Pesticides in water and sediments from natural protected areas of Spain and their associated ecological risk.

In the last years, issues related to intensive agriculture have been found in protected areas potentially harming wildlife. This study aimed to analyze a wide range of pesticides in water and sediments of two protected areas namely Doñana Natural Park (DNP) and Tablas de Daimiel National Park (TDNP) performing an environmental risk assessment in order to highlight potential risks to living organisms derived from pesticide burden. Higher pesticide load was found in DNP than TDNP with similar distribution profiles, with pyrethroid insecticides (PYRs) the main detected class. Particularly problematic are two PYRs, cyhalothrin and fenvalerate, which were detected at high concentrations that can pose a high risk to aquatic organisms. In addition, despite being detected at lower concentrations, the presence of chlorpyrifos, cypermethrin, and permethrin in water, and of chlorpyrifos, dicofol, and diflufenican in sediments, must be taken into account due to their potential risks for aquatic organisms. Moreover, some banned pesticides such as dimethoate, terbutryn, diazinon, and tricyclazol were detected in water at levels which deserve further investigation to assess their potential sources, including potential illegal practices.

Pesticide residue determination in surface waters by stir bar sorptive extraction and liquid chromatography/tandem mass spectrometry.

In this stir bar sorptive extraction (SBSE) method, 16 pesticides were extracted from surface water samples by sorption onto 1 mm polydimethylsiloxane layer coated on a 10-mm-length stir bar magnet. After liquid desorption of the analytes with 1 ml of methanol, the detection was performed on a liquid chromatography-tandem mass spectrometry with a triple quadrupole (QqQ) analyzer using selected reaction monitoring mode via electrospray ionization. Parameters affecting SBSE operation, including sample volume, salt addition, extraction time, stirring rate, and desorption conditions, have been evaluated. The optimized SBSE method required two 50 ml aliquots of surface water samples, one aliquot was added of 30% NaCl and stirred at 900 rpm during 1 h for testing five pesticides with log K(o/w) < 3, and the other aliquot was directly extracted following the same procedure for the rest of the pesticides with log K(o/w) > 3. The method was validated in spiked surface water samples at limits of quantifications (LOQs) and ten times the LOQs showing recoveries <62%, and the LOQs reached were from 0.03 microg l(-1) for diazinon to 3 microg l(-1) for simazine. The proposed methodology was applied to the determination of these compounds in samples from Albufera Lake and surrounding channels, showing that SBSE is a powerful tool for routine control analysis of pesticide residues in surface water.

Contaminants of emerging concern in freshwater fish from four Spanish Rivers.

This study investigated the occurrence of 135 contaminants of emerging concern (CECs) - pharmaceuticals, pesticides, a set of endocrine disrupting compounds (EDCs) (parabens, bisphenols, hormones, triazoles, organophosphorus flame retardants and triclosan), UV-filters, perfluoroalkyl substances (PFASs) and halogenated flame retardants (HFRs) - in 59 fish samples, collected in 2010 in 4 Spanish Rivers (Guadalquivir, Júcar, Ebro and Llobregat). Of the 135 CECs, 76 including 8 pharmaceuticals, 25 pesticides, 10 EDCs, 5 UV-filters, 15 PFASs and 13 HFRs were detected. Pharmaceuticals were the less frequently found and at lower concentrations. Pesticides, EDCs, UV-filters, PFASs and HFRs were detected more frequently (>50% of the samples). The maximum concentrations were 15 ng/g dry weight (dw) for pharmaceuticals (diclofenac), 840 ng/g dw for pesticides (chlorpyrifos), 224 ng/g dw for EDCs (bisphenol A), 242 ng/g dw for UV-filters (EHMC), 1738 ng/g dw for PFASs (PFHxA) and 64 ng/g dw for HFRs (Dec 602). The contaminants detected in fish are commonly detected also in sediments. In light of current knowledge, the risk assessment revealed that there was no risk for humans related to the exposure to CECs via freshwater fish consumption. However, results provide detailed information on the mixtures of CECs accumulated that would be very useful to identify their effects on aquatic biota.

Quantification of Listeria monocytogenes in salads by real time quantitative PCR.

A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 10(5)L. monocytogenes colony forming units (CFU), was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 10(5)L. monocytogenes CFU and the coefficient of determination (r2) was >0.98. RTQ-PCR presented an efficiency of >85%. The accuracy of the PCR-based assay, expressed as % bias, ranged from 9% to 26% and the precision, expressed as % CV, ranged 9-22%. Intraday and interday variabilities were studied at 10(2) CFU/g and resulted in 12% and 14%, respectively. The proposed RTQ-PCR method and classical cultural methods were applied to analyse 77 salads from restaurants in Valencia (Spain). All culture positive samples were also RTQ-PCR positive.

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Presence of pharmaceuticals and heavy metals in the waters of a Mediterranean coastal wetland: Potential interactions and the influence of the environment.

The occurrence of 17 relevant pharmaceuticals and 7 heavy metals in the waters of the Pego-Oliva Marsh Natural Park (Valencia Community, Spain) were monitored. Thirty four zones (including the lagoon and the most important irrigation channels), covering the main land uses and water sources, were selected for sampling. Thirty three of them were contaminated with at least one pharmaceutical. Ibuprofen and codeine were the pharmaceuticals more frequently detected, in concentrations between 4.8 and 1.2 ng/L and a maximum of 59 ng/L and 63 ng/L, respectively. Regarding metals, Zn showed values under the detection limit in all the samples, while Cd, Co, Cr, Cu, Ni and Pb were detected at concentrations lower than the WHO and EU maximum levels for drinking waters. Ni showed significant direct correlations with diazepam, norfloxacin, ofloxacin and fenofibrate, and inverse relationships with ibuprofen, at 99 and 95% of significance. Cu, Co and Cr also showed significant correlations with some of the pharmaceuticals. These interactions could favor the synergistic/antagonistic interactions among pharmaceuticals and metals in the marsh, which can affect its aquatic fauna and flora or even human health. The influences of the water sources, land uses and spatial distribution of both types of contaminants were also studied.

Shared effects of organic microcontaminants and environmental stressors on biofilms and invertebrates in impaired rivers.

Land use type, physical and chemical stressors, and organic microcontaminants were investigated for their effects on the biological communities (biofilms and invertebrates) in several Mediterranean rivers. The diversity of invertebrates, and the scores of the first principal component of a PCA performed with the diatom communities were the best descriptors of the distribution patterns of the biological communities against the river stressors. These two metrics decreased according to the progressive site impairment (associated to higher area of agricultural and urban-industrial, high water conductivity, higher dissolved organic carbon and dissolved inorganic nitrogen concentrations, and higher concentration of organic microcontaminants, particularly pharmaceutical and industrial compounds). The variance partition analyses (RDAs) attributed the major share (10%) of the biological communities' response to the environmental stressors (nutrients, altered discharge, dissolved organic matter), followed by the land use occupation (6%) and of the organic microcontaminants (2%). However, the variance shared by the three groups of descriptors was very high (41%), indicating that their simultaneous occurrence determined most of the variation in the biological communities.

Invertebrate community responses to emerging water pollutants in Iberian river basins.

Chemical pollution is one of the greatest threats to freshwater ecosystems, especially in Mediterranean watersheds, characterized by periodical low flows that may exacerbate chemical exposure. Different groups of emerging pollutants have been detected in these basins during the last decade. This study aims to identify the relationships between the presence and levels of prioritary and emerging pollutants (pesticides, pharmaceutical active compounds--PhACs, Endocrine Disrupting Compounds EDCs and Perfluorinated Compounds--PFCs) and the invertebrate community in four Mediterranean basins: the Ebro, the Llobregat, the Júcar and the Guadalquivir. Structural (species composition and density) and functional (catalase activity of the tricopteran Hydropsyche exocellata and the feeding activity of the cladoceran Daphnia magna) variables were analyzed to determine which of the pollutants would greatly influence invertebrate responses. EDCs and conductivity, followed by PhACs, were the most important variables explaining the invertebrate density changes in the studied basins, showing a gradient of urban and industrial pollutions. Despite this general pattern observed in the four studied basins - impoverishment of species diversity and abundance change with pollution - some basins maintained certain differences. In the case of the Llobregat River, analgesics and anti-inflammatories were the significant pollutants explaining the invertebrate community distribution. In the Júcar River, fungicides were the main group of pollutants that were determining the structure of the invertebrate community. Functional biomarkers tended to decrease downstream in the four basins. Two groups of pollutants appeared to be significant predictors of the catalase activity in the model: EDCs and PhACs. This study provides evidence that the information given by functional biomarkers may complement the results found for the structural community descriptors, and allowed us to detect two emerging contaminant groups that are mainly affecting the invertebrate community in these basins.

Determination of dithiocarbamates and metabolites in plants by liquid chromatography-mass spectrometry.

A quantitative matrix solid-phase dispersion and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method is outlined for the simultaneous analysis of dithiocarbamates (DTCs) and their degradation products in plants. Compounds analyzed are dazomet, disulfiram, thiram and the metabolites ethylenthiourea and propylenthiourea. The performance of two different sample preparation protocols, the proposed one and other based on solid-phase extraction, as well as, of both atmospheric pressure ionization sources, APCI and electrospray, were compared. The effect of several parameters on the extraction, separation and detection was studied. Dithiocarbamates and metabolites were dispersed with carbograph, eluted with a mixture of dichloromethane-methanol, and then, identified by monitoring the base peak of the spectra corresponding to [M + H]+. The method was validated for avocados, cherries, lemons, nuts, oat, oranges, peaches, rice and tomatoes. Average recoveries varied from 33 to 109%, and relative standard deviation were between 4 and 21% with limits of quantification ranged from 0.25 to 2.5 mg kg(-1), except for thiram and disulfiram, which were not recovered from fruits with high acid content. The procedure was applied to the determination of DTCs and their metabolites in fruits, vegetables and cereals taken from different markets of Valencia, Spain.

Determination of carbamate residues in fruits and vegetables by matrix solid-phase dispersion and liquid chromatography-mass spectrometry.

Thirteen carbamates were analysed in orange, grape, onion and tomatoes by matrix solid-phase dispersion (MSPD) followed by liquid chromatography-mass spectrometry (LC-MS). Electrospray (ES) and atmospheric pressure chemical ionisation (APCI) were compared and both gave similar results in terms of sensitivity and structural information because at 20 V fragmentor voltages the fragmentation is minimal. The efficiency of different solid-phases (C18, C8, cyano, amine and phenyl) for the MSPD was compared. Mean recoveries using C8 varied from 64 to 106% with relative standard deviations of 5-15% in the concentration range of 0.01-10 mg kg(-1). Matrix constituents did not interfere significantly with the ionisation process of carbamates. The limits of detection were typically in the 0.001-0.01 mg kg(-1) range, which were between 10 and 100 times lower than the maximum residue levels (MRLs) established by the European Union (EU). The method was applied to residue detection in fruit and vegetable samples taken from Valencian markets, in which carbamates were detected at low concentrations.

Comparison of microextraction procedures to determine pesticides in oranges by liquid chromatography-mass spectrometry.

A liquid chromatographic-mass spectrometric method has been developed for the determination of bitertanol, carbendazim, fenthion, flusilazole, hexythiazox, imidacloprid, methidathion, methiocarb, pyriproxyfen and trichlorfon. Two procedures, based on stir bar sorptive extraction (SBSE) and matrix solid-phase dispersion (MSPD), have been evaluated for the extraction of these compounds in oranges. Their respective advantages and disadvantages are also discussed. The recoveries obtained by MSPD ranged from 47 to 96% and the relative standard deviations (RSDs) ranged from 1 to 15%, whereas with the SBSE method the recoveries were between 8 and 84% and the RSDs between 4 and 16%. Although, the limits of quantitation of most compounds are much better (0.001-0.05 mg kg(-1)) by SBSE, it is not suitable to determine some polar pesticides as carbendazim, imidacloprid and trichlorfon. Results obtained by both methods were compared, in terms of sensitivity and selectivity, with a classical ethyl acetate extraction method, and the three methods were applied to analyze real samples. As MSPD is easier to perform, faster than the organic solvent extraction, and shows equal accuracy and resolution, its application for analyzing pesticides in oranges is recommended.

Improving the solid-phase extraction of "quat" pesticides from water samples. Removal of interferences.

A novel strategy, based on the addition of a cationic surfactant, for preventing the interferences associated with a diminution in the efficacy of solid-phase extraction (SPE) with silica cartridges of diquat, paraquat and difenzoquat in water is developed. Conditions for extraction are optimised with respect to pH, cationic surfactant and its concentration. Humic acids, anionic surfactants, inorganic salts and other organic contaminants like pesticides, phenols, polycyclic aromatic hydrocarbons and polychlorinated biphenyls produce the studied interferences. The best performance is shown in the improvement of the "quats" recovery from waters with high levels of humic acids and anionic surfactants (recovery is increased from ca. 30% to more than 80%). Unfortunately, the strong interference from inorganic salts remains. The presence in the water sample of other organic contaminants only affected the extraction efficiency of difenzoquat at high concentrations (more than 1 mg/l). Analytic utility is illustrated by selective measurements of the three herbicides, in real water samples. Overall, the results show that in spite of its drawbacks, SPE is a useful technique that allows the detection and quantification of the "quats" at limits below 100 ng/l as established by the European Union.

Comparison of solid-phase microextraction and stir bar sorptive extraction for determining six organophosphorus insecticides in honey by liquid chromatography-mass spectrometry.

Two approaches based on sorptive extraction, solid-phase microextraction (SPME) and stir bar sorptive extraction (SBSE), in combination with liquid chromatography (LC)-atmospheric pressure chemical ionization mass spectrometry (MS) have been assayed for analyzing chlorpyriphos methyl, diazinon, fonofos, phenthoate, phosalone, and pirimiphos ethyl in honey. In both, SPME and SBSE, enrichment was performed using a poly(dimethylsiloxane) coating. Significant parameters affecting sorption process such as sample volume, sorption and desorption times, ionic strength, elution solvent, and dilution (water/honey) proportion were optimized and discussed. Performance of both methods has been compared through the determination of linearity, extraction efficiencies, and limits of quantification. Relative standard deviations for the studied compounds were from 3 to 10% by SPME and from 5 to 9% by SBSE. Both methods were linear in a range of at least two orders of magnitude, and the limits of quantification reached ranging from 0.04 to 0.4 mg kg(-1) by SBSE, and from 0.8 to 2 mg kg(-1) by SPME. The two procedures were applied for analyzing 15 commercial honeys of different botanical origin. SPME and SBSE in combination with LC-MS enabled a rapid and simple determination of organophosphorus pesticides in honey. SBSE showed higher concentration capability (large quantities of sample can be handled) and greater accuracy (between 5 and 20 times) and sensitivity (between 10 and 50 times) than SPME: thus, under equal conditions, SBSE is the recommended technique for pesticide analysis in honey.

Analysis of post-harvest fungicides by micellar electrokinetic chromatography.

A method based on solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC) was developed for the simultaneous determination of carbendazim, imazalil, methylthiophanate, O-phenylphenol, prochloraz, procimidone, thiabendazole and triadimefon residues in grape, lettuce, orange and tomato. Selectivity and resolution were studied changing the pH and the concentration of the buffer, the type and concentration of surfactant and the methanol content in the mobile phase. A buffer consisting of 4 mM borate with 75 mM sodium cholate (pH 9.2) gave the best results. The recoveries of the fungicides in spiked fruit and vegetable samples ranged from 30 to 105%, and the limits of detection were between 0.1 and 1 mg kg(-1). The reproducibility and repeatability of the combination of SPE pretreatment and MEKC were good for all the compounds, except for imazalil and O-phenylphenol in oranges, due to some matrix compounds interfering with the separation. The method was applied to post harvest treated samples, and the fungicides were sometimes detected at concentration levels lower than maximum residue limits (MRLs).

Analysis of thiabendazole and procymidone in fruits and vegetables by capillary electrophoresis-electrospray mass spectrometry.

A capillary electrophoresis-mass spectrometry method for determining procymidone and thiabendazole in apples, grapes, oranges, pears, strawberries and tomatoes is described. Separation is achieved using a buffer of formic acid-ammonium formate at pH 3.5 with 2% of methanol. Fungicide residues present in the sample are preconcentrated by both solid-phase extraction and injection of large sample volumes into the capillary by a stacking technique, to obtain lower detection limits. Ionization is performed at atmospheric pressure in an electrospray type source and detection is carried out using positive ionization and selected ion monitoring modes. The quantitation limits are 0.005 and 0.05 mg kg(-1), and the mean recoveries are 64 and 75% for thiabendazole and procymidone, respectively, with relative standard deviations below 12% (n=5). Real fruit and vegetable samples are analyzed by the proposed method showing that residues of both fungicides are frequently present.

Liquid chromatographic-mass spectrometric determination of post-harvest fungicides in citrus fruits.

Liquid chromatography (LC)-atmospheric pressure ionisation (API)-mass spectrometry (MS) has been used to determine residues of five fungicides in oranges with a minimum sample cleanup. Atmospheric pressure chemical ionisation (APCI) and electrospray (ES) were compared and both gave similar results in terms of sensitivity and structural information. The main ions were [M+H]+ for carbendazim, imazalil, thiophanate methyl and thiabendazole, and [M+H-C4H9NHCO]+ for benomyl. Samples were extracted with sodium sulphate and ethyl acetate. Although benomyl and thiophanate methyl were transformed through the extraction procedure to carbendazim, the method showed good precision (<13%) and recovery (>70%), except for thiophanate methyl (50%), whilst also yielding limits of detection (<0.03 mg kg(-1)) that are adequate for the determination of the studied fungicides in oranges.

Comparison of octadecylsilica and graphitized carbon black as materials for solid-phase extraction of fungicide and insecticide residues from fruit and vegetables.

Methods for the determination of thirteen fungicide and insecticide residues by solid-phase extraction with C18 and graphitized carbon black (GCB) were evaluated. The extraction of the residues was achieved by using matrix solid-phase dispersion (MSPD) and more conventional polar solvent extraction followed by liquid-solid phase partitioning. Determination was carried out by capillary gas chromatography with electron-capture and mass spectrometry detectors. The recoveries were determined by fortifying six different crops (apples, oranges, pears, tomatoes, lettuces and paprikas) with the pesticides studied (bromopropylate, chlorpyrifos methyl, cypermethryn, deltamethryn, fenarimol, fenvalerate, imazalil, lindane, permethryn, phentoate, procymidone, propiconazole and vinclozoline). Although, the data showed that the two extraction methods and both sorbents were able to isolate the pesticide residues from fruit and vegetables, the best results were obtained using MSPD with C18 which gave recoveries ranging from 70 to 105% and practical detection limits between 5.0 and 50.0 micrograms/kg for all the compounds. Ten of these pesticides have been detected in samples taken from Valencia markets, at levels of 0.02-20.50 mg/kg using the described methodology.

Determination of pesticide residues in fruit and vegetables.

A review concerning the determination of pesticide residues in fruit and vegetables is presented. The basic principles and recent developments in the extraction and quantitation of pesticides are discussed. Consideration is given to solid phase and supercritical extraction techniques, automation and robotic systems, and immunoassay procedures.

Determination of fungicide residues in fruits and vegetables by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A liquid chromatography (LC) method for the quantitative determination of five fungicide residues (dichloran, flutriafol, o-phenylphenol, prochloraz and tolclofos methyl) in oranges, lemons, bananas, peppers, chards and onions is described. The residues were extracted by matrix solid-phase dispersion (MSPD) using C8. Quantitative analysis was performed by isocratic LC coupled to quadrupole mass spectrometer using atmospheric pressure chemical ionization in the negative ionization mode. The limit of quantification was 0.01 mg kgmicro for flutriafol, o-phenylphenol and dichloran, and 0.1 mg kg(-1) for prochloraz and tolclofos methyl. The MSPD method is also suitable for LC-UV analysis but higher limits of quantification (between 1 and 5 mg kg(-1)) were obtained. Validation of the method was performed between 0.01 and 25 mg kg(-1). Recoveries for fungicides ranged from 52.5 to 91.1% with relative standard deviations between 6.1 and 11.9%. The method was applied to the determination of residues in samples taken from agricultural cooperatives. The fungicides most often detected were o-phenylphenol and prochloraz.

Application of matrix solid-phase dispersion to the determination of a new generation of fungicides in fruits and vegetables.

A method based on matrix solid-phase dispersion (MSPD) and gas chromatography to determine eight fungicides in fruits and vegetables is described. Fungicide residues were identified and quantified using nitrogen-phosphorus detection and electron-capture detection connected in parallel and confirmed by mass spectrometric detection. The method required 0.5 g of sample, C18 bonded silica as dispersant sorbent, silica as clean-up sorbent and ethyl acetate as eluting solvent. Recoveries from spiked orange, apple, tomato, artichoke, carrot and courgette samples ranged from 62 to 102% and relative standard deviations were less than 15% in the concentration range 0.05-10 mg kg(-1). Detection and quantitation limits ranged 3-30 microg kg(-1) and 10-100 microg kg(-1), respectively, with linear calibration curves up to 10 mg kg(-1). The analytical characteristics of MSPD compared very favourably with the results of a classical multiresidue method, which uses ethyl acetate and anhydrous sodium sulphate for the extraction.