and rapid formation of a lot of small molecules, which were quickly degraded to complete mineralization. The most effective oxidation methods were those using titanium dioxide photo-catalysis enhanced either by ozonation or hydrogen peroxide. Most of all, these last processes were useful to avoid the development of dangerous by-products.

Passive sampling of selected pesticides in aquatic environment using polar organic chemical integrative samplers.

PURPOSE: Polar chemical integrative samplers (POCIS) were examined for their sampling efficiency of 12 pesticides and one metabolite commonly detected in surface waters. Laboratory-based calibration experiments of POCISs were conducted. The determined passive sampling rates were applied for the monitoring of pesticides levels in Lake Amvrakia, Western Greece. Spot sampling was also performed for comparison purposes. METHODS: Calibration experiments were performed on the basis of static renewal exposure of POCIS under stirred conditions for different time periods of up to 28 days. The analytical procedures were based on the coupling of POCIS and solid phase extraction by Oasis HLB cartridges with gas chromatography-mass spectrometry. RESULTS: The recovery of the target pesticides from the POCIS was generally >79% with relative standard deviation (RSD) <16%. The calibration results revealed an integrative uptake of all pesticides for 28 days and the calculated sampling rates ranged from 0.025 to 0.388 L day(-1) with RSD <29%. Low nanogram/liter levels of pesticides such as diazinon, alachlor, and s-metolachlor were detected during the monitoring campaign using both passive and spot sampling whereas higher concentrations were measured by spot sampling in most cases. CONCLUSIONS: Passive sampling by POCIS provides a useful tool for the monitoring of pesticides in aquatic systems since integrative sampling at rates sufficient for analytical quantitation of ambient levels was observed. Calibration data are in demand for a greater number of compounds in order to extend the use in environmental monitoring.

2-Hydroxypyridine photolytic degradation by 254 nm UV irradiation at different conditions.

The degradation and mineralisation of (400 mL, 3.15 mM) aqueous solutions of 2-hydroxypyridine (2-HPY), a primary intermediate formed during the photolytic degradation of 2-chloropyridine (2-CPY), 2-bromopyridine (2-BPY) and 2-iodopyridine (2-IPY), was studied at 50 degrees C, under 254 nm UV irradiation in a range of conditions. The rate of 2-HPY disappearance was found to heavily depend on agitation, dissolved oxygen, pH and hydroxyl radical scavenger presence. pH has a pronounced effect on the phototreatment of 2-HPY, i.e. 2-HPY removal is faster at lower pH, but except for their influence on the solution pH, Cl(-) or F(-) do not appear to affect 2-HPY rate of removal or mineralisation. 2-HPY removal rate increases with dissolved oxygen, thus indicating a prominent 2-HPY photooxidation pathway. Helium purging of the solution before and during the measurement reduced 2-HPY removal rate, thus supporting the existence of a photooxidation pathway. Pure photolytic 2-HPY removal, i.e. when photooxidation is limited, fits pseudo-first order kinetics, and a kinetic model was developed for 2-HPY formation and removal during 2-CPY, 2-BPY and 2-IPY photolytic degradation under laminar flow. The addition of radical scavenger (tert-butanol) had a detrimental effect on the photolytic degradation of 2-HPY. Complete total organic carbon removal was achieved.

Photolytic removal and mineralisation of 2-halogenated pyridines.

The photolytic destruction and mineralisation of 2-halogenated pyridines (2-HalPYs) was studied in a range of conditions by means of ultraviolet irradiation at 254 nm. In all cases pseudo-first order kinetics satisfactorily describe the degradation process. Key decomposition products were identified and primary reaction pathways are proposed. 2-HalPYs were rapidly dehalogenated with a subsequent sharp pH drop. Complete Total Organic Carbon (TOC) removal was achieved. Their photolytic removal rate is not affected by pH or aeration. The fastest TOC removal was observed in 2-fluoropyridine (2-FPY) photolysis. 2-Hydroxypyridine (2-HPY) was identified as the primary intermediate formed by destruction of 2-chloropyridine (2-CPY), 2-bromopyridine (2-BPY) and 2-iodopyridine (2-IPY). 2-HPY is further destructed to form Dewar pyridinone. 2-FPY only marginally reacted to form 2-HPY. Instead, its major intermediate product is Dewar pyridinone. 2-IPY results in a, most likely aliphatic, photostable product, the production of which appears to increase at low pH and high 2-HPY concentration.

Effects of dissolved carboxylates and carbonates on the adsorption properties of thiuram disulfate pesticides.

The adsorption of thiram and disulfiram onto alpha-Al2O3 and montmorillonite clay has been studied in the presence of small carboxylate anions, bicarbonate, formate, and oxalate. At natural concentrations, HCO3- enhances dramatically the adsorption of both pesticides on alpha-Al2O3 and clay. An analogous significant enhancement of pesticide adsorption is also observed in the presence of formate and oxalate. Density functional theory calculations demonstrate that in solution a stable molecular complex between one molecule of thiram and one molecule of HCO3- is formed with interaction energy -35.6 kcal/mol. In addition, two H20 molecules further stabilize it by an interaction energy of -3.6 kcal/mol. This clustering [thiram- HCO3- -2H2O] leads to a change of the electronic structure and the ultraviolet-visible spectrum of thiram that is observed experimentally. Surface complexation modeling shows that the molecular cluster [thiram-HCO3- -2H2O], which bears a total net charge of -1, is responsible for the observed enhanced adsorption on the charged surface of alumina and clay at pH below their points of zero surface charge. The results reveal a novel pervasive role of carboxylate anions and particularly HCO3- on the adsorption of dithiocarbamate pesticides in natural waters.

The status of pesticide pollution in surface waters (rivers and lakes) of Greece. Part I. Review on occurrence and levels.

