Identification of Green-Leaf Volatiles Released from Cabbage Palms (Sabal palmetto) Infected with the Lethal Bronzing Phytoplasma.

Lethal bronzing (LB) is a fatal infection that affects over 20 species of palms (Arecaceae) and is caused by the phytoplasma 'Candidatus Phytoplasma aculeata'. This pathogen causes significant economic losses to landscape and nursery companies in Florida, USA. Recently, the vector was determined to be the planthopper Haplaxius crudus, which was more abundant on LB-infected palms. Herein, the volatile chemicals emitted from LB-infected palms were characterized using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS). Infected Sabal palmetto were identified and confirmed as positive for LB via quantitative PCR. Healthy controls of each species were selected for comparison. All infected palms exhibited elevated levels of hexanal and E-2-hexenal. Threatened palms showed high releasing concentrations of 3-hexenal and Z-3-hexen-1-ol. The volatiles characterized herein are common green-leaf volatiles (GLVs) emitted by plants under stress. This study considers the first documented case of GLVs in palms attributed to phytoplasma infection. Due to the apparent attraction of LB-infected palms to the vector, one or several of the GLVs identified in this study could serve as a lure for the vector and supplement management programs.

Study of insecticide translocation in coconut palm trees after using pressurized endotherapy.

Alternative techniques for applying agricultural products, such as pressurized endotherapy, have shown promise in pest and disease control in coconut palms (Cocos nucifera Linn.). In this work, azadirachtin and abamectin were applied by pressurized endotherapy to control Brassolis sophorae. Twelve different treatments were carried out, and in all of them, at least one plant had the larvae completely dead four and five days after endotherapeutic applications, and all the others died within the next few hours. Leaf and fruit analyses were performed to determine the concentration over time. High concentrations of abamectin were observed after 15 and 30 days on leaves when applied in larger volumes. In fruits, no residue was found regardless of the applied concentration. Analytical methods were developed and validated for leaves and fruits to analyze insecticide residues using LC-MS/MS and modified QuEChERS acetate according to SANTE/11813/2017 guidelines. The insecticide translocation tests in the leaves and the high mortality of insects showed that pressurized endotherapy is a technique to be considered for future studies in controlling B. sophorae in coconut palm trees.

Exposure of tilapia (Oreochromis niloticus) to the antibiotic florfenicol in water: determination of the bioconcentration factor and the withdrawal period.

The demand for healthier foods with high nutritional value has resulted in intensive fish farming. In this production system, high-frequency infections occur, and antibiotics are administrated for control. Only two antibiotics are allowed for use in Brazilian aquaculture, one of which is florfenicol. In this work, a bioconcentration assay was performed to assess the accumulation of florfenicol in the muscle of Nile tilapia (Oreochromis niloticus). Tilapia was evaluated as it is the most produced fish species in Brazil. The fish were exposed to florfenicol at a nominal concentration of 10 mg/L, through the water. Muscle and water were collected at 0, 1.5, 3, 6, 24, and 48 h during the exposure phase and at 1.5, 3, 6, 24, 48, and 120 h during the depuration phase. Quantification was performed using an LC-MS/MS. The results showed rapid absorption and elimination of the antibiotic (half-life, t1/2 = 5 h), with low potential for accumulation of florfenicol in tilapia muscles. The study was performed to determine the bioconcentration factor (BCF) and withdrawal period of florfenicol, being 0.05 mL/µg and 1.8 h, respectively. The results contribute to set protocols for the safe use of florfenicol in tilapia transport, avoiding residues in fish that may pose risks to human health.

Role of bioactive metabolites from Acremonium camptosporum associated with the marine sponge Aplysina fulva.

Acremonium camptosporum, a fungus associated with the marine sponge Aplysina fulva, was collected from the isolated mid-Atlantic Saint Peter and Saint Paul Archipelago, Brazil, and was found to produce secondary metabolites that displayed antibacterial activities. Mass spectra data obtained by UPLC-ESI-MS/MS analyses of these extracts were compared to several databases and revealed the presence of several different cytotoxic acremonidins and acremoxanthones. The close association between the sponge and the fungi with its compounds could be of strategic importance in defending both from the high predation pressure and spatial competition in the warm-water scarps of the islands.

Effects of sublethal and realistic concentrations of the commercial herbicide atrazine in Pacu (Piaractus mesopotamicus): Long-term exposure and recovery assays.

BACKGROUND AND AIM: The commercial formulations of the herbicide atrazine (cATZ) are widely employed in Brazilian agriculture, and, as a consequence, ATZ has been found at levels above that established by law in the river basins in Brazil. Although the toxicity of ATZ in fish is well documented, there are few studies on the recovery capacity after cATZ exposure. This work aimed to evaluate, using several biomarkers, the toxic effects of long-term exposure to the sublethal (3.57 mg/L) and nonlethal realistic (3.00 µg/L) cATZ concentrations followed by a recovery assay, in fingerlings of a Brazilian teleost, the Piaractus mesopotamicus (pacu). MATERIALS AND METHODS: Pacu fingerlings were housed in glass tanks and divided into the following experimental groups (two tanks/group): Exposure control = EC, recovery control = RC, the sublethal groups exposed to 3.57 mg/L of cATZ, (sublethal exposure group = SLE and sublethal recovery group = SLR) and the nonlethal groups treated with 3.00 µg/L of cATZ (nonlethal exposure group = NLE and nonlethal recovery group = NLR). The exposure assay was semi-static with a duration of 30 days and the recovery assay (after cATZ withdrawal) lasted 14 days. Several biomarkers were evaluated in fingerlings from all groups: The swimming behavior, the body weight gain, the micronucleus formation and nuclear alterations in erythrocytes, and the hepatic and renal histopathology analyzed by qualitative and semi-quantitative morphological methods (using light and electron microscopy). RESULTS: No significant difference in weight gain was observed among the groups after the exposure and recovery assays. The sublethal exposure induced impaired swimming movements, significant histopathological alterations, including necrosis in the liver and kidney, and a significant increase in the frequency of micronuclei in erythrocytes. The nonlethal exposure induced only subtle histopathological changes in the liver and kidney. After recovery assay, no genotoxic alteration was noted in pacu exposed to sublethal concentration, while the cATZ-induced kidney damage was partially reversed but not the hepatic injury. CONCLUSION: cATZ exhibits long-term toxic effects on pacu, even at relatively low concentrations, affecting mainly the liver and the kidney, and the effects of sublethal concentration are only partially reversed after cATZ withdrawal.

Bioconcentrations of herbicides used in sugarcane crops in tilapia (Oreochromis niloticus) and the risk for human consumption.

The practice of intensive herbicide use in the sugarcane industry has a high risk of compromising the quality of the water and the organisms that live there due to losses through runoff, leaching and other processes. In this work, the dynamics of four herbicides present in three different mixtures were evaluated through their incorporation and elimination in the muscle tissue of tilapia (Oreochromis niloticus). The highest mean values of bioconcentration factors were 1.730 for ametryn, 0.891 for tebuthiuron, 0.322 for hexazinone and 4.783 for diuron. Diuron presented the highest risk regarding the consumption of tilapia fillets by the population. However, considering that the fish would reach maximum levels of diuron when exposed to extremely high concentrations, an individual weighing 70 kg would need to ingest approximately 1.5 kg of this food product to surpass the acceptable daily intake of 0.007 mg kg-1 body weight. It was concluded that the risk of injury to the population consuming tilapia fillets from fish exposed to herbicides in water arising from sugarcane activities is very low. According to the risk estimation performed in this work, which is substantiated by the assumptions of the World Health Organization and the International Life Sciences Institute, there is a low risk of injury to the population consuming tilapia fillets from fish exposed to water containing herbicides in concentrations arising from sugarcane activities. However, as the risk was estimated from laboratory conditions, caution should be taken where herbicide applications are carried out with high frequency near water bodies, as the consumption of fish from these areas is quite common.

Removal of the endocrine disruptors ethinyl estradiol, bisphenol A, and levonorgestrel by subsurface constructed wetlands.

The present work aimed to evaluate the removal efficiency of the endocrine disruptors ethinyl estradiol (EE2), the progestin levonorgestrel (LNG), and bisphenol A (BPA), considered to be contaminants of major concern, by using four laboratory scale constructed wetlands (CW) - three containing gravel as support media, one cultivated with Cyperus isocladus (WL1), other with Eichhornia crassipes (WL3), and one without macrophyte (WL2). The fourth unit contained gravel and bamboo charcoal as support medium, also cultivated with Cyperus isocladus (WLC). Two hydraulic retention times (HRT) were tested, 2 and 4 days. Average removals ranged from 9.0 to 95.6% for EE2, from 29.5 to 91.2% for BPA and from 39.1 to 100.0% for LNG. The results showed that the most efficient CW for removal of EE2 and BPA was WLC, and for LNG removal was WL3. The 2 days HRT was statistically more efficient in removing EE2, and the 4 days HRT was more efficient in the LNG removal. The other endocrine disruptors and concentration ranges were not influenced by HRT. It was concluded that WLC was the most suitable CW for removal of these three compounds, and it possibly is efficient also for the removal of other endocrine disruptors with similar physical-chemical characteristics.

Bioactivity and action mechanism of green propolis against Pythium aphanidermatum.

We have established how natural compounds from green propolis collected by the species Apis mellifera act against the growth of Pythium aphanidermatum. On the basis of mass spectrometry (Q-ToF MS), we determined that Artepillin C, the major constituent of green propolis, underlies the effect and displays activity against P. aphanidermatum at a minimal inhibitory concentration of 750 µg.mL-1. Biophysical studies based on model membranes showed that this inhibitory effect may be linked with a membrane-related phenomenon: Artepillin C increases the permeability of membranes with relatively high fluidity in their lateral structure, a feature that is in line with the lipid composition reported for the cytoplasmic membrane of P. aphanidermatum. Therefore, the present study supports the use of the effective and inexpensive green propolis to control the impact of the dangerous phytopathogen P. aphanidermatum on agriculture.

Multiresidue Method for Quantification of Sulfonamides and Trimethoprim in Tilapia Fillet by Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry Using QuEChERS for Sample Preparation.

A multiresidue method for detecting and quantifying sulfonamides (sulfapyridine, sulfamerazine, sulfathiazole, sulfamethazine, sulfadimethoxine, sulfamethoxazole, and sulfamethoxypyridazine) and trimethoprim in tilapia fillet (Oreochromis niloticus) using liquid chromatography coupled to mass spectrometry was developed and validated. The sample preparation was optimized using the QuEChERS approach. The chromatographic separation was performed using a C18 column and 0.1% formic acid in water and acetonitrile as the mobile phase in the isocratic elution mode. Method validation was performed based on the Commission Decision 2002/657/EC and Brazilian guideline. The validation parameters evaluated were linearity (r ≥ 0.99); limits of detection (LOD) and quantification (LOQ), 1 ng·g-1 and 5 ng·g-1, respectively; intraday and interdays precision (CV lower than 19.4%). The decision limit (CCα 102.6-120.0 ng·g-1 and 70 ng·g-1 for sulfonamides and trimethoprim, respectively) and detection capability (CCß 111.7-140.1 ng·g-1 and 89.9 ng·g-1 for sulfonamides and trimethoprim, respectively) were determined. Analyses of tilapia fillet samples from fish exposed to sulfamethazine through feed (incurred samples) were conducted in order to evaluate the method. This new method was demonstrated to be fast, sensitive, and suitable for monitoring sulfonamides and trimethoprim in tilapia fillet in health surveillance programs, as well as to be used in pharmacokinetics and residue depletion studies.

Depletion study, withdrawal period calculation and bioaccumulation of sulfamethazine in tilapia (Oreochromis niloticus) treated with medicated feed.

The residue depletion of sulfamethazine (SMZ) was evaluated in tilapia (Oreochromis niloticus) after 11 days of administration of medicated feed containing SMZ, at the dose of 422 mg/kg body weight (bw). The determination of SMZ in feed and tilapia fillet was carried out using the QuEChERS approach for sample preparation, and high performance liquid chromatography with diode array detector (HPLC-DAD) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) for quantitation, respectively. Both methods were validated based on international and Brazilian guidelines and shown to be suitable for the intended purposes. The withdrawal period to reach the maximum residue level (MRL) of 100 µg/kg, according to the European Union (EU) legislative framework to all substances belonging to the sulfonamide (SA) group (EU, 2010), was 10 days (260 °C-day). After treatment, the maximum level of SMZ accumulation in the tilapia muscle was 1.6 mg/kg. SMZ was shown to be quickly excreted by tilapia. Thus, considering the acceptable daily intake of SMZ established by the Codex Commission (0-0.05 mg/kg bw), and a factor of 5 times the upper amount of fish consumption in Brazil (38 kg/year), this study showed that there is a low risk of adverse effects to consumers. This study offers subsidies not only for the establishment of public policies with regard to the use of veterinary drugs currently not allowed in a country by their legal legislative framework for fish farming, but also to fish producers for the proper handling to ensure safe fish fillets.

Antibacterial Compounds from Marine Bacteria, 2010-2015.

This review summarizes the reports on antibacterial compounds that have been obtained from marine-derived bacteria during the period 2010-2015. Over 50 active compounds were isolated during this period, most of which (69%) were obtained from Actinobacteria. Several compounds were already known, such as etamycin A (11) and nosiheptide (65), and new experiments with them showed some previously undetected antibacterial activities, highlighting the fact that known natural products may be an important source of new antibacterial leads. New broad-spectrum antibacterial compounds were reported with activity against antibiotic resistant Gram-positive and Gram-negative bacteria. Anthracimycin (33), kocurin (66), gageotetrins A-C (72-74), and gageomacrolactins 1-3 (86-88) are examples of compounds that display promising properties and could be leads to new antibiotics. A number of microbes produced mixtures of metabolites sharing similar chemical scaffolds, and structure-activity relationships are discussed.

A method for determination of imazapic and imazethapyr residues in soil using an ultrasonic assisted extraction and LC-MS/MS.

At least 52 % of the planted rice area in Rio Grande do Sul, a major rice producing state in Brazil, employs Clearfield(®) production system, corresponding to 580,000 ha of cultivated area. To grow rice with Clearfield(®) technology, producers combine imazethapyr and imazapic herbicides. However, these herbicides leave residual activity in soil; consequently, the repeated application of imazethapyr and imazapic on Brazilian Clearfield(®) rice fields has increased these herbicides persistence in treated soils. In this study, a method has been developed for removal and quantification of imazethapyr and imazapic residues in soil through ultrasonic assisted extraction using methanol-phosphoric acid aqueous solution (pH 2.0). The detected response was linear for both herbicides within the range of 0.25-5 ng mL(-1) with correlations coefficients >0.99. The quantification limit was limit of quantification 0.2 µg Kg(-1) for both pesticides. The good recovery rate from all pesticides, which ranges between 70 % and 120 %, demonstrates the method's validity.

Isolation and biological activities of an endophytic Mortierella alpina strain from the Antarctic moss Schistidium antarctici.

The Antarctic endophytic fungus (strain ITA1-CCMA 952) was isolated from the moss Schistidium antarctici found in Admiralty Bay, King George Island, Antarctica. Strain ITA1-CCMA 952 was assigned to the specie Mortierella alpina by phylogenetic analysis based on 18S rRNA gene sequences. This strain produces high levels of polyunsaturated fatty acids (PUFAs), including y-(gamma) linolenic acid and arachidonic acid, which when combined represents 48.3% of the total fatty acid content. Fungal extracts demonstrated strong antioxidant activity with the EC50 value of 48.7 µg mL(-1) and also a strong antibacterial activity, mainly against the following bacteria: Escherichia coli, with a MIC of 26.9 µg mL(-1) and Pseudomonas aeruginosa and Enterococcus faecalis, both with a MIC of 107 µg mL(-1). A GC-MS analysis of the chloroform fraction obtained from the crude extract revealed the presence of potential antimicrobials (Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)) as the major compounds. Therefore, the M. alpina strain ITA1-CCMA 952 is a promising fungus for the biotechnological production of antibiotics, antioxidant substances and PUFAs. This study highlights the need for more research in extreme environments, such as Antarctica.

A simple method for the determination of malachite green and leucomalachite green residues in fish by a modified QuEChERS extraction and LC/MS/MS.

A simple method using LC/MS/MS was developed and validated to determine residues of malachite green (MG) and leucomalachite green (LMG) in fish fillet. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) technique was used to perform the sample preparation. The optimal extraction and cleanup conditions were established using an experimental design. The validation parameters obtained to determine both MG and LMG complied with the requirements established by regulatory agencies for the presence of such substances in fish, which establish that the method must attain, at least, a minimum required performance limit of 2 ng/g. The accuracy values ranged between 95 and 107%, and the precision values were lower than 11.2%. The method was used in the analysis of tilapia samples (n = 20) commercialized in Campinas, SP, Brazil. None of the samples presented detectable levels of MG or LMG residues.

Bioassay-directed isolation and identification of phytotoxic and fungitoxic acetylenes from Conyza canadensis.

Conyza canadensis (L.) Cronquist syn. (horseweed) is a problematic and invasive weed with reported allelopathic properties. To identify the phytotoxic constituents of the aerial parts, a systematic bioactivity-guided fractionation of the dichloromethane extract was performed. Three active enyne derivatives, (2Z,8Z)-matricaria acid methyl ester, (4Z,8Z)-matricaria lactone, and (4Z)-lachnophyllum lactone, were identified. The lactones inhibited growth of the monocot Agrostis stolonifera (bentgrass) and the dicot Lactuca sativa (lettuce) at 1 mg mL(-1), while the (2Z,8Z)-matricaria acid methyl ester was less active. In a dose-response screening of the lactones for growth inhibitory activity against Lemna paucicostata , (4Z)-lachnophyllum lactone was the most active with an IC50 of 104 µM, while the (4Z,8Z)-matricaria lactone was less active (IC50 of 220 µM). In a fungal direct bioautography assay, the two lactones at 10 and 100 µg/spot inhibited growth of the plant pathogenic fungi Colletotrichum acutatum , Colletotrichum fragariae , and Colletotrichum gloeosporioides . In a dose-response screening of the lactones against six different plant pathogenic fungi, (4Z,8Z)-matricaria lactone was more active than the commercial fungicide azoxystrobin on Col. acutatum , Col. fragariae , and Col. gloeosporioides at 30 µM and about as active as the commercial fungicide captan against Col. gloeosporioides , while (4Z)-lachnophyllum lactone was less active.

Studies on degradation of glyphosate by several oxidative chemical processes: ozonation, photolysis and heterogeneous photocatalysis.

Several different Advanced Oxidation Processes (AOPs) including ozonation at pH 6.5 and 10, photolysis and heterogeneous photocatalysis using TiO(2) as semiconductor and dissolved oxygen as electron acceptor were applied to study the degradation of glyphosate (N-phosphonomethyl glycine) in water. The degree of glyphosate degradation, the reactions kinetic and the formation of the major metabolite, aminomethyl phosphonic acid (AMPA), were evaluated. Ozonation at pH 10 resulted in the maximum mineralization of glyphosate. It was observed that under the experimental conditions used in this study the degradation of glyphosate followed the first-order kinetics. The half-life obtained for glyphosate degradation in the O(3)/pH 10 process was 1.8 minutes.

Leaching and half-life of the herbicide tebuthiuron on a recharge area of Guarany aquifer in sugarcane fields in Brazil.

This study was undertaken to evaluate the degradation and mobility of the herbicide tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N'-dimethylurea) in soil under field conditions, and its potential for leaching and groundwater contamination. A watershed, Espraiado, located over a recharge area in Brazil, was chosen for soil and water studies. At Espraiado, water samples were collected from seven wells at intervals of three months from March 2004 to June 2006 and analyzed for tebuthiuron. Other samples were taken from city wells located outside of the recharge area. To assess the potential movement to the aquifer, tebuthiuron was also applied to trial plots at the recommended label rate of 1.0 kg/ha a.i. in May of 2004, with and without sugarcane coverage, on sandy soil. Soil samples were collected during the years of 2004 and 2005, at depths intervals of 20 cm from soil surface down to 120 cm and analyzed for tebuthiuron at zero, 3, 30, 60, 90, 120, 150, 180, 240, and 300 days after application. There was no clear effect of sugarcane coverage on the tebuthiuron degradation in soils, but it moved faster into the soil where there was no cover. After 180 days there were no measurable residues in the soil, and tebuthiuron was not found below 40 cm depth in any time. Tebuthiuron had a half-life of 20 days under those conditions. No tebuthiuron residue was found in ground water samples at any sampling time.

Application of new high-performance liquid chromatography and solid-phase extraction materials to the analysis of pesticides in human urine.

A method for the simultaneous determination of diuron and linuron pesticides in human urine was developed, using both solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) phases made in our own laboratory. These materials were prepared by sorption of polysiloxanes onto a silica surface, followed by immobilization. The HPLC columns were prepared from poly(methyloctylsiloxane), PMOS, immobilized onto silica with microwave radiation while the SPE cartridges where made with poly(methyloctadecylsiloxane), immobilized thermally. Method validation was performed for diuron and linuron for three fortification levels. The recoveries obtained were 85-103%, the inter- and intra-assay precisions were less than 1.6 and 1.8%, respectively. The limits of quantitation and detection for diuron were 2.4 and 8.0 microg/l and for linuron were 5.0 and 12 microg/l, respectively.

Poly(methyloctadecylsiloxane) immobilized on silica by gamma radiation for use in solid-phase extraction.

This work presents a fast, easy and effective procedure to obtain silica-based C18 type sorbents for use in solid-phase extraction (SPE). The solid-phase was made by depositing a polymer, poly(methyloctadecylsiloxane) (PMODS), on the silica support followed by immobilization using gamma irradiation. The method presents an application of green chemistry in the analytical laboratory, since there are no toxic residues after the synthesis. Water samples spiked with a mixture of pesticides (benomyl, tebuthiuron, diuron, simazine, atrazine and ametryn) were extracted in the off-line mode in order to evaluate the performance of this sorbent.