Validation of an Analytical Method for the Determination of Pesticide Residues in Vine Leaves by GC-MS/MS.

A modified and miniaturized SweEt/QuEChERS method for pesticide residue analysis applied to vine leaves is presented. The deep-frozen plant material was cryogenically processed. A 2 g analytical portion was hydrated for 30 min and extracted with acidified ethyl acetate after buffering with NaHCO3 and adding Na2SO4. A dispersive solid-phase (d-SPE) cleanup step with primary-secondary amine (PSA) was performed. The pesticide residues were determined using GC-MS/MS. The whole procedure was validated for 54-59 pesticides at 0.01, 0.02, 0.1, and 0.2 mg/kg in fresh vine leaves ( Vitis vinifera). The key method performance parameters investigated were specificity, linearity, trueness, within laboratory repeatability and reproducibility, limit of detection, limit of quantitation, and matrix effects. Recoveries for the 59 pesticides tested ranged from 60 to 110%, and the RSDs were lower than 20% for the majority of the pesticides studied.

Occurrence of pesticide residues in fish from south American rainfed agroecosystems.

Environmental sustainability of South American rainfed agroecosystems is of current concern. In this work, we evaluate the occurrence of multiple pesticide residues in muscle tissue of wild fish species from two large rivers in South America (Uruguay and Negro Rivers). Two sampling campaigns (representing summer and winter crops) were performed during 2015 targeting a wide biodiversity of fish species used for human consumption (ranging from migratory to non-migratory and from detritivorous to top-predators). Three different localities associated to rainfed agriculture were assessed, two of them enclosed to a RAMSAR site (National Park "Esteros de Farrapos e Islas del Rio Uruguay"). Pesticide residues occurred in muscle tissue of 143 from 149 sampled fishes (96%). Thirty different pesticides were detected at concentrations from <1 to 194µgkg-1. Incidence of pesticides in fish were tightly related to: i) features of the contaminant: (Kow, environmental persistence and mobility) and ii) intensity of use of particular pesticides and land dedicated to rainfed agriculture. Trifloxystrobin, metolachlor and pyraclostrobin showed the highest rates of occurrence. Of great concern is that strobirulins have highest toxicity to fish from those detected compounds. From the pattern of pesticides occurring for non-migratory fish species it was possible to trend important spatial differences related to the intensity of rainfed agriculture. Results suggest a regular exposition of aquatic wild biota to sublethal concentrations of multiple semi-polar pesticides.

Matrix Effects and Interferences of Different Citrus Fruit Coextractives in Pesticide Residue Analysis Using Ultrahigh-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

The matrix effects of ethyl acetate extracts from seven different citrus fruits on the determination of 80 pesticide residues using liquid chromatography coupled to high-resolution time-of-flight mass spectrometry (UHPLC-(ESI)-HR-TOF) at 4 GHz resolution mode were studied. Only 20% of the evaluated pesticides showed noticeable matrix effects (ME) due to coelution with natural products between tR = 3 and 11 min. Principal component analysis (PCA) of the detected coextractives grouped the mandarins and the orange varieties, but separated lemon, oranges, and mandarins from each other. Matrix effects were different among species but similar between varieties, forcing the determination of pesticide residues through matrix-matched calibration curves with the same fruit. Twenty-three natural products (synephrine, naringin, poncirin, glycosides of hesperitin, limonin, nomilin, and a few fatty acids, among others) were identified in the analyzed extracts. Twelve of the identified compounds coeluted with 28 of the pesticides under study, causing different matrix effects.

Neonicotinoids transference from the field to the hive by honey bees: Towards a pesticide residues biomonitor.

The beehive as a quantitative monitor of pesticide residues applied over a soybean crop was studied through a semi field experiment of controlled exposure of honey bees to pesticides in macro tunnels. The distribution within exposed beehives of pesticides commonly used in soybean plantation, was assessed. Residue levels of insecticides in soybean leaves, honey bees, wax, honey and pollen were analyzed. The transference from pesticides present in the environment into the beehive was evidenced. The obtained results allow relating pesticide concentrations present in the environment with traces found in foraging bees. Therefore, pesticide transference ratios could be calculated for each detected compound (acetamiprid, imidacloprid and thiamethoxam) which showed a linear inverse trend with their 1-octanol/water partition coefficient (Kow). The least transferred pesticide to the hive (acetamiprid) has the highest vapor pressure (Vp). This study gives new insights on the usefulness of monitoring the environment through beehives aiming to evaluate if agroecosystems remain sustainable. It also contributes to generate valuable information for model building aiming to predict environmental quality through beehive's analysis.