This review evaluates and summarizes the results of long-term research projects, monitoring programs and published papers concerning the pollution of surface waters (rivers and lakes) of Greece by pesticides. Pesticide classes mostly detected involve herbicides used extensively in corn, cotton and rice production, organophosphorus insecticides as well as the banned organochlorines insecticides due to their persistence in the aquatic environment. The compounds most frequently detected were atrazine, simazine, alachlor, metolachlor and trifluralin of the herbicides, diazinon, parathion methyl of the insecticides and lindane, endosulfan and aldrin of the organochlorine pesticides. Rivers were found to be more polluted than lakes. The detected concentrations of most pesticides follow a seasonal variation, with maximum values occurring during the late spring and summer period followed by a decrease during winter. Nationwide, in many cases the reported concentrations ranged in low ppb levels. However, elevated concentrations were recorded in areas of high pesticide use and intense agricultural practices. Generally, similar trends and levels of pesticides were found in Greek rivers compared to pesticide contamination in other European rivers. Monitoring of the Greek water resources for pesticide residues must continue, especially in agricultural regions, because the nationwide patterns of pesticide use are constantly changing. Moreover, emphasis should be placed on degradation products not sufficiently studied so far.

Environmental monitoring and ecological risk assessment for pesticide contamination and effects in Lake Pamvotis, northwestern Greece.

Monitoring of pesticide residues in water and sediments was conducted as a basis for subsequent ecotoxicological risk assessment for the shallow eutrophic Lake Pamvotis, northwestern Greece. During a one-year study period, atrazine, desethylatrazine (DEA), simazine, diazinon, malathion, oxamyl, carbofuran, and ethion were detected in water and atrazine, desethylatrazine, diazinon, and s-ethyl dipropylthiocarbamate (EPTC) in sediments, all at ppb concentration level. Temporal variation in pesticide concentrations was observed. Highest residue levels for most pesticides in both water and sediment matrices occurred in the May to July period with the exception of atrazine and DEA, which show highest levels in water during the September to November period. The ecological risk associated with pesticide contamination was assessed using two different methods: The toxic unit method, which provides a first indication of the relative contribution of detected pesticides to the total toxicity and a probabilistic approach, and the inverse method of Van Straalen and Denneman, which is used to quantify the ecological risk. The maximum percentage of the ecological risk was 10.3 and 51.8% for water and 17.2 and 70.6% for sediment, based on acute and chronic level, respectively. These results show that pesticides exert a significant pressure on the aquatic system of Lake Pamvotis, especially for the chronic-effect level. Simple quotient methods should be coupled with higher-tier risk assessment, especially if restoration activities on lake ecosystems are to be undertaken for sustainable development.

Pirimiphos-methyl and benalaxyl losses in surface runoff from plots cultivated with potatoes.

Abstract: Losses of pirimiphos-methyl and benalaxyl in runoff water from clay soil plots cultivated with potatoes and of differing soil surface slopes were determined over approximately 120 days (1 October 1999-28 January 2000). The plot slopes were 0, 1, 2.5 and 5%, and soil erosion increased with the slope from 610 to 1760kgha(-1). The runoff of surface water was between 3.1 and 16.6% of the rainfall. Surface runoff was highest for the fifth and seventh runoff events due to rainfall, 51 days and 72 days after the first pesticide application. The maximum concentrations of the two pesticides in runoff occurred in the plots with the greatest slope (5%) during the fifth runoff event, November 21, 1999 reaching 8.4 and 12.3 microg litre(-1) for pirimiphos-methyl and 17.8 and 20.2 microg litre(-1) for benalaxyl in tilled and untilled plots respectively. The cumulative losses of pirimiphos-methyl in surface runoff from tilled and untilled plots with a slope 5% were estimated at only 0.37 and 0.59% of the initial applied active ingredient, respectively, while for plots with a slope 0% the percentages were 0.013 and 0.018%. For benalaxyl the corresponding values from tilled and untilled plots were 1.69 and 1.76% (slope 5%), and 0.062 and 0.085 (slope 0%). Degradation of the pesticides in the topsoil was monitored from October 1999 and May 2000. Cultivation of potatoes decreased the half-life of the two pesticides compared to the untilled fields, for pirimiphos-methyl from 16.7 to 9.2 days and for benalaxyl from 26.7 to 12.6 days. The slope of soil surface and the different sorption capacities for the compounds are the main parameters which influenced the transportation of studied pesticides, pirimiphos-methyl and benalaxyl residues via surface water in soil-water systems.

Application of solid-phase microextraction in the monitoring of priority pesticides in the Kalamas River (N.W. Greece).

A solid-phase microextraction (SPME) method was applied to an extended monitoring survey of priority pesticides for the European Union for a period of 12 months in water of the Kalamas River (Epirus region of northwestern Greece) in order to determine their concentrations and seasonal variations. Polydimethylsiloxane-coated fiber (100 microm) was used. The samples were screened using gas chromatography with flame thermionic detection. Detection was confirmed by gas chromatographymass spectroscopy. The most frequently detected pesticides were some of the more commonly used herbicides, such as S-ethyl-N,N-di-n-propylthiol carbamate (EPTC), trifluralin, atrazine, deethylatrazine, terbuthylazine and alachlor, and insecticides, such as carbofuran, diazinon, disulfoton, parathion methyl, parathion ethyl, fenthion and ethion. Concentrations of individual compounds ranged from 0.020 to 0.3 microg/L. Greater pesticide concentrations occurred during the seasons of application. A comparison with a well-established solid-phase extraction (C18 disks) procedure was performed for samples of high-season application (May-September) in order to confirm the effectiveness of the SPME technique. The results demonstrate the suitability of the SPME method for routine screening multiresidue analysis in natural waters.