Degradation of imazalil, orthophenylphenol and pyrimethanil in Clementine mandarins under conventional postharvest industrial conditions at 4°C.

The degradation of the postharvest fungicides imazalil, orthophenylphenol, and pyrimethanil was studied on Clementine mandarins during packinghouse storage for a 28day period at 4°C. Fruits to which orthophenylphenol was applied, were treated with imazalil and pyrimethanil at doses of 1000 and 2000mgL(-1), using cascade application for the later and cascade and wax for the former. The decay of the three fungicides was evaluated using an in-house validated analytical procedure that includes the extraction and dispersive clean up of the samples followed by the GC-MS determination of the pesticide residues. The impact of fruit storage time on pesticide residues concentration was assessed. The residues found for the different application technologies were always below the established Maximum Residue Limits by the Codex Alimentarius and the European Union (5mgkg(-1) for imazalil, 7 and 8mgkg(-1) for pyrimethanil, and 10mgkg(-1) and 5mgkg(-1) for orthophenylphenol). The fungicides dissipated differentially. Pyrimethanil showed little degradation, if any, at both tested concentrations, but the half-life of imazalil on the fruit was 15-18days, independent of the application technology. Orthophenylphenol dissipated with a half-life of 15days. The initial imazalil residue found after cascade treatment was not significantly different between the doses studied (p<0.5), whereas when the fungicide was included in wax as an emulsifiable concentrate the initial and final imazalil residues were significantly different. Final residue levels after 28days of storage were 0.12-0.24mgkg(-1) for imazalil, 0.68mgkg(-1) for 2-phenylphenol and 0.56mgkg(-1) for pyrimethanil for all the evaluated treatments.

QuEChERS Adaptability for the Analysis of Pesticide Residues in Beehive Products Seeking the Development of an Agroecosystem Sustainability Monitor.

Beehive products could be powerful monitors of pesticide residues originating in agroecosystems during production cycles. Their ready availability provides enough samples to perform analytical determinations, but their chemical complexity makes residue analysis a real challenge. Taking advantage of the plasticity of QuEChERS coupled to LC-MS/MS, validated methodologies were developed for bees, honey, beeswax, and pollen and applied to real samples for the simultaneous determination of 19 of the most employed pesticides in intensive cropping fields. Beehives placed in Uruguayan agroecosystems accumulated the pesticides thiacloprid, imidacloprid, methomyl, carbaryl, hexythiazox, azoxystrobin, pyraclostrobin, tebuconazole, and haloxyfop-methyl at 0.0001-0.01 mg/kg levels. The oscillations on the amount and occurrence of residue findings for specific apiaries were correlated statistically with the sampling season and the agroecosystem where the beehives were located, showing the potential of bees and bee products to record relevant information to survey the chemicals applied in their surroundings.

Viability of Cabralea canjerana extracts to control the South American fruit fly, Anastrepha fraterculus.

Several representatives of Meliaceae contain biologically active compounds that are toxic to insects with few negative effects on the environment and humans. Our study evaluated the activity of ethyl acetate and ethanol extracts from the fruit and seeds of Cabralea canjerana (Vellozo) Mart (Sapindales: Meliaceae) on Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae). Limonoids and triterpenes were detected in fruit and seed extracts. Each extract was added to an artificial diet at three concentrations and tested after 24, 48, and 72 hr of extract application. Ethyl acetate extracts were the most active ones and showed the effect of both dose and time elapses after application on the insects. The highest toxic effect on A. fraterculus adults was from ethyl acetate extracts from fruit, followed by extracts from seeds. These extracts showed antifeedant activities. Extract solutions sprinkled on fruits of Carica papaya (L.) (Brassicales: Caricaceae) caused oviposition repellency and negatively affected the biological development of A. fraterculus. Ethyl acetate extracts highly hampered oviposition, but seed extracts showed lesser oviposition deterrence. The fruit and seed extracts diminished pupal viability. Particularly, the ethyl acetate fruit extract caused malformed adults. The sex ratio was also affected, resulting in female predominance for the fruit extract, while the seed extract showed a dose-dependent effect. Low doses caused male abundance, but at higher concentrations the effect was reversed. These encouraging results showed that the C. canjerana extracts have great potential as new tools to be used in integrated pest management programs to protect fruits against A. fraterculus.

QuEChERS-based method for the multiresidue analysis of pesticides in beeswax by LC-MS/MS and GC×GC-TOF.

The validation of an analytical procedure for the determination of pesticide residues in beeswax, an interesting matrix for environmental pollution monitoring, is presented. Using the QuEChERS template, the impacts of wax particle size, sample amount, and cleanup procedure (water addition, dispersive solid phase extraction, freeze-out, and combinations thereof) on extraction yield and coextractive load were studied. Sample preparation through liquid-liquid partitioning between acetonitrile and melted wax (∼80 °C), followed by freeze-out and primary-secondary amine dispersive cleanup, was performed on incurred and pesticide-free samples for 51 residues. Determinations were made through LC-MS/MS and GC×GC-TOF, and the whole procedure was validated. Matrix effects were evaluated, with recoveries between 70 and 120% and RSDs below 20% in almost all cases. LC-MS/MS LOQs ranged from 0.01 to 0.1 mg/kg for most pesticides, but for GC-amenable pesticides, GC×GC-TOF sensitivity was lower (0.1-0.2 mg/kg). This methodology can be applied for routine analysis of pesticide residues in beeswax.

Infusion, decoction and hydroalcoholic extracts of leaves from artichoke (Cynara cardunculus L. subsp. cardunculus) are effective scavengers of physiologically relevant ROS and RNS.

The globe artichoke (Cynara cardunculus L. subsp. cardunculus) is a perennial plant cultivated in the Mediterranean region and Americas for its edible young flower heads and as an interesting source of bioactive compounds. The present study was undertaken to evaluate scavenging capacity against the most physiologically relevant reactive oxygen species (ROS) and reactive nitrogen species (RNS) of three different extracts from artichoke leaves (infusion, decoction and hydroalcoholic) using different solvents, commonly accepted for human consumption (water and a mixture of ethanol/water). Additionally, the phenolic compounds in each extract were identified and quantified by high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). Chlorogenic acid was the major phenolic compound identified in all extracts, followed by 1,3-dicaffeoylquinic acid (cynarin), luteolin-7-rutinoside and the infusion extract presented the highest phenolic content (108mg/g extract, dry basis). In general, the extracts of artichoke leaves presented a remarkable capacity to scavenge ROS and RNS with IC50 values in a low µg/mL range (3.4-43µg/mL). These findings suggest that artichoke could be a potential source of natural antioxidants and has an undeniable nutraceutical value.

Anticonvulsant, neuroprotective and behavioral effects of organic and conventional yerba mate (Ilex paraguariensis St. Hil.) on pentylenetetrazol-induced seizures in Wistar rats.

Epilepsy, which is one of the most common neurological disorders, involves the occurrence of spontaneous and recurrent seizures that alter the performance of the brain and affect several sensory and behavioral functions. Oxidative damage has been associated with post-seizure neuronal injury, thereby increasing an individual's susceptibility to the occurrence of neurodegenerative disorders. The present study investigated the possible anticonvulsive and neuroprotective effects of organic and conventional yerba mate (Ilex paraguariensis), a plant rich in polyphenols, on pentylenetetrazol (PTZ)-induced seizures in Wistar rats. The behavioral and polyphenolic profiles of the yerba mate samples were also evaluated. Infusions of yerba mate (50mg/kg) or distilled water were given to rats for fifteen days by oral gavage. On the 15th day the animals were subjected to open field test, and exploratory behavior was assessed. Subsequently, 60mg/kg PTZ (i.p.) was administered, and animals were observed for the appearance of convulsions for 30min. Latency for the first seizure, tonic-clonic and generalized seizures time, frequency of seizures and mortality induced by PTZ were recorded. The animals were then sacrificed, and the cerebellum, cerebral cortex and hippocampus were quickly removed and frozen to study the neuroprotective effects of yerba mate. The oxidative damage in lipids and proteins, nitric oxide levels, the activities of the antioxidant enzymes superoxide dismutase (Sod) and catalase (Cat) and non-enzymatic cellular defense (sulfhydryl protein) were quantified in all the tissues. The results showed that organic and conventional yerba mate infusions were able to reduce the frequency of seizures when compared to the PTZ group. Besides, organic yerba mate infusion decreases the tonic-clonic seizures time in relation to the PTZ group. It was also shown that organic and conventional yerba mate infusions reduced the oxidative damage in lipids and proteins and nitric oxide levels and prevented the decrease in Sod and Cat activities and sulfhydryl protein content when compared to the PTZ group in all the CNS tissues assayed. Organic and conventional yerba mate commercial samples did not change the behavior (locomotion, exploration or anxiety) of the treated animals. In both organic and conventional infusions, the presence of the polyphenols rutin, chlorogenic acid and their acyl derivatives were detected, which could be associated with the biological effects observed. These data indicate that yerba mate may provide new perspectives for the development of therapeutic approaches with natural compounds in the pharmaceutical area, both to reduce the convulsions' frequency and to minimize the neuronal damage associated with recurrent seizures.

Organic and Conventional Yerba Mate (Ilex paraguariensis A. St. Hil) Improves Metabolic Redox Status of Liver and Serum in Wistar Rats.

Organic and conventional yerba mate (Ilex paraguariensis) is widely used in South America to prepare nonalcoholic drinks rich in polyphenols. These compounds are able to prevent the generation of reactive species, thus minimizing the incidence of several diseases. In this perspective, we hypothesized that yerba mate may have protective effects against pentylenetetrazol (PTZ)-induced oxidative damage in liver and serum of rats. Animals (n = 42) received distilled water (control) or yerba mate (organic or conventional) for fifteen days. Then, half of the rats of each group received 60 mg/kg PTZ intraperitoneally or saline solution. After 30 min the animals were euthanized and the liver and blood were collected. The results showed that organic and conventional yerba mate avoided PTZ-induced oxidative damage and nitric oxide production in the liver and serum of the rats. Moreover, both kinds of yerba mate prevented the decrease in enzymatic (superoxide dismutase and catalase) and non-enzymatic (sulfhydryl protein content) defenses in the liver and serum. In addition, histopathologic analysis of the liver showed that yerba mate reduced PTZ-induced cell damage. These findings indicate that yerba mate provides hepatoprotection and improves antioxidant status in the serum, which may contribute to the development of new therapeutic strategies using nutraceuticals drinks.

Analytical methodology for the study of endosulfan bioremediation under controlled conditions with white rot fungi.

A general procedure to study the biodegradation of endosulfan under laboratory conditions by white rot fungi isolated from native sources growing in YNB (yeast nitrogen base) media with 1% of glucose is presented. The evaluation of endosulfan biodegradation as well as endosulfan sulfate, endosulfan ether and endosulfan alcohol production throughout the whole bioremedation process was performed using an original and straightforward validated analytical procedure with recoveries between 78 and 112% at all concentration levels studied except for endosulfan sulfate at 50 mg L(-1) that yielded 128% and RSDs<20%. Under the developed conditions, the basidiomycete Bjerkandera adusta was able to degrade 83% of (alpha+beta) endosulfan after 27 days, 6 mg kg(-1) of endosulfan diol were determined; endosulfan ether and endosulfan sulfate were produced below 1 mg kg(-1) (LOQ, limit of quantitation).

Occurrence and distribution study of residues from pesticides applied under controlled conditions in the field during rice processing.

The results of an experiment to study the occurrence and distribution of pesticide residues during rice cropping and processing are reported. Four herbicides, nine fungicides, and two insecticides (azoxystrobin, byspiribac-sodium, carbendazim, clomazone, difenoconazole, epoxiconazole, isoprothiolane, kresoxim-methyl, propanil, quinclorac, tebuconazole, thiamethoxam, tricyclazole, trifloxystrobin, λ-cyhalotrin) were applied to an isolated rice-crop plot under controlled conditions, during the 2009-2010 cropping season in Uruguay. Paddy rice was harvested and industrially processed to brown rice, white rice, and rice bran, which were analyzed for pesticide residues using the original QuEChERS methodology and its citrate variation by LC-MS/MS and GC-MS. The distribution of pesticide residues was uneven among the different matrices. Ten different pesticide residues were found in paddy rice, seven in brown rice, and eight in rice bran. The highest concentrations were detected in paddy rice. These results provide information regarding the fate of pesticides in the rice food chain and its safety for consumers.

Photolytic and photocatalytic degradation of quinclorac in ultrapure and paddy field water: identification of transformation products and pathways.

Quinclorac (QNC) is an effective but rather persistent herbicide commonly used in rice production. This herbicide presents a mean persistence in the environment so its residues are considered of environmental relevance. However, few studies have been conducted to investigate its environmental behavior and degradation. In the present work, direct photolysis and TiO(2) photocatalysis of the target compound in ultrapure and paddy field water were investigated. After 10h photolysis in ultrapure water, the concentration of QNC declined 26% and 54% at 250 and 700 W m(-2), respectively. However, the amount of quinclorac in paddy field water remained almost constant under the same irradiation conditions. QNC dissipated completely after 40 min of TiO(2) photocatalysis in ultrapure water, whereas 130 min were necessary to degrade 98% of the initial concentration in paddy field water. Possible QNC photolytic and photocatalytic degradation pathways are proposed after structure elucidation of the main transformation products, through liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and exact mass measurements. Pyridine ring hydroxylation at C-9 followed by ring opening and/or oxidative dechlorination were the key steps of QNC degradation.

Trace analysis of pesticides in paddy field water by direct injection using liquid chromatography-quadrupole-linear ion trap-mass spectrometry.

A multiresidue method was developed for the quantification and confirmation of 70 pesticides in paddy field water. After its filtration, water was injected directly in a liquid chromatograph coupled to a hybrid triple quadrupole-linear ion trap-mass spectrometer (QqLIT). The list of target analytes included organophosphates, phenylureas, sulfonylureas, carbamates, conazoles, imidazolinones and others compounds widely used in different countries where rice is cropped. Detection and quantification limits achieved were in the range from 0.4 to 80 ng L(-1) and from 2 to 150 ng L(-1), respectively. Correlation coefficients for the calibration curves in the range 0.1-50 µg L(-1) were higher than 0.99 except for diazinon (0.1-25 µg L(-1)). Only 9 pesticides presented more than 20% of signal suppression/enhancement, no matrix effect was observed in the studied conditions for the rest of the target pesticides. The method developed was used to investigate the occurrence of pesticides in 59 water samples collected in paddy fields located in Spain and Uruguay. The study shows the presence of bensulfuron methyl, tricyclazole, carbendazim, imidacloprid, tebuconazole and quinclorac in a concentration range from 0.08 to 7.20 µg L(-1).

Evaluation of various QuEChERS based methods for the analysis of herbicides and other commonly used pesticides in polished rice by LC-MS/MS.

Four different extraction and clean-up protocols based on the QuEChERS method were compared for the development of an optimized sample preparation procedure for the multiresidue analysis of 16 commonly applied herbicides in rice crops using LC-QqQ/MS. Additionally the methods were evaluated for the analysis of 26 insecticides and fungicides currently used in rice crops. The methods comprise, in general, the hydratation of the sample with water followed by the extraction with acetonitrile, phase separation with the addition of different salts and finally a clean-up step with various sorbents. Matrix effects were evaluated for the 4 studied methods using LC-QqQ/MS. Additionally LC-TOF/MS was used to compare the co-extractants obtained with the four assayed methodologies. Thirty-six pesticides presented good performance with recoveries in the range 70-120% and relative standard deviations below 20% using 7.5 g of milled polished rice and the buffered acetate QuEChERS method without clean-up at both fortification levels: 10 and 300 µg kg(-1). The other six pesticides presented low recovery rates, nevertheless all these analytes could be analyzed with at least one of the other three studied procedures.

Transfer of pesticides to the brew during mate drinking process and their relationship with physicochemical properties.

In order to evaluate the extraction of pesticide residues that are transferred to the brew during mate drinking process of P.U.1 yerba mate leaves (Ilex paraguariensis), a special device to simulate the way in which mate is drunk in Uruguay was developed. The transfer to the brew of 12 organophosphates, 5 synthethic pyrethroids and one organochlorine pesticide from spiked samples was studied. The relationship between the transfer data thus obtained and physicochemical properties like water solubility (Ws), octanol-water coefficient (Kow) and Henry's constant (H) was evaluated. The extractability of the pesticide residues from yerba mate can be correlated with log Ws and log Kow. These transfer values allowed the calculation of ARLs (acceptable residue level) for the pesticides following Food and Agriculture Organization (FAO), World Health Organizaion (WHO) guidelines. These results can help the future establishment of maximum residue levels (MRLs).

Morphological and chemical diversity in the Type IV glandular trichomes of Solananeae (S. sisymbrifolium and N. glauca) as germplasm resources for agricultural and food uses

Morphological variation in type IV trichomes in Ss and Ng was studied through SEM. The differences can be related to chemical differences in the excreted sugar esters. Ng trichomes exude two fractions, one of glucose tri-esters and the other one of sucrose tetra-esters, in a 3:7 ratio. The main acid found forming these esthers, is 3-methylvalerianic acid, in consonance to those secreted by other Solanaceae. Esters from Ss are novel structures, which can also be separated into three fractions, two of arabinoxylans, and the other one of arabinose, all glycosilated with â-hydroxipalmitic acid and sterified with the C12-C16 acids. All five fractions have antifungic activity at ìg/cm² concentrations, both against common and mycotoxigenic fungi, such as A. niger, A. flavus, P. chrysogenum y P. expansum. A. flavus does not grow in the presence of the SE of Ss and is not insensible to those from Ng, but these last are more effective in the inhibition of P. expansum, the other mycotoxigenic fungi studied. The differential antifungal activity observed gives the plant protection against a wide spectrum of fungi, resulting in a better adaptation to the environment. Both plants are common weeds, with the potential of contributing to germplams lines in the improvement programmes of crops such as L. esculentum, and their extracts can be used as natural fungicides to protect crops and plantations